Cryo-EM structure of native honey bee vitellogenin

Cryo-EM structure of native honey bee vitellogenin | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Cryo-EM structure of native honey bee vitellogenin Hartmut Luecke, Mateu Montserrat-Canals, Kilian Schnelle, Arne Moeller, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4768326/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Jul, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Vitellogenin (Vg) is the main yolk precursor lipoprotein in almost all egg-laying animals. In addition, along its evolutionary history, Vg has developed a range of new functions in different taxa. In the honey bee, Vg has functions related to immunity, antioxidant protection, social behavior and longevity. However, the molecular mechanisms underlying Vg functionalities are still poorly understood. Here, we report the cryo-EM structure of full-length honey bee Vg, onestep purified directly from hemolymph. The structure provides structural insights into the overall domain architecture, including the lipid binding cavity and the previously uncharacterized von Willebrand factor type D domain. A domain of unknown function has been identified as a C-terminal cystine knot domain based on structural homology. Information about post-translational modifications, cleavage products, metal and lipid binding allow an improved understanding of the mechanisms underlying the range of Vg functionalities. The findings have numerous implications for the structure-function relationship of vitellogenins of other species as well as members of the same protein superfamily, which share the same structural elements. Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy Biological sciences/Molecular biology/Post-translational modifications/Glycosylation Biological sciences/Zoology/Entomology Biological sciences/Biochemistry/Proteins/Lipoproteins Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Pleiotropy – or the ability of a single gene to affect multiple phenotypic traits – is a common phenomenon but is generally poorly understood at the molecular level. Interesting examples of pleiotropy are found in the large lipid transfer protein (LLTP) superfamily. LLTPs are primarily responsible for the circulatory transport of lipids in animals, with their emergence linked to the increased need for lipid transport associated with multicellularity 1 . Members of the superfamily include the mammalian apolipoprotein B (apoB), the microsomal triglyceride transfer protein (MTP), vitellogenin (Vg) and insect apolipophorins II/I (apoLp-II/I). Interestingly, some LLTPs have acquired new functionalities along their evolutionary history. Given their circulation in body fluids in relatively high concentrations, these new functions are often linked to immunity. These include involvement in antigen presentation 2 , blood clotting (reviewed in Chap. 2 of Hoeger and Harris 3 ) and a plethora of other functions for apoLp-II/I (reviewed in Chaps. 4 and 5 of Hoeger and Harris 3 ). In particular, pleiotropy appears to be most developed for the egg yolk precursor protein lipo-glyco-metallo-phosphoprotein Vg. Vg is present in almost all egg-laying animals and has been traditionally studied as a female-specific protein in the context of vitellogenesis 4 . During vitellogenesis, Vg synthesis by somatic cell lineages is boosted and the protein is released into circulation, from where it is internalized by the oocytes as the main precursor of yolk proteins. Thus, Vg provides the developing egg with amino acids, ions, lipids and fat-soluble vitamins and hormones. In the last 20 years, data about the immune functions of Vg have emerged in taxa as different as corals 5 , mollusks 6 , 7 , arthropods 8 – 10 and fishes 11 – 17 . Vg has been found to have antibacterial 5 – 9 , 11 – 15 , 17 , 18 and antiviral 16 activities. Vg achieves these by recognizing a range of pathogen-associated molecular patterns (PAMPs) 6 , 18 , directly causing the death of the pathogen 6 , 12 or opsonizing for phagocytosis by immune cells 5 , 11 , 17 – 19 . Interestingly, it has been found that trans-generational immune priming can occur through Vg 10 , suggesting a link between Vg immune and reproductive functions. Vg has also been shown to recognize membranes 20 , 21 and protect from oxidative stress through different mechanisms 21 – 23 . Additionally, in insects, Vg has been found to regulate and functionally interact with different hormones 24 , 25 . For social insects such as bees and ants, Vg governs social roles for sterile workers as a function of nutritional and metabolic status, while controlling the highly variable lifespan of different castes 25 – 29 , most likely through its ability to protect from oxidative damage. More recently, it has been suggested that a cleavage product of Vg can translocate to the nucleus and thus regulate gene expression 30 , hinting at a possible mechanism by which Vg can exert some of its various functions. However, there is limited understanding of the molecular basis for the functional data regarding Vg pleiotropy. Structurally, Vg is characterized by a lipid binding module common for the LLTP superfamily 1 . At the turn of the millennium, a crystal structure of lipovitellin (Lv), the proteolytically processed Vg product obtained from silver lamprey eggs ( Ichthyomyzon unicuspis ), provided a first glimpse of the LLTP lipid binding module, with particular insight on the Vg lipid binding cavity 31 , 32 and its reproductive role as a nutrient source for the developing embryo. However, the structure covered only about 75% of the Vg sequence, with entire domains missing and several flexible stretches. In addition to lipovitellin from silver lamprey (IuLv), the LLTP lipid binding module was experimentally observed within the crystal structure of human MTP, showing a much smaller lipid binding cavity that adapts to the role of the protein, which is not a transporter but a lipid-loading protein for other LLTPs 33 . Recently, cryo-EM also allowed the reconstruction of human MTP from native raw liver lysate 34 . Among vitellogenins, a wide range of structural variation is produced by taxa-specific loops and domain additions 35 . Additionally, different proteolytic cleavage events generate separate chains 35 , 36 that may or may not keep working together as a single unit, as observed for IuLv 31 . In silver lamprey lipovitellin, some domains are missing from the lipid binding module, which is formed by separate protein chains. This molecular complexity is further increased by the presence of splicing variants 9 , multiple nonidentical gene copies in a single species and the existence of Vg-like proteins in some species. Pleiotropy through neo- and sub-functionalization in different taxa seems to be an intrinsic characteristic of Vg evolutionary history 3 7 . Much of the research around Vg pleiotropy has been carried out for the honey bee ( Apis mellifera and Apis cerana ). These widely distributed species highlight the importance of Vg for animal health, with extensive ecologic and economic implications. Here, we report the 3.2 Å resolution cryogenic electron microscopy (cryo-EM) structure of honey bee Vg (AmVg) purified from its hemolymph, the first structure from a non-vertebrate species with nearly full-length coverage. This includes the von Willebrand factor type D (vWD) domain, present also in some other LLTPs 1 , with unknown function and never reported before for a protein of the LLTP superfamily. We also identified a new putative dimerization site in the C-terminal domain, which we classify as a C-terminal cystine knot (CTCK) domain. In addition, the structures reported herein provide insight into vitellogenin post-translational modifications, binding to metals and lipids. The structure of a previously uncharacterized cleavage product with unclear biological significance was also solved at 3.0 Å. All these findings represent a leap forward in our understanding of the multiple molecular mechanisms that underlie Vg pleiotropy in general and for the honey bee in particular. Furthermore, many of the structural elements and domains of vitellogenin here described are shared by other members of the LLTP superfamily. Results The cryo-EM structures of vitellogenin from a native source In order to better understand the molecular mechanisms that allow AmVg to exert its many functions, we determined the cryo-EM structure of AmVg purified from hemolymph of the honey bee to 3.2 Å (Fig. 1 ). The sample was heterogeneous and contained the full-length protein along with a roughly 150 kDa AmVg cleavage product at an abundance similar to that of the full-length protein. Particles of the cleavage product yielded maps of a resolution of 3.0 Å. For both particle classes, AmVg was observed as a monomer, and there was no evidence for dimerization. A general sequence and structure comparison of AmVg with other LLTP structures is shown in Fig. 2 . The lipid binding module As expected, AmVg contains an LLTP lipid binding module, which is characterized by several subdomains, the N sheet, responsible for receptor binding 38 , 39 , the lipid binding cavity itself formed by the A and C-sheets, and the a-helical domain that wraps around the A and C-sheets. The N-sheet is found at the N-terminus and formed by an antiparallel b-sheet wrapped around a central a-helix. The sheet is one strand short of forming a barrel and has strands of very different length, allowing for an overlap between the N-sheet and the A-sheet from the lipid binding cavity to form a b-sandwich, as already observed for IuLv 31 . The N-sheet contains loops of varying length, some of which contain short helices. In one of the loops, a disulphide bridge (C178-C222) conserved in IuLv and human MTP (HsMTP) seems to stabilize a short b-strand that integrates with the A-sheet. Interestingly, after this short b-strand, density is not observed for the rest of the loop (residues 232–245), most likely due to flexibility. The equivalent loop is well resolved for IuLv, containing a second disulphide not conserved for honey bee Vg. This loop might be stabilized upon zinc binding in AmVg, as discussed later. The long and solvent-exposed section at residues 147–160 is only well-resolved for the AmVg cleavage product, where it forms a short helix. The region comprised of residues 340–384 between the N-sheet and the a-helical domain corresponds to a polyserine region (polyS) that is characteristic of insect vitellogenins 35 . This region was studied before using nuclear magnetic resonance (NMR) spectroscopy and was highly disordered, with predicted protease binding sites and multiple phosphorylated serine residues preventing its cleavage 40 . Not surprisingly, cryo-EM density for this region is not observed for AmVg. However, at high contour levels, poorly-defined densities can be observed next to the protein at the interface where the loop is found. From the ordered regions of the protein, basic side chains point towards the poorly defined cryo-EM densities (H20, K264, H265, K112, H113, K601 and H602), which is to be expected given the presence of phosphorylated residues with negative charge in the flexible polyserine region. The a-helical subdomain is formed by 17 long a-helices arranged in two layers forming a super-helical right-handed coil. The helices are parallel within each layer and antiparallel between layers. The interface between layers is hydrophobic while the solvent-exposed surface is highly positively charged containing up to 34 positively charged amino acids (Fig. 4 E). The highly charged surface of the subdomain has been associated with membrane binding 21 . Some of the positively charged amino acids contribute to stabilizing the subdomain by means of salt bridges (E435-K450, R498-E526, K514-E551, R547-E609, E617-R629, R664-E695 and R700-D727). Here, some loops between helices are significantly longer than for IuLv. These include the stretch before the domain starts that contains two short helices (residues 387–421), the loop between long helices 3 and 4 (residues 473–488) and the insect-specific loop 21 containing two short helices between helices 9 and 10 (residues 581–604). The structure of the C-sheet is similar to that of the IuLv C-sheet. The helix-containing loop corresponding to residues 842–873 is involved in inter-domain stabilization by wrapping around the vWD domain. This loop is flexible in the absence of the vWD domain, as is the case for IuLv and the AmVg cleavage product we observed. The A-sheet is concave-shaped and significantly larger than the C-sheet. The A-sheet defines most of the lipid binding cavity. Interestingly, the region of the Asheet closest to the front (as defined in Fig. 1 ) of the lipid binding cavity has a different configuration in AmVg compared to IuLv. In IuLv, a semi-disordered and highly polar glutamine-rich mini domain of unknown function 3 1 is present in the region, creating significantly different openings to the lipid binding cavity. At the top of the lipid binding cavity, vertebrate vitellogenins have a loop corresponding to the phosvitin chain. This serine-rich region, similar but not functionally equivalent to the polyserine region in insect vitellogenins, is released by proteolytic cleavage and not observed associated to IuLv 3 1 . In the loop where phosvitin is observed for the IuLv structure, AmVg contains a long helix with non-structured stretches at both ends that sit on top of the A-sheet (residues 1144–1189). Compared to IuLv, the loops extending towards the vWD domain are longer. The loop comprising residues 1291–1336 even contains three a-helices with their positions stabilized by a disulphide bond (C1310-C1324). These loops, together with slightly longer b-strands, effectively decrease the exposure of the lipid cavity to the solvent and allow an increased interaction surface between the vWD domain and the A-sheet. The Asheet is capped off by a long a-helix that establishes the edge of the lipid binding cavity and is conserved for lamprey Lv. The A- and C-sheet are connected by an a-helix running perpendicular to the sheets that establishes the back of the lipid binding cavity (green arrowhead in Fig. 1 c, darker orange in Figs. 1 a and 2 a). The vWD and CTCK domains At the C-terminal end of the lipid binding module, we observe the vWD domain. It is connected to the lipid binding module through a flexible region that shows no density in our maps (residues 1411–1432). The vWD domain sits at the top-back of the lipid cavity opening between the A-sheet and the C-sheet. It is characterized by a beta sandwich, with a long loop (residues 1573 − 1492) involved in interaction with the C-sheet and a long, mostly unstructured region at the C-terminal end including 3 short helices. The vWD domain is stabilized by a range of disulphide bonds that are conserved from its functionally unrelated homologs studied in humans including mucins and the von Willebrand factor (vWF) (C1444-C1598, C1466-C1634, C1615-C1650) 41 , 42 . In mucins and vWF, the vWD domain is found with the accompanying modules C8-3 and trypsin inhibitor-like (TIL), responsible for homodimerization stabilized through intermolecular disulphide bond formation. Importantly, in all vitellogenins, C8-3 and TIL3 modules are absent along with the vWD domain (Fig. 3 ). Furthermore, there are no cysteines that are not involved in intramolecular disulphide bonds. Therefore, no cysteines are available to form additional intermolecular disulphide bonds. No density is observed for AmVg after the last disulphide bond (residues C1615-C1650, where the protein is predicted to have a small C-terminal domain of unknown function linked to the vWD domain through a long (35 residue) flexible linker (Supplementary Figure S1). The C-terminal domain has been suggested to participate in gating the lipid binding cavity 43 . Structural alignment based on the AlphaFold 2 prediction of AmVg for the C-terminal domain using Foldseek 44 shows it is a C-terminal cystine knot domain (CTCK) (Fig. 3 b). CTCK domains are often found for vWD domain-containing proteins such as mucins and the vWF. CTCK domains stabilize homodimerization through intermolecular disulphide bonds. The combination of homodimerization through vWF and CTCK domains leads to the formation of long concatemers for mucins and the vWF (Fig. 3 c) 41 , 45 . Glycosylation Conservation of glycosylation among different vitellogenins is generally low. Of the three N ‑glycosylations predicted from the sequence for honey bee Vg from sequence analysis 49 , only one glycosylation has been identified: a covalently linked carbohydrate at N296 in the N-sheet (Fig. 4 D). Vg N-linked glycans contain the fundamental unit of high-mannose oligosaccharides from both vertebrates and invertebrates, with varying amounts of glucose residues at the ends 50 – 52 . The exact identity of the glycan tree is unknown, but the identity of the residues modelled at the base of the tree is clear based on the cryo-EM density, matching the NMR studies by Osir et al. 51 . Our structures include the first two N-acetyl glucosamines (GlcNAc) as well as three mannose residues. The rest of the residues are flexible without cryo-EM map density. The base of the glycosylation tree is stabilized by long structured loops of the N-sheet. The loops are not present in the vertebrate IuLv structure, where no equivalent asparagine residue that could be subject to N -glycosylation can be found at the surface of the N-sheet. Lipid binding Vitellogenins contain approximately 16% of their mass in lipids 32 , 52 . These include mostly but not only phospholipids. The binding is generally non-specific with the lipid binding residues showing very low conservation 32 , 33 . The headgroup of only one phospholipid can be modelled in AmVg, while small density blobs likely belonging to part of lipid chains are observed in the lipid binding cavity (Fig. 4 a to c). This general lack of lipid cryo-EM density in AmVg shows the generally disordered and dynamic nature of lipid interactions under native conditions. The lipid binding cavities of AmVg (33,506 Å 3 ) and IuLv (33,179 Å 3 ) have similar volumes, although the typical funnel shape of the IuLv cavity is not the same for AmVg, with the narrow part of the funnel being completely cut off from solvent access. The shape of the cavity for the cleavage product of AmVg is similar to that of the uncleaved protein, while the volume (16,168 Å 3 ) is significantly smaller as a result of a shorter A-sheet that is wrapped around itself. The polarity of the lipid binding cavity is also similar between AmVg and IuLv. Positively charged residues at the base of the cavity are thought to interact with phospholipid headgroups while the less polar sides and top of the cavity are able to accommodate non-polar lipids, as was originally described for IuLv 32 . The most obvious difference between the lipid binding cavities of AmVg, its cleavage product and IuLv is their accessibility and solvent exposure, as described later in the section on the cleavage product of AmVg. Our analysis of the dynamics of AmVg using cryo-EM data processing methods (described in the methods section) did not yield an indication of large opening/closing motions for the lipid binding cavity. In IuLv, lipids not only bind in the main lipid cavity but also in a small cavity in the N-sheet domain. In the case of AmVg, no such cavity is observed as the equivalent region contains extra protein density. Such density is present due to the longer linker between the polyserine region and the a-helical domain. Putative metal ion binding sites Vg is known to provide zinc ions for developing oocytes and the metal ions have been suggested to play a role in its antioxidant activities 21 . The number of zinc ions carried by Vg varies in different species 53 – 55 , with histidine residues known to play a central role in zinc binding 56 . For the honey bee, it was recently determined that an average of 3.5 zinc ions bind to each Vg molecule 57 . Importantly, no metal chelators were used during the purification of AmVg from honey bee hemolymph. We identified three putative binding sites in our structures, however, there is no clear density for metal ions at most of these positions. Further information on the binding of metals to AmVg can be obtained from the latest release of AlphaFold 58 . The putative metal ion binding sites are shown in Fig. 5 . Site 1 is in the loop of the N-sheet that forms a short b-strand and interacts with the A-sheet, involving residues H229 and H926, where the two histidine side chains are in close proximity (Fig. 5 b). Although there is no clear density for a metal ion, the flexible loop from residues 232–245 may be stabilized by the presence of a Zn 2+ ion, where E239 would contribute to the coordination of the cation (Fig. 5 a). Site 2 is very close to site 1 and formed by H587 and H593, in an insect-specific loop of the ahelical domain that contains two short helices (Fig. 5 c). The helices are oriented such that the histidine side chains are positioned to coordinate a metal cation. The cryo-EM density of the cleavage product of AmVg seems to indicate the presence of a coordinating water next to the poor density for the putative zinc ion. No other residues in the proximity seem to be available to complete the coordination sphere of a zinc cation. Site 2 is also predicted in the AlphaFold 3 model as a zinc-binding site, with pLDDT score for the ion of 78.24. Site 3 is formed by H990 and H1045 and again seems to involve only two histidine side chains, although there is density between the side chains likely indicating the presence of a cation (Fig. 5 d). All putative sites described are solvent exposed, either at the protein surface or in an aqueous cavity easily accessible through loop motion (Site 3). Given the incomplete coordination spheres and the quality of the cryoEM densities we have not included any metal cations in our models. None of the putative sites herein described are conserved for lamprey Lv. An additional and highly conserved Ca 2+ binding site is believed to be part of the vWD domain based on sequence alignments with known structures of functionally unrelated homologues 59 as well as the AlphaFold 3 prediction (pLDDT score for the ion of 65.15). The quality of the cryo-EM density in the area, however, is not sufficient to draw any conclusions regarding metal binding, although the position of the protein backbone allows ion coordination as predicted (Figure S2). The AlphaFold predictions for AmVg Since its development, AlphaFold 2 60 has revolutionized the world of structural biology. For honey bee Vg, the AlphaFold 2 structure prediction is surprisingly accurate (Supplementary Figure S1). AlphaFold 2 was able to correctly predict the overall domain organization of the protein, completely unknown for the vWD domain with respect to the lipid binding module. In addition, it predicted the fold of individual domains and long structured loops with astonishing accuracy. The root mean square deviation (RMSD) value of the main chain comparing our cryo-EM AmVg structure and the AlphaFold 2 model is 2.35 Å. The AI-generated model also provides information about the C-terminal domain (CTCK) that is not observed in our experimental structure. However, a lipoprotein rich in post-translational modifications such as Vg also highlights the current limitations of AlphaFold 2. No information about cleavage products, metal binding sites –including side chain conformations and densities for the metals– or glycosylations can be obtained from the AlphaFold 2 model. The recent development of AlphaFold 3 58 addresses some of these limitations and provides extra insight into the binding of metals by AmVg. The AlphaFold 3 model for AmVg was generated using the AlphaFold Server (alphafoldserver.com). By manually adding the desired metal ions as input for the prediction, the model predicts a calcium ion that is not observed experimentally in the vWD domain and provides extra support for zinc binding in one of the binding sites as discussed in the previous section. Compared to the cryo-EM structure, the AlphaFold 3 model shows a root mean square deviation (RMSD) in the main chain of 1.74 Å, showing an improvement compared to AlphaFold 2. The presence of a 150-kDa cleavage product of AmVg During cryo-EM data processing, we also determined the structure of a cleavage product of AmVg found in numbers similar to those of the full-length protein. A 150-kDa cleavage product in honey bee hemolymph has been described previously for samples obtained from the hemolymph of the insect 49 , 52 . However, most of the cleavage of AmVg occurred after purification, with the presence of the cleavage product increasing over time as observed with denaturing polyacrylamide gel electrophoresis. For regions that are common for the cleavage product and the full-length protein, the fold is identical, with a main-chain RMSD of 0.65 Å. The biggest deviations are close to the cleavage site, where the A-sheet of the cleavage product is substantially more wrapped around itself, effectively decreasing the volume of the lipid binding cavity. Although smaller, the lipid binding cavity is almost intact, with density up to residue 1276 in the A-sheet, with some arguable evidence at low contouring levels for some residues of the next strand being present (residues 1285–1292) (Fig. 6 b). Therefore, a small part of the A-sheet and the whole vWD domain are missing in the cleavage product of AmVg. This effectively increases the accessibility of the lipid cavity when compared to full-length AmVg, both at the front and at the top-back (Fig. 2 b). Interestingly, through loss of the vWD domain the cleavage product displays a domain composition more similar to that of lipovitellin. Insect vitellogenins are known to be processed post-translationally by subtilisin-like proteases recognizing the motif R/K XX R/K 35 . However, no evidence for cleavage at such motifs has been described previously for the suborder apocrita, to which bees and ants belong. Honey bee Vg contains several such motifs, any of which could constitute the cleavage site. One of these motifs —Motif 1— is found where the density for the cleavage product ends near residue 1276 (Motif 1: R YG K , residues 1274 to 1277). A second motif can be found immediately after (Motif 2: K GE R , residues 1281 to 1284), although no density is observed for the residues between the two motifs. The residues corresponding to these two motifs are at the beginning and end of a short loop in the A-sheet (Fig. 6 A). Interestingly, the only disulphide bond in the b-strands of the A-sheet of the full-length AmVg is found stabilizing this short loop (C1242-C1279). The disulphide bond is conserved for IuLv but not observed at all for the cleavage product of AmVg. However, weak density for the b-strand immediately adjacent could be evidence that a partly flexible stretch of amino acids is still present. Two overlapping protease-recognition motifs in a loop shortly after are therefore also cleavage site candidates (Motif 3: R GN K , residues 1316 to 1319 and Motif 4: K IL R , residues 1319 to 1322). Discussion The cryo-EM structures presented herein represent the first of a non-vertebrate vitellogenin and the first of a full-length LLTP containing a vWD domain. Furthermore, due to the native source of the protein, we have been able to identify post-translational modifications, metal binding sites and a cleavage product of AmVg. However, a lot is still unknown about the molecular details of AmVg. The function of the vWD domain and the C-terminal domains of vitellogenins and other LLTPs remains elusive. Recently, copper binding was reported for the vWD domain D1 of human mucin 2 61 . However, the residues involved in copper coordination are not conserved in AmVg. As presented in the results section, proteins containing vWD and CTCK domains are able to form extensive concatemers through dimerization and subsequent linkage by disulphides both close to the N-terminus (vWD domain and accompanying modules) and the C-terminus (CTCK domains) (Fig. 3 a). Such covalent concatemers confer mechanical stress resistance to secreted mucins and vWF in blood, which is required for their functions 41 , 47 . However, AmVg lacks the cysteine residues that are involved in intermolecular disulphide formation, both within the vWD domain 59 and in the CTCK domain newly identified here (Fig. 3 c). Thus, AmVg contains the two domains required for formation of concatemers but lacks the main structural features needed for their stabilization. It cannot be ruled out that there are inaccuracies in the AlphaFold 2 prediction of the CTCK domain and that some cysteines predicted to be involved in intramolecular interactions might in fact be available for intermolecular interactions. However, some cystine knot containing cytokines such as chorionic gonadotropin (CG) form dimers without any intermolecular disulphide bond stabilization (Fig. 3 b) 48 . Vg is known to be able to form dimers 59 , although their biological significance is unknown. The CTCK domain is therefore a strong candidate for a dimerization domain for vitellogenin (Fig. 3 c). In our experimental structures, none of the putative Zn 2+ binding sites shows a complete coordination sphere. We only observe clear cryo-EM density for the pairs of histidine side chains involved in metal binding. There is, however, density that seems to correspond to a metal cation in two of the sites and density for a third coordinating group, likely water, in one of the sites. Therefore, AmVg likely binds Zn 2+ with low affinity. Loose binding of Zn 2+ might be desirable for the specific needs of Vg as a transporter, where the zinc ions need to be able to dissociate easily when required in the oocyte. Low affinities for metal ions might also be statistically compensated for by the presence of several binding sites. The presence of multiple binding sites might also allow the evolutionary plasticity required for the protein to retain its Zn 2+ binding functions in different species without specific binding sites being highly conserved. In addition, keeping a low number of coordinating residues might allow the binding not only of Zn 2+ but also of other metal cations. The biological relevance of the cleavage product of honey bee Vg remains to be established. The presence of a 150-kDa cleavage product in honey bee hemolymph has been described previously for samples obtained from the hemolymph of the insect 49 , 52 . A 150-kDa AmVg fragment has also been identified as more abundant in samples purified from the fat body of the insect 49 , compared to those from hemolymph. The fragment was named fat-body vitellogenin (fbVg) and was described as lacking the N-sheet subdomain based on a lower concentration of matching hits in the N-sheet from liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) 49 . However, in the same study fbVg was shown to contain Nlinked glycosylations 4 9 , which our cryo-EM study only observed in the N-sheet subdomain, supporting the fact that fbVg contains the N-sheet. The cleavage fragment of AmVg observed in this study could correspond to fbVg, which could thus be the 150-kDa fragment that co-purifies at low abundance together with full-length AmVg obtained from honey bee hemolymph. The relative abundance of the cleavage product could increase over time in storage through the action of small amounts of proteases remaining after purification. The exact cleavage site remains unknown, with all the motifs presented in the results section as possible candidates. The AmVg cleavage product described here has its lipid binding cavity almost intact and more accessible than full-length AmVg. If a biological significance is demonstrated, it is tempting to assume that the fragment is mostly involved in lipid binding related functions such as storage of lipids in the fat body. Since the cleavage fragment is also present in the hemolymph, it could also be involved in vitellogenesis. The cleavage product contains all the structural elements related to vitellogenesis functionalities. These include receptor binding for internalization in the oocyte, lipid and full Zn 2+ binding used as a nutrient source and PAMP recognition for trans-generational immune priming. Remarkably, the cleavage product seems to be structurally analogous to the type-C vitellogenins from fish, which lack the vWD and the CTCK domains (named b-component in vertebrate vitellogenins) as well as the phosvitin chain, which is not present in invertebrate Vg 3 6 . The function of fish type-C vitellogenins and their role in vitellogenesis – if any – remain unknown. An alternative interpretation is that the 150 kDa AmVg fragment from the hemolymph and fbVg are different, though of similar size. Indeed, protein product formation can proceed differently in intra or extra cellular compartments that have different biochemical environments 62 , 63 . By such mechanisms, AmVg can be processed differently in hemolymph vs. fat body cells. For example, different cleavage products are identified in fat body and eggs for cockroach 35 . A sequence analysis of insect vitellogenins identified cleavage motifs (R/KXXR/K) at both the N- and C-terminal, often in the same molecule 35 , 64 . Glycosylation is a modification that affects the availability of protease cleavage sites 64 , and more than one potential glycosylation site has been identified for AmVg 49 . Glycosylation, moreover, is highly variable and non-permanent 65 changing with respect to time, tissue, subcellular location 66 , organismal health status 65 , 67 , as well as with protein subtypes 68 . Through the lens of this cumulative information, we cannot rule out that AmVg has more than one fragmentation and glycosylation possibility. The 150 kDa fragment observed in hemolymph, thereby, can be an alternative cleavage product compared to fbVg 49 . The relevance of Vg as a target for AI-based technologies to predict protein structures is highlighted by an article where it was used to exemplify the upcoming revolution in structural biology 69 . Although some important features of such a complex protein are missing in the AlphaFold predictions, the quality of the AlphaFold 2 model compared to our experimental structures shows high accuracy for the structure prediction of species-specific structural features of Vg. This suggests that AlphaFold is useful for structural phylogenetic studies of Vg and other Vg-like proteins. The presence of certain domains and subdomains, together with their architecture and functional data can easily provide important information on neo-functionalization. AI-based structure prediction for vitellogenins will become even more relevant after the advent of AlphaFold 3 58 , given its reported ability to predict protein structures with non-protein ligands such as metal ions, lipids and covalent glycosylation. Vitellogenin from the honey bee is a target in the ongoing CASP 16 competition (target T1210). Overall, the structures described herein significantly advance our understanding of vitellogenins and their molecular complexity, both for all egg-laying animals but for vertebrates and the honey bee in particular. New insight has been obtained into AmVg domain arrangement for the previously uncharacterized vWD domain as well as for several new structural features not observed in the vertebrate IuLv structure. The molecular complexity of vitellogenins is the basis of its pleiotropy, with taxa-specific variations underlying the different neo- and sub-functionalizations the protein has developed over time in widely different animal groups. Furthermore, valuable information about the honey bee specific glycosylation profile, metal binding abilities and cleavage products has been obtained. The results presented here provide the molecular basis for further indepth investigation of the particular details of Vg functionalities and binding abilities, including comparative studies among multiple taxa and for other related LLTPs. Methods Protein extraction and purification To obtain purified AmVg, 1–10 µL of honey bee hemolymph were collected and diluted 1/10 in 0.5 M Tris/HCl pH 7.6, using BD needles (30 G) as described earlier 70 . The diluted protein was filtered using a 0.2 µm syringe filter. Vg was then subjected to ion-exchange chromatography using a HiTrap Q FF 1 mL column equilibrated in 0.5 M Tris/HCl pH 7.6 and eluted with a NaCl gradient in 0.5 M Tris/HCl pH 7.6 up to 0.45 M NaCl. 400–450 µL of diluted hemolymph were manually injected and Vg eluted at a conductivity of 15–22 mS·cm − 1 . Fractions were collected, pooled and concentrated using an Amicon Ultracel 100 kDa membrane centrifuge filter (Merck KGaA, Darmstadt, Germany). Fraction purity was verified by running SDS-PAGE. Protein concentration was measured with Qubit. Cryo-EM grid preparation and data collection Cryo-EM grids preparation and data collection was carried out at the cryo-EM Sweden National facility at SciLife lab in Stockholm according to standard procedures. Grids were prepared and plunge frozen in liquid ethane using a FEI Vitrobot Mark IV. AmVg at a concentration of 1.2 mg/mL was applied to a set of different grids with different treatments in order to overcome the preferential orientation problem we had observed previously for the sample. From those, two datasets were collected, one for a glow discharged Quantifoil R2/1 300 mesh holey carbon cooper grid (Quantifoil Micro Tools, Germany) and one for a Au 1.2/1.3 300 mesh holey carbon grid that was not glow discharged. Using the EPU control software, 28,406 movies were recorded on a FEI Titan Krios operating at 300 kV. The nominal magnification was 105,000x, corresponding to a physical pixel size of 0.8464 Å. The dose rate was set to 15.8 e − /px/s, and the total exposure time was 2.83 s, resulting in a total dose of 62.4 e − /Å 2 . Each movie was split into 40 frames of 0.07075 s. Nominal defocus range was − 0.8 µm to -2.8 µm in 0.2 µm steps. Image processing and model building The dataset underwent processing utilizing CryoSPARC 4.1 71 as detailed in the Supplementary Figure S3. The preprocessing of the movies involved patch-based motion correction and patch-based contrast transfer function (CTF) estimation. Micrographs exhibiting a CTF fit greater than 3.5 Å were discarded, leaving a final dataset of 23,127 images. Initial 2D classes were derived and used for subsequent template-based particle picking, generating a stack of 23.3 million particles. These were extracted within a box size of 320 and then subjected to Fourier cropping down to 80 pixels. To further refine the stack, two additional rounds of 2D classification were performed prior to re-extraction of the particles at a box size of 320 and cropping to 160 pixels. The remaining 10.9 million particles underwent two additional rounds of 2D classification, yielding a residual stack of 9.4 million particles. The final extraction was carried out with a box size of 320 pixels without additional cropping. These particles were then utilized for ab-initio 3D reconstruction, with the most promising classes chosen for further heterogeneous refinement. This step yielded two distinct classes: one for the smaller cleavage product and one for the full-length protein. These stacks were then further refined through two cycles of heterogeneous refinement, followed by non-uniform refinement to evaluate potential improvements. A final local refinement was performed to enhance the quality of the consensus maps further, resulting in final maps with resolutions of 3.2 Å for the full protein and 3.0 Å for the smaller cleavage product. The estimated local resolution for the two reconstructed maps is shown in Figure S4. The flexibility and dynamics of AmVg, both for the full-length protein and the cleavage product were also analyzed in cryoSPARC 4.1 71 . 3D classification and 3D variability jobs did not provide obvious clues into alternative conformations, while flexible refinement did not produce final densities of better quality than local and non-uniform refinement. The AlphaFold 2 structure prediction for AmVg (Uniprot ID: Q868N5) was docked in the cryo-EM map in UCSF Chimera 72 . From there, the model was manually fitted and rebuilt in Coot 73 (see supplementary information for details on map quality, model building and regions not included in the final models). The smaller cleavage product was modelled manually from the full-length AmVg model. Iterative cycles of real-space refinement were done in Phenix 74 with further model rebuilding in Coot. Final geometries were assessed with Molprobity 75 . Glycan geometries were assessed with Privateer 76 . Structural analysis, comparison and figures were done in PyMOL (Schrödinger, LLC). The volumes of the lipid binding cavities were calculated with CASTp 77 using a probe of 2.5 Å radius optimized to exclude areas outside of the lipid binding cavities. The cryo-EM maps were deposited in the Electron Microscopy Data Bank with accession codes EMD-19842 and EMD-19843 for full-length AmVg and the 150 kDa cleavage product of AmVg, respectively. The atomic coordinates of the models were deposited in the Protein Data Bank with accession numbers 9ENR for full-length AmVg and 9ENS for the cleavage product of AmVg. Alphafold 3 model generation The Alphafold 3 model was generated using release 23/6/2024 of the AlphaFold server (alphafoldserver.com). The sequence for AmVg (Uniprot ID: Q868N5) was used as input, together with three Zn 2+ ions and one Ca 2+ ion. Additionally, the glycosylation observed in our cryo-EM density was input to occur at residue N298. Abbreviations AmVg honey bee vitellogenin apoB mammalian apolipoprotein B apoLp-II/I insect apolipophorins II/I CG chorionic gonadotropin cryo-EM cryogenic electron microscopy CTCK domain C-terminal cystine knot domain fbVg fat-body vitellogenin GlcNAc N-acetyl glucosamine HsCG human chorionic gonadotropin HsMTP human microsomal triglyceride transfer protein HsvWF human von Willebrand factor IuVg silver lamprey vitellogenin IuLv silver lamprey lipovitellin LC-MS/MS liquid chromatography coupled to tandem mass spectrometry LLTP superfamily large lipid transfer protein superfamily Lv lipovitellin MTP microsomal triglyceride transfer protein NMR nuclear magnetic resonance PAMP pathogen-associated molecular pattern polyS region polyserine region TIL domain trypsin inhibitor-like domain Vg vitellogenin vWF von Willebrand factor vWD domain von Willebrand factor type D domain Declarations Acknowledgments Cryo-EM data were collected at the Cryo-EM Swedish National Facility funded by the Knut and Alice Wallenberg, Family Erling Persson and Kempe Foundations, SciLifeLab, Stockholm University and Umeå University. We thank Marta Carroni and Dustin Morado for facilitating grid freezing, remote data collection and initial assistance with data processing. We thank Gabriele Cordara for his help during model building and Marta Sanz-Gaitero for her work during the initial negative-staining EM studies of AmVg. We acknowledge The Research Council of Norway grant number 262137 and 335244 for funding toward running costs and positions. This project received funding from the European Union's Horizon Europe Research and Innovation Programme under grant agreement number 101087571 (H.L). Arne Moeller was supported by SFB 944 and SFB 1557 and the DFG INST190/196-1 FUGG. Author contributions M.M.-C. performed cryo-EM data processing, built the structural models and performed structural analysis under the supervision of H.L. and E.C. E.C. also performed initial cryo-EM data processing. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4768326","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":336348069,"identity":"0e25c7da-8021-4ee6-a02b-390a3112ff05","order_by":0,"name":"Hartmut Luecke","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsklEQVRIiWNgGAWjYBACAxB6UCEH4T0gWkvCGWMGHhAvgYFBgjgtiW2kaDGXSN74IHGegbw9A4/ZgwSGO3UEtVjOSCs2SNxmYNjDwGNukMDwjAiH3cgxk0jc9ieBB2iLRALDYaK0mP9InGNAmhYzhsQGkrSceVYskXAM6JfDbGUSCQbPJBsIajmevPHDhxoDefb25m0SHyru8BO0BQGYwSYcIEEHFJChZRSMglEwCoY9AABbrjY3r4uCNAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-4938-0775","institution":"Nova University Lisbon","correspondingAuthor":true,"prefix":"","firstName":"Hartmut","middleName":"","lastName":"Luecke","suffix":""},{"id":336348070,"identity":"7d143da5-1e3f-4134-ab9e-d0eedbd9794e","order_by":1,"name":"Mateu Montserrat-Canals","email":"","orcid":"","institution":"University of Oslo","correspondingAuthor":false,"prefix":"","firstName":"Mateu","middleName":"","lastName":"Montserrat-Canals","suffix":""},{"id":336348071,"identity":"2905653e-81f1-4a7b-b096-722671053061","order_by":2,"name":"Kilian Schnelle","email":"","orcid":"https://orcid.org/0000-0001-8808-594X","institution":"University of Osnabrück","correspondingAuthor":false,"prefix":"","firstName":"Kilian","middleName":"","lastName":"Schnelle","suffix":""},{"id":336348072,"identity":"5416b1a1-53bc-49c6-bc1e-e7e93670c46d","order_by":3,"name":"Arne Moeller","email":"","orcid":"https://orcid.org/0000-0003-1101-5366","institution":"Osnabrueck University","correspondingAuthor":false,"prefix":"","firstName":"Arne","middleName":"","lastName":"Moeller","suffix":""},{"id":336348073,"identity":"8cbb0c07-10c9-4be6-a36d-964d34fc41e2","order_by":4,"name":"Gro Amdam","email":"","orcid":"","institution":"Arizona State University","correspondingAuthor":false,"prefix":"","firstName":"Gro","middleName":"","lastName":"Amdam","suffix":""},{"id":336348074,"identity":"03326935-ec45-47fb-8478-e931d66a6b55","order_by":5,"name":"Øyvind Halskau","email":"","orcid":"","institution":"University of Bergen","correspondingAuthor":false,"prefix":"","firstName":"Øyvind","middleName":"","lastName":"Halskau","suffix":""},{"id":336348075,"identity":"415ac616-c1a4-457c-811d-ae431ef1c93a","order_by":6,"name":"Vilde Leipart","email":"","orcid":"","institution":"Norwegian University of Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Vilde","middleName":"","lastName":"Leipart","suffix":""},{"id":336348076,"identity":"809a01b2-a62a-4983-8ff6-fe725c067c35","order_by":7,"name":"Eva S. Cunha","email":"","orcid":"","institution":"Proteros Biostructures GmbH","correspondingAuthor":false,"prefix":"","firstName":"Eva","middleName":"S.","lastName":"Cunha","suffix":""}],"badges":[],"createdAt":"2024-07-19 14:50:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4768326/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4768326/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-58575-y","type":"published","date":"2025-07-01T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":61940812,"identity":"241f74f9-0055-437c-bdb4-41866235f20a","added_by":"auto","created_at":"2024-08-07 10:18:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1018664,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eThe cryo-EM structure of native AmVg\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. Domain architecture of full-length Vg from Apis mellifera (AmVg). Different domains and subdomains are colored differently, while flexible regions not observed in the experimental structure are shaded in gray. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. Cryo-EM density map of native AmVg at 3.2 Å resolution in different orientations, colored by domain and subdomain. The black arrowhead points to where the flexible polyserine region is located. The red arrowhead points to the C-sheet, while the blue arrowheads point to the A-sheet, which, together with the C-sheet, forms the lipid binding cavity with its concave shape. The green arrowhead points to the helix separating the C and A-sheets at the back of the lipid binding cavity, shown with a darker shade of orange in panel a. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ec\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. Ribbon model of the AmVg structure colored by domain and subdomain as in panel b and a.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-4768326/v1/6fd2cfb841974ea22f71988c.png"},{"id":61940818,"identity":"eb02c28b-afd1-461b-b14d-82619d55563a","added_by":"auto","created_at":"2024-08-07 10:18:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":694653,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eDomain organization and experimental structures of LLTP superfamily members.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. Domain architecture of full-length Vg from the honey bee (AmVg), with a second representation showing flexible regions not observed in the cryo-EM structure shaded in light gray. The domain architecture of the cleavage product of AmVg we observed in our sample is also shown. Below, the domain architecture of Vg from silver lamprey (IuVg) and its cleavage product Lv (IuLv) are shown. Regions not observed experimentally due to flexibility are shaded light gray for the crystal structure of IuLv\u003c/em\u003e\u003csup\u003e\u003cem\u003e32\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e. Finally, the domain architecture of human MTP (HsMTP) for which the crystal structure has been reported is also shown\u003c/em\u003e\u003csup\u003e\u003cem\u003e33\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e.\u0026nbsp;Atomic models of all experimentally solved LLTPs, colored by domain and subdomain.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-4768326/v1/59eb97272b2919421adc6efa.png"},{"id":61940813,"identity":"5ed03dc1-54c0-484d-a295-a0a4120569f1","added_by":"auto","created_at":"2024-08-07 10:18:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":560521,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eThe vWD and CTCK domains\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. Structures of vWD (including the accompanying modules C8-3, TIL3 and E3) and CTCK domains of the human von Willebrand factor (HsvWF)\u003c/em\u003e\u003csup\u003e42\u003c/sup\u003e\u003cem\u003e. Intramolecular disulphide bridges are shown as yellow sticks and cysteines involved in intermolecular disulphide bonds are shown as spheres. A schematic of how such domains allow the formation of long concatemers through intermolecular disulphide bonds is also shown. Such concatemers achieve specific mechanical properties relevant for biological function. Equivalent interactions have been observed for mucins\u003c/em\u003e\u003csup\u003e46,47\u003c/sup\u003e\u003cem\u003e. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e.\u0026nbsp;CTCK domain of the human hormone chorionic gonadotropin (HsCG)\u003c/em\u003e\u003csup\u003e48\u003c/sup\u003e\u003cem\u003e. This CTCK domain can from stable dimers without stabilizing intermolecular disulphide bonds. Intermolecular disulphide bonds are shown as yellow sticks and regions involved in dimerization shown as yellow stretches. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ec\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. Experimental structure of the vWD domain and AlphaFold 2 prediction of the CTCK domain from AmVg. No cysteines are available for the formation of intermolecular disulphide bridges as in the case of vWF or mucins. Vg could achieve dimerization through non-covalent interactions between the CTCK domains, as in the case of the CG CTCK domain (panel b). The PDB IDs of the relevant structures presented are shown in a light purple box.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-4768326/v1/3e42450141a62e98e4875ed0.png"},{"id":61941803,"identity":"a3a6df6f-4abc-4fc7-889f-9e4365a21f91","added_by":"auto","created_at":"2024-08-07 10:34:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":763411,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eDetails of the AmVg structure.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ea \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eand\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e b\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. Unmodelled partial lipid densities in the interior and at the edge of the lipid binding cavity contoured at 7σ. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ec\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. Phospholipid head group identified at the edge of the lipid binding cavity contoured at 7σ. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ed\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. Glycosylation at N296. The quality of the density allows the identification of the N-acetyl groups of the first two N-acetylglucosamine units and the identification of up to three more mannose units at lower contouring levels. Density shown for the higher-resolution cleavage product of AmVg contoured at 1σ. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ee\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. Solvent-facing view of the a-helical domain with the side chains of basic amino acids colored in blue, which have been associated with the ability of Vg to bind membranes\u003c/em\u003e\u003csup\u003e21\u003c/sup\u003e\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-4768326/v1/16fef20bd18e0c3e4824860d.png"},{"id":61940816,"identity":"6bc0c0c8-877a-4bba-b432-c85a89f5ecfe","added_by":"auto","created_at":"2024-08-07 10:18:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":506322,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003ePutative Zn\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e2+\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e\u003cstrong\u003e binding sites of AmVg.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. General overview of the putative zinc ion binding region, at the interface between the N- and A-sheets and the a-helical domain. Arrows point to the ends of the loop not observed between residues 232-245 that might be stabilized upon zinc binding. Panels \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e, \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ec\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e and \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ed\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e show the three putative binding sites in detail. Densities are shown at a contouring level of 7σ. Spherical density can be observed between histidine side chains for Sites 2 and 3, while density for a third ligand (likely water) can be observed in Site 2. The densities shown here represent the higher resolution map obtained for the AmVg cleavage product.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-4768326/v1/9cc5ee4b5ba7b6b829a05adf.png"},{"id":61941326,"identity":"03cd459d-3470-4d3c-b93b-00b1c0d518c8","added_by":"auto","created_at":"2024-08-07 10:26:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":866636,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eThe AmVg cleavage product\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. Location of possible cleavage sites of AmVg in its structure and sequence. Possible motifs for cleavage are colored gray, with basic residues shown in ball‑and‑stick representation and disulphide bonds colored yellow. Numbers have been given to the cleavage sites and letters to the b-strands in the cleavage area. The secondary structure of the segments is indicated under the sequence. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. Density around the cleavage motifs for the cleavage product at two contour levels representing different confidence levels. Strands a and b are clearly visible at normal contour levels (7σ). What could be residual density for strand c (shown as a dashed orange line) is visible only at low contour levels (1σ). The positions of the different possible cleavage motifs are also shown. Map quality around the cleavage area is quite poor and some regions are modelled as alanine sidechains except for glycine and proline residues. More details regarding the areas modelled as poly-alanine can be found in the supplementary data.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-4768326/v1/fd6735542d5eff269651dcdd.png"},{"id":85829234,"identity":"f1f9497e-7a7b-4194-9843-b6ddd7286461","added_by":"auto","created_at":"2025-07-02 07:35:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5506445,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4768326/v1/0084f02c-dc8f-45cd-8197-bb7af9161695.pdf"},{"id":61941324,"identity":"55a40e78-9f83-49c7-9652-1036e3cd286f","added_by":"auto","created_at":"2024-08-07 10:26:01","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4536505,"visible":true,"origin":"","legend":"","description":"","filename":"SUPPLEMENTARYDATA.docx","url":"https://assets-eu.researchsquare.com/files/rs-4768326/v1/3ed9ae5cf41c55d355f7d49c.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Cryo-EM structure of native honey bee vitellogenin","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePleiotropy \u0026ndash; or the ability of a single gene to affect multiple phenotypic traits \u0026ndash; is a common phenomenon but is generally poorly understood at the molecular level. Interesting examples of pleiotropy are found in the large lipid transfer protein (LLTP) superfamily. LLTPs are primarily responsible for the circulatory transport of lipids in animals, with their emergence linked to the increased need for lipid transport associated with multicellularity\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Members of the superfamily include the mammalian apolipoprotein B (apoB), the microsomal triglyceride transfer protein (MTP), vitellogenin (Vg) and insect apolipophorins II/I (apoLp-II/I). Interestingly, some LLTPs have acquired new functionalities along their evolutionary history. Given their circulation in body fluids in relatively high concentrations, these new functions are often linked to immunity. These include involvement in antigen presentation\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, blood clotting (reviewed in Chap.\u0026nbsp;2 of Hoeger and Harris\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e) and a plethora of other functions for apoLp-II/I (reviewed in Chaps.\u0026nbsp;4 and 5 of Hoeger and Harris\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e). In particular, pleiotropy appears to be most developed for the egg yolk precursor protein lipo-glyco-metallo-phosphoprotein Vg.\u003c/p\u003e \u003cp\u003eVg is present in almost all egg-laying animals and has been traditionally studied as a female-specific protein in the context of vitellogenesis\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. During vitellogenesis, Vg synthesis by somatic cell lineages is boosted and the protein is released into circulation, from where it is internalized by the oocytes as the main precursor of yolk proteins. Thus, Vg provides the developing egg with amino acids, ions, lipids and fat-soluble vitamins and hormones. In the last 20 years, data about the immune functions of Vg have emerged in taxa as different as corals\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, mollusks\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, arthropods\u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e and fishes\u003csup\u003e\u003cspan additionalcitationids=\"CR12 CR13 CR14 CR15 CR16\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Vg has been found to have antibacterial\u003csup\u003e\u003cspan additionalcitationids=\"CR6 CR7 CR8\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan additionalcitationids=\"CR12 CR13 CR14\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e and antiviral \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e activities. Vg achieves these by recognizing a range of pathogen-associated molecular patterns (PAMPs)\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, directly causing the death of the pathogen\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e or opsonizing for phagocytosis by immune cells\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Interestingly, it has been found that trans-generational immune priming can occur through Vg\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, suggesting a link between Vg immune and reproductive functions. Vg has also been shown to recognize membranes\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e and protect from oxidative stress through different mechanisms\u003csup\u003e\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Additionally, in insects, Vg has been found to regulate and functionally interact with different hormones\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. For social insects such as bees and ants, Vg governs social roles for sterile workers as a function of nutritional and metabolic status, while controlling the highly variable lifespan of different castes\u003csup\u003e\u003cspan additionalcitationids=\"CR26 CR27 CR28\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, most likely through its ability to protect from oxidative damage. More recently, it has been suggested that a cleavage product of Vg can translocate to the nucleus and thus regulate gene expression\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, hinting at a possible mechanism by which Vg can exert some of its various functions. However, there is limited understanding of the molecular basis for the functional data regarding Vg pleiotropy.\u003c/p\u003e \u003cp\u003eStructurally, Vg is characterized by a lipid binding module common for the LLTP superfamily\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. At the turn of the millennium, a crystal structure of lipovitellin (Lv), the proteolytically processed Vg product obtained from silver lamprey eggs (\u003cem\u003eIchthyomyzon unicuspis\u003c/em\u003e), provided a first glimpse of the LLTP lipid binding module, with particular insight on the Vg lipid binding cavity\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e and its reproductive role as a nutrient source for the developing embryo. However, the structure covered only about 75% of the Vg sequence, with entire domains missing and several flexible stretches. In addition to lipovitellin from silver lamprey (IuLv), the LLTP lipid binding module was experimentally observed within the crystal structure of human MTP, showing a much smaller lipid binding cavity that adapts to the role of the protein, which is not a transporter but a lipid-loading protein for other LLTPs\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Recently, cryo-EM also allowed the reconstruction of human MTP from native raw liver lysate\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Among vitellogenins, a wide range of structural variation is produced by taxa-specific loops and domain additions\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Additionally, different proteolytic cleavage events generate separate chains\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e that may or may not keep working together as a single unit, as observed for IuLv\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. In silver lamprey lipovitellin, some domains are missing from the lipid binding module, which is formed by separate protein chains. This molecular complexity is further increased by the presence of splicing variants\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, multiple nonidentical gene copies in a single species and the existence of Vg-like proteins in some species. Pleiotropy through neo- and sub-functionalization in different taxa seems to be an intrinsic characteristic of Vg evolutionary history\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e7\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMuch of the research around Vg pleiotropy has been carried out for the honey bee (\u003cem\u003eApis mellifera\u003c/em\u003e and \u003cem\u003eApis cerana\u003c/em\u003e). These widely distributed species highlight the importance of Vg for animal health, with extensive ecologic and economic implications. Here, we report the 3.2 \u0026Aring; resolution cryogenic electron microscopy (cryo-EM) structure of honey bee Vg (AmVg) purified from its hemolymph, the first structure from a non-vertebrate species with nearly full-length coverage. This includes the von Willebrand factor type D (vWD) domain, present also in some other LLTPs\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, with unknown function and never reported before for a protein of the LLTP superfamily. We also identified a new putative dimerization site in the C-terminal domain, which we classify as a C-terminal cystine knot (CTCK) domain. In addition, the structures reported herein provide insight into vitellogenin post-translational modifications, binding to metals and lipids. The structure of a previously uncharacterized cleavage product with unclear biological significance was also solved at 3.0 \u0026Aring;. All these findings represent a leap forward in our understanding of the multiple molecular mechanisms that underlie Vg pleiotropy in general and for the honey bee in particular. Furthermore, many of the structural elements and domains of vitellogenin here described are shared by other members of the LLTP superfamily.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eThe cryo-EM structures of vitellogenin from a native source\u003c/h2\u003e \u003cp\u003eIn order to better understand the molecular mechanisms that allow AmVg to exert its many functions, we determined the cryo-EM structure of AmVg purified from hemolymph of the honey bee to 3.2 \u0026Aring; (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The sample was heterogeneous and contained the full-length protein along with a roughly 150 kDa AmVg cleavage product at an abundance similar to that of the full-length protein. Particles of the cleavage product yielded maps of a resolution of 3.0 \u0026Aring;. For both particle classes, AmVg was observed as a monomer, and there was no evidence for dimerization. A general sequence and structure comparison of AmVg with other LLTP structures is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eThe lipid binding module\u003c/h2\u003e \u003cp\u003eAs expected, AmVg contains an LLTP lipid binding module, which is characterized by several subdomains, the N sheet, responsible for receptor binding\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, the lipid binding cavity itself formed by the A and C-sheets, and the a-helical domain that wraps around the A and C-sheets. The N-sheet is found at the N-terminus and formed by an antiparallel b-sheet wrapped around a central a-helix. The sheet is one strand short of forming a barrel and has strands of very different length, allowing for an overlap between the N-sheet and the A-sheet from the lipid binding cavity to form a b-sandwich, as already observed for IuLv\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. The N-sheet contains loops of varying length, some of which contain short helices. In one of the loops, a disulphide bridge (C178-C222) conserved in IuLv and human MTP (HsMTP) seems to stabilize a short b-strand that integrates with the A-sheet. Interestingly, after this short b-strand, density is not observed for the rest of the loop (residues 232\u0026ndash;245), most likely due to flexibility. The equivalent loop is well resolved for IuLv, containing a second disulphide not conserved for honey bee Vg. This loop might be stabilized upon zinc binding in AmVg, as discussed later. The long and solvent-exposed section at residues 147\u0026ndash;160 is only well-resolved for the AmVg cleavage product, where it forms a short helix.\u003c/p\u003e \u003cp\u003eThe region comprised of residues 340\u0026ndash;384 between the N-sheet and the a-helical domain corresponds to a polyserine region (polyS) that is characteristic of insect vitellogenins\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. This region was studied before using nuclear magnetic resonance (NMR) spectroscopy and was highly disordered, with predicted protease binding sites and multiple phosphorylated serine residues preventing its cleavage\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Not surprisingly, cryo-EM density for this region is not observed for AmVg. However, at high contour levels, poorly-defined densities can be observed next to the protein at the interface where the loop is found. From the ordered regions of the protein, basic side chains point towards the poorly defined cryo-EM densities (H20, K264, H265, K112, H113, K601 and H602), which is to be expected given the presence of phosphorylated residues with negative charge in the flexible polyserine region.\u003c/p\u003e \u003cp\u003eThe a-helical subdomain is formed by 17 long a-helices arranged in two layers forming a super-helical right-handed coil. The helices are parallel within each layer and antiparallel between layers. The interface between layers is hydrophobic while the solvent-exposed surface is highly positively charged containing up to 34 positively charged amino acids (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). The highly charged surface of the subdomain has been associated with membrane binding\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Some of the positively charged amino acids contribute to stabilizing the subdomain by means of salt bridges (E435-K450, R498-E526, K514-E551, R547-E609, E617-R629, R664-E695 and R700-D727). Here, some loops between helices are significantly longer than for IuLv. These include the stretch before the domain starts that contains two short helices (residues 387\u0026ndash;421), the loop between long helices 3 and 4 (residues 473\u0026ndash;488) and the insect-specific loop\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e containing two short helices between helices 9 and 10 (residues 581\u0026ndash;604).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe structure of the C-sheet is similar to that of the IuLv C-sheet. The helix-containing loop corresponding to residues 842\u0026ndash;873 is involved in inter-domain stabilization by wrapping around the vWD domain. This loop is flexible in the absence of the vWD domain, as is the case for IuLv and the AmVg cleavage product we observed. The A-sheet is concave-shaped and significantly larger than the C-sheet. The A-sheet defines most of the lipid binding cavity. Interestingly, the region of the Asheet closest to the front (as defined in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) of the lipid binding cavity has a different configuration in AmVg compared to IuLv. In IuLv, a semi-disordered and highly polar glutamine-rich mini domain of unknown function\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e1\u003c/sup\u003e is present in the region, creating significantly different openings to the lipid binding cavity. At the top of the lipid binding cavity, vertebrate vitellogenins have a loop corresponding to the phosvitin chain. This serine-rich region, similar but not functionally equivalent to the polyserine region in insect vitellogenins, is released by proteolytic cleavage and not observed associated to IuLv\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e1\u003c/sup\u003e. In the loop where phosvitin is observed for the IuLv structure, AmVg contains a long helix with non-structured stretches at both ends that sit on top of the A-sheet (residues 1144\u0026ndash;1189). Compared to IuLv, the loops extending towards the vWD domain are longer. The loop comprising residues 1291\u0026ndash;1336 even contains three a-helices with their positions stabilized by a disulphide bond (C1310-C1324). These loops, together with slightly longer b-strands, effectively decrease the exposure of the lipid cavity to the solvent and allow an increased interaction surface between the vWD domain and the A-sheet. The Asheet is capped off by a long a-helix that establishes the edge of the lipid binding cavity and is conserved for lamprey Lv. The A- and C-sheet are connected by an a-helix running perpendicular to the sheets that establishes the back of the lipid binding cavity (green arrowhead in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, darker orange in Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eThe vWD and CTCK domains\u003c/h2\u003e \u003cp\u003eAt the C-terminal end of the lipid binding module, we observe the vWD domain. It is connected to the lipid binding module through a flexible region that shows no density in our maps (residues 1411\u0026ndash;1432). The vWD domain sits at the top-back of the lipid cavity opening between the A-sheet and the C-sheet. It is characterized by a beta sandwich, with a long loop (residues 1573\u0026thinsp;\u0026minus;\u0026thinsp;1492) involved in interaction with the C-sheet and a long, mostly unstructured region at the C-terminal end including 3 short helices. The vWD domain is stabilized by a range of disulphide bonds that are conserved from its functionally unrelated homologs studied in humans including mucins and the von Willebrand factor (vWF) (C1444-C1598, C1466-C1634, C1615-C1650)\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. In mucins and vWF, the vWD domain is found with the accompanying modules C8-3 and trypsin inhibitor-like (TIL), responsible for homodimerization stabilized through intermolecular disulphide bond formation. Importantly, in all vitellogenins, C8-3 and TIL3 modules are absent along with the vWD domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Furthermore, there are no cysteines that are not involved in intramolecular disulphide bonds. Therefore, no cysteines are available to form additional intermolecular disulphide bonds.\u003c/p\u003e \u003cp\u003eNo density is observed for AmVg after the last disulphide bond (residues C1615-C1650, where the protein is predicted to have a small C-terminal domain of unknown function linked to the vWD domain through a long (35 residue) flexible linker (Supplementary Figure S1). The C-terminal domain has been suggested to participate in gating the lipid binding cavity\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Structural alignment based on the AlphaFold 2 prediction of AmVg for the C-terminal domain using Foldseek\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e shows it is a C-terminal cystine knot domain (CTCK) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). CTCK domains are often found for vWD domain-containing proteins such as mucins and the vWF. CTCK domains stabilize homodimerization through intermolecular disulphide bonds. The combination of homodimerization through vWF and CTCK domains leads to the formation of long concatemers for mucins and the vWF (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec)\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eGlycosylation\u003c/h2\u003e \u003cp\u003eConservation of glycosylation among different vitellogenins is generally low. Of the three \u003cem\u003eN\u003c/em\u003e‑glycosylations predicted from the sequence for honey bee Vg from sequence analysis\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, only one glycosylation has been identified: a covalently linked carbohydrate at N296 in the N-sheet (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Vg N-linked glycans contain the fundamental unit of high-mannose oligosaccharides from both vertebrates and invertebrates, with varying amounts of glucose residues at the ends\u003csup\u003e\u003cspan additionalcitationids=\"CR51\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. The exact identity of the glycan tree is unknown, but the identity of the residues modelled at the base of the tree is clear based on the cryo-EM density, matching the NMR studies by Osir \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Our structures include the first two N-acetyl glucosamines (GlcNAc) as well as three mannose residues. The rest of the residues are flexible without cryo-EM map density. The base of the glycosylation tree is stabilized by long structured loops of the N-sheet. The loops are not present in the vertebrate IuLv structure, where no equivalent asparagine residue that could be subject to \u003cem\u003eN\u003c/em\u003e-glycosylation can be found at the surface of the N-sheet.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eLipid binding\u003c/h2\u003e \u003cp\u003eVitellogenins contain approximately 16% of their mass in lipids\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. These include mostly but not only phospholipids. The binding is generally non-specific with the lipid binding residues showing very low conservation\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. The headgroup of only one phospholipid can be modelled in AmVg, while small density blobs likely belonging to part of lipid chains are observed in the lipid binding cavity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea to c). This general lack of lipid cryo-EM density in AmVg shows the generally disordered and dynamic nature of lipid interactions under native conditions. The lipid binding cavities of AmVg (33,506 \u0026Aring;\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e) and IuLv (33,179 \u0026Aring;\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e) have similar volumes, although the typical funnel shape of the IuLv cavity is not the same for AmVg, with the narrow part of the funnel being completely cut off from solvent access. The shape of the cavity for the cleavage product of AmVg is similar to that of the uncleaved protein, while the volume (16,168 \u0026Aring;\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e) is significantly smaller as a result of a shorter A-sheet that is wrapped around itself. The polarity of the lipid binding cavity is also similar between AmVg and IuLv. Positively charged residues at the base of the cavity are thought to interact with phospholipid headgroups while the less polar sides and top of the cavity are able to accommodate non-polar lipids, as was originally described for IuLv\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. The most obvious difference between the lipid binding cavities of AmVg, its cleavage product and IuLv is their accessibility and solvent exposure, as described later in the section on the cleavage product of AmVg. Our analysis of the dynamics of AmVg using cryo-EM data processing methods (described in the \u003cspan refid=\"Sec12\" class=\"InternalRef\"\u003emethods\u003c/span\u003e section) did not yield an indication of large opening/closing motions for the lipid binding cavity.\u003c/p\u003e \u003cp\u003eIn IuLv, lipids not only bind in the main lipid cavity but also in a small cavity in the N-sheet domain. In the case of AmVg, no such cavity is observed as the equivalent region contains extra protein density. Such density is present due to the longer linker between the polyserine region and the a-helical domain.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePutative metal ion binding sites\u003c/h2\u003e \u003cp\u003eVg is known to provide zinc ions for developing oocytes and the metal ions have been suggested to play a role in its antioxidant activities\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. The number of zinc ions carried by Vg varies in different species\u003csup\u003e\u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e, with histidine residues known to play a central role in zinc binding\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. For the honey bee, it was recently determined that an average of 3.5 zinc ions bind to each Vg molecule\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Importantly, no metal chelators were used during the purification of AmVg from honey bee hemolymph. We identified three putative binding sites in our structures, however, there is no clear density for metal ions at most of these positions. Further information on the binding of metals to AmVg can be obtained from the latest release of AlphaFold\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. The putative metal ion binding sites are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eSite 1 is in the loop of the N-sheet that forms a short b-strand and interacts with the A-sheet, involving residues H229 and H926, where the two histidine side chains are in close proximity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Although there is no clear density for a metal ion, the flexible loop from residues 232\u0026ndash;245 may be stabilized by the presence of a Zn\u003csup\u003e2+\u003c/sup\u003e ion, where E239 would contribute to the coordination of the cation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Site 2 is very close to site 1 and formed by H587 and H593, in an insect-specific loop of the ahelical domain that contains two short helices (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). The helices are oriented such that the histidine side chains are positioned to coordinate a metal cation. The cryo-EM density of the cleavage product of AmVg seems to indicate the presence of a coordinating water next to the poor density for the putative zinc ion. No other residues in the proximity seem to be available to complete the coordination sphere of a zinc cation. Site 2 is also predicted in the AlphaFold 3 model as a zinc-binding site, with pLDDT score for the ion of 78.24. Site 3 is formed by H990 and H1045 and again seems to involve only two histidine side chains, although there is density between the side chains likely indicating the presence of a cation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). All putative sites described are solvent exposed, either at the protein surface or in an aqueous cavity easily accessible through loop motion (Site 3). Given the incomplete coordination spheres and the quality of the cryoEM densities we have not included any metal cations in our models. None of the putative sites herein described are conserved for lamprey Lv.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAn additional and highly conserved Ca\u003csup\u003e2+\u003c/sup\u003e binding site is believed to be part of the vWD domain based on sequence alignments with known structures of functionally unrelated homologues\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e as well as the AlphaFold 3 prediction (pLDDT score for the ion of 65.15). The quality of the cryo-EM density in the area, however, is not sufficient to draw any conclusions regarding metal binding, although the position of the protein backbone allows ion coordination as predicted (Figure S2).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eThe AlphaFold predictions for AmVg\u003c/h3\u003e\n\u003cp\u003eSince its development, AlphaFold 2\u003csup\u003e60\u003c/sup\u003e has revolutionized the world of structural biology. For honey bee Vg, the AlphaFold 2 structure prediction is surprisingly accurate (Supplementary Figure S1). AlphaFold 2 was able to correctly predict the overall domain organization of the protein, completely unknown for the vWD domain with respect to the lipid binding module. In addition, it predicted the fold of individual domains and long structured loops with astonishing accuracy. The root mean square deviation (RMSD) value of the main chain comparing our cryo-EM AmVg structure and the AlphaFold 2 model is 2.35 \u0026Aring;. The AI-generated model also provides information about the C-terminal domain (CTCK) that is not observed in our experimental structure. However, a lipoprotein rich in post-translational modifications such as Vg also highlights the current limitations of AlphaFold 2. No information about cleavage products, metal binding sites \u0026ndash;including side chain conformations and densities for the metals\u0026ndash; or glycosylations can be obtained from the AlphaFold 2 model. The recent development of AlphaFold 3\u003csup\u003e58\u003c/sup\u003e addresses some of these limitations and provides extra insight into the binding of metals by AmVg. The AlphaFold 3 model for AmVg was generated using the AlphaFold Server (alphafoldserver.com). By manually adding the desired metal ions as input for the prediction, the model predicts a calcium ion that is not observed experimentally in the vWD domain and provides extra support for zinc binding in one of the binding sites as discussed in the previous section. Compared to the cryo-EM structure, the AlphaFold 3 model shows a root mean square deviation (RMSD) in the main chain of 1.74 \u0026Aring;, showing an improvement compared to AlphaFold 2.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eThe presence of a 150-kDa cleavage product of AmVg\u003c/h2\u003e \u003cp\u003eDuring cryo-EM data processing, we also determined the structure of a cleavage product of AmVg found in numbers similar to those of the full-length protein. A 150-kDa cleavage product in honey bee hemolymph has been described previously for samples obtained from the hemolymph of the insect\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. However, most of the cleavage of AmVg occurred after purification, with the presence of the cleavage product increasing over time as observed with denaturing polyacrylamide gel electrophoresis. For regions that are common for the cleavage product and the full-length protein, the fold is identical, with a main-chain RMSD of 0.65 \u0026Aring;. The biggest deviations are close to the cleavage site, where the A-sheet of the cleavage product is substantially more wrapped around itself, effectively decreasing the volume of the lipid binding cavity. Although smaller, the lipid binding cavity is almost intact, with density up to residue 1276 in the A-sheet, with some arguable evidence at low contouring levels for some residues of the next strand being present (residues 1285\u0026ndash;1292) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Therefore, a small part of the A-sheet and the whole vWD domain are missing in the cleavage product of AmVg. This effectively increases the accessibility of the lipid cavity when compared to full-length AmVg, both at the front and at the top-back (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Interestingly, through loss of the vWD domain the cleavage product displays a domain composition more similar to that of lipovitellin.\u003c/p\u003e \u003cp\u003eInsect vitellogenins are known to be processed post-translationally by subtilisin-like proteases recognizing the motif \u003cb\u003eR/K\u003c/b\u003eXX\u003cb\u003eR/K\u003c/b\u003e\u003csup\u003e35\u003c/sup\u003e. However, no evidence for cleavage at such motifs has been described previously for the suborder apocrita, to which bees and ants belong. Honey bee Vg contains several such motifs, any of which could constitute the cleavage site. One of these motifs \u0026mdash;Motif 1\u0026mdash; is found where the density for the cleavage product ends near residue 1276 (Motif 1: \u003cb\u003eR\u003c/b\u003eYG\u003cb\u003eK\u003c/b\u003e, residues 1274 to 1277). A second motif can be found immediately after (Motif 2: \u003cb\u003eK\u003c/b\u003eGE\u003cb\u003eR\u003c/b\u003e, residues 1281 to 1284), although no density is observed for the residues between the two motifs. The residues corresponding to these two motifs are at the beginning and end of a short loop in the A-sheet (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Interestingly, the only disulphide bond in the b-strands of the A-sheet of the full-length AmVg is found stabilizing this short loop (C1242-C1279). The disulphide bond is conserved for IuLv but not observed at all for the cleavage product of AmVg. However, weak density for the b-strand immediately adjacent could be evidence that a partly flexible stretch of amino acids is still present. Two overlapping protease-recognition motifs in a loop shortly after are therefore also cleavage site candidates (Motif 3: \u003cb\u003eR\u003c/b\u003eGN\u003cb\u003eK\u003c/b\u003e, residues 1316 to 1319 and Motif 4: \u003cb\u003eK\u003c/b\u003eIL\u003cb\u003eR\u003c/b\u003e, residues 1319 to 1322).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe cryo-EM structures presented herein represent the first of a non-vertebrate vitellogenin and the first of a full-length LLTP containing a vWD domain. Furthermore, due to the native source of the protein, we have been able to identify post-translational modifications, metal binding sites and a cleavage product of AmVg. However, a lot is still unknown about the molecular details of AmVg.\u003c/p\u003e \u003cp\u003eThe function of the vWD domain and the C-terminal domains of vitellogenins and other LLTPs remains elusive. Recently, copper binding was reported for the vWD domain D1 of human mucin 2\u003csup\u003e61\u003c/sup\u003e. However, the residues involved in copper coordination are not conserved in AmVg. As presented in the \u003cspan refid=\"Sec2\" class=\"InternalRef\"\u003eresults\u003c/span\u003e section, proteins containing vWD and CTCK domains are able to form extensive concatemers through dimerization and subsequent linkage by disulphides both close to the N-terminus (vWD domain and accompanying modules) and the C-terminus (CTCK domains) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Such covalent concatemers confer mechanical stress resistance to secreted mucins and vWF in blood, which is required for their functions\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. However, AmVg lacks the cysteine residues that are involved in intermolecular disulphide formation, both within the vWD domain\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e and in the CTCK domain newly identified here (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Thus, AmVg contains the two domains required for formation of concatemers but lacks the main structural features needed for their stabilization. It cannot be ruled out that there are inaccuracies in the AlphaFold 2 prediction of the CTCK domain and that some cysteines predicted to be involved in intramolecular interactions might in fact be available for intermolecular interactions. However, some cystine knot containing cytokines such as chorionic gonadotropin (CG) form dimers without any intermolecular disulphide bond stabilization (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb)\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Vg is known to be able to form dimers\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e, although their biological significance is unknown. The CTCK domain is therefore a strong candidate for a dimerization domain for vitellogenin (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eIn our experimental structures, none of the putative Zn\u003csup\u003e2+\u003c/sup\u003e binding sites shows a complete coordination sphere. We only observe clear cryo-EM density for the pairs of histidine side chains involved in metal binding. There is, however, density that seems to correspond to a metal cation in two of the sites and density for a third coordinating group, likely water, in one of the sites. Therefore, AmVg likely binds Zn\u003csup\u003e2+\u003c/sup\u003e with low affinity. Loose binding of Zn\u003csup\u003e2+\u003c/sup\u003e might be desirable for the specific needs of Vg as a transporter, where the zinc ions need to be able to dissociate easily when required in the oocyte. Low affinities for metal ions might also be statistically compensated for by the presence of several binding sites. The presence of multiple binding sites might also allow the evolutionary plasticity required for the protein to retain its Zn\u003csup\u003e2+\u003c/sup\u003e binding functions in different species without specific binding sites being highly conserved. In addition, keeping a low number of coordinating residues might allow the binding not only of Zn\u003csup\u003e2+\u003c/sup\u003e but also of other metal cations.\u003c/p\u003e \u003cp\u003eThe biological relevance of the cleavage product of honey bee Vg remains to be established. The presence of a 150-kDa cleavage product in honey bee hemolymph has been described previously for samples obtained from the hemolymph of the insect\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. A 150-kDa AmVg fragment has also been identified as more abundant in samples purified from the fat body of the insect\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, compared to those from hemolymph. The fragment was named fat-body vitellogenin (fbVg) and was described as lacking the N-sheet subdomain based on a lower concentration of matching hits in the N-sheet from liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS)\u003csup\u003e49\u003c/sup\u003e. However, in the same study fbVg was shown to contain Nlinked glycosylations\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e9\u003c/sup\u003e, which our cryo-EM study only observed in the N-sheet subdomain, supporting the fact that fbVg contains the N-sheet. The cleavage fragment of AmVg observed in this study could correspond to fbVg, which could thus be the 150-kDa fragment that co-purifies at low abundance together with full-length AmVg obtained from honey bee hemolymph. The relative abundance of the cleavage product could increase over time in storage through the action of small amounts of proteases remaining after purification. The exact cleavage site remains unknown, with all the motifs presented in the \u003cspan refid=\"Sec2\" class=\"InternalRef\"\u003eresults\u003c/span\u003e section as possible candidates. The AmVg cleavage product described here has its lipid binding cavity almost intact and more accessible than full-length AmVg. If a biological significance is demonstrated, it is tempting to assume that the fragment is mostly involved in lipid binding related functions such as storage of lipids in the fat body. Since the cleavage fragment is also present in the hemolymph, it could also be involved in vitellogenesis. The cleavage product contains all the structural elements related to vitellogenesis functionalities. These include receptor binding for internalization in the oocyte, lipid and full Zn\u003csup\u003e2+\u003c/sup\u003e binding used as a nutrient source and PAMP recognition for trans-generational immune priming. Remarkably, the cleavage product seems to be structurally analogous to the type-C vitellogenins from fish, which lack the vWD and the CTCK domains (named b-component in vertebrate vitellogenins) as well as the phosvitin chain, which is not present in invertebrate Vg\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e6\u003c/sup\u003e. The function of fish type-C vitellogenins and their role in vitellogenesis \u0026ndash; if any \u0026ndash; remain unknown.\u003c/p\u003e \u003cp\u003eAn alternative interpretation is that the 150 kDa AmVg fragment from the hemolymph and fbVg are different, though of similar size. Indeed, protein product formation can proceed differently in intra or extra cellular compartments that have different biochemical environments\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e,\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. By such mechanisms, AmVg can be processed differently in hemolymph vs. fat body cells. For example, different cleavage products are identified in fat body and eggs for cockroach\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. A sequence analysis of insect vitellogenins identified cleavage motifs (R/KXXR/K) at both the N- and C-terminal, often in the same molecule \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. Glycosylation is a modification that affects the availability of protease cleavage sites\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e, and more than one potential glycosylation site has been identified for AmVg\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Glycosylation, moreover, is highly variable and non-permanent\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e changing with respect to time, tissue, subcellular location\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e, organismal health status\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e,\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e, as well as with protein subtypes\u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. Through the lens of this cumulative information, we cannot rule out that AmVg has more than one fragmentation and glycosylation possibility. The 150 kDa fragment observed in hemolymph, thereby, can be an alternative cleavage product compared to fbVg\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe relevance of Vg as a target for AI-based technologies to predict protein structures is highlighted by an article where it was used to exemplify the upcoming revolution in structural biology\u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. Although some important features of such a complex protein are missing in the AlphaFold predictions, the quality of the AlphaFold 2 model compared to our experimental structures shows high accuracy for the structure prediction of species-specific structural features of Vg. This suggests that AlphaFold is useful for structural phylogenetic studies of Vg and other Vg-like proteins. The presence of certain domains and subdomains, together with their architecture and functional data can easily provide important information on neo-functionalization. AI-based structure prediction for vitellogenins will become even more relevant after the advent of AlphaFold 3\u003csup\u003e58\u003c/sup\u003e, given its reported ability to predict protein structures with non-protein ligands such as metal ions, lipids and covalent glycosylation. Vitellogenin from the honey bee is a target in the ongoing CASP 16 competition (target T1210).\u003c/p\u003e \u003cp\u003eOverall, the structures described herein significantly advance our understanding of vitellogenins and their molecular complexity, both for all egg-laying animals but for vertebrates and the honey bee in particular. New insight has been obtained into AmVg domain arrangement for the previously uncharacterized vWD domain as well as for several new structural features not observed in the vertebrate IuLv structure. The molecular complexity of vitellogenins is the basis of its pleiotropy, with taxa-specific variations underlying the different neo- and sub-functionalizations the protein has developed over time in widely different animal groups. Furthermore, valuable information about the honey bee specific glycosylation profile, metal binding abilities and cleavage products has been obtained. The results presented here provide the molecular basis for further indepth investigation of the particular details of Vg functionalities and binding abilities, including comparative studies among multiple taxa and for other related LLTPs.\u003c/p\u003e "},{"header":"Methods","content":"\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003eProtein extraction and purification\u003c/h2\u003e \u003cp\u003eTo obtain purified AmVg, 1\u0026ndash;10 \u0026micro;L of honey bee hemolymph were collected and diluted 1/10 in 0.5 M Tris/HCl pH 7.6, using BD needles (30 G) as described earlier\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. The diluted protein was filtered using a 0.2 \u0026micro;m syringe filter. Vg was then subjected to ion-exchange chromatography using a HiTrap Q FF 1 mL column equilibrated in 0.5 M Tris/HCl pH 7.6 and eluted with a NaCl gradient in 0.5 M Tris/HCl pH 7.6 up to 0.45 M NaCl. 400\u0026ndash;450 \u0026micro;L of diluted hemolymph were manually injected and Vg eluted at a conductivity of 15\u0026ndash;22 mS\u0026middot;cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Fractions were collected, pooled and concentrated using an Amicon Ultracel 100 kDa membrane centrifuge filter (Merck KGaA, Darmstadt, Germany). Fraction purity was verified by running SDS-PAGE. Protein concentration was measured with Qubit.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCryo-EM grid preparation and data collection\u003c/h2\u003e \u003cp\u003eCryo-EM grids preparation and data collection was carried out at the cryo-EM Sweden National facility at SciLife lab in Stockholm according to standard procedures. Grids were prepared and plunge frozen in liquid ethane using a FEI Vitrobot Mark IV. AmVg at a concentration of 1.2 mg/mL was applied to a set of different grids with different treatments in order to overcome the preferential orientation problem we had observed previously for the sample. From those, two datasets were collected, one for a glow discharged Quantifoil R2/1 300 mesh holey carbon cooper grid (Quantifoil Micro Tools, Germany) and one for a Au 1.2/1.3 300 mesh holey carbon grid that was not glow discharged. Using the EPU control software, 28,406 movies were recorded on a FEI Titan Krios operating at 300 kV. The nominal magnification was 105,000x, corresponding to a physical pixel size of 0.8464 \u0026Aring;. The dose rate was set to 15.8 e\u003csup\u003e\u0026minus;\u003c/sup\u003e/px/s, and the total exposure time was 2.83 s, resulting in a total dose of 62.4 e\u003csup\u003e\u0026minus;\u003c/sup\u003e/\u0026Aring;\u003csup\u003e2\u003c/sup\u003e. Each movie was split into 40 frames of 0.07075 s. Nominal defocus range was \u0026minus;\u0026thinsp;0.8 \u0026micro;m to -2.8 \u0026micro;m in 0.2 \u0026micro;m steps.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eImage processing and model building\u003c/h2\u003e \u003cp\u003eThe dataset underwent processing utilizing CryoSPARC 4.1\u003csup\u003e71\u003c/sup\u003e as detailed in the Supplementary Figure S3. The preprocessing of the movies involved patch-based motion correction and patch-based contrast transfer function (CTF) estimation. Micrographs exhibiting a CTF fit greater than 3.5 \u0026Aring; were discarded, leaving a final dataset of 23,127 images. Initial 2D classes were derived and used for subsequent template-based particle picking, generating a stack of 23.3\u0026nbsp;million particles. These were extracted within a box size of 320 and then subjected to Fourier cropping down to 80 pixels. To further refine the stack, two additional rounds of 2D classification were performed prior to re-extraction of the particles at a box size of 320 and cropping to 160 pixels. The remaining 10.9\u0026nbsp;million particles underwent two additional rounds of 2D classification, yielding a residual stack of 9.4\u0026nbsp;million particles.\u003c/p\u003e \u003cp\u003eThe final extraction was carried out with a box size of 320 pixels without additional cropping. These particles were then utilized for ab-initio 3D reconstruction, with the most promising classes chosen for further heterogeneous refinement. This step yielded two distinct classes: one for the smaller cleavage product and one for the full-length protein. These stacks were then further refined through two cycles of heterogeneous refinement, followed by non-uniform refinement to evaluate potential improvements. A final local refinement was performed to enhance the quality of the consensus maps further, resulting in final maps with resolutions of 3.2 \u0026Aring; for the full protein and 3.0 \u0026Aring; for the smaller cleavage product. The estimated local resolution for the two reconstructed maps is shown in Figure S4.\u003c/p\u003e \u003cp\u003eThe flexibility and dynamics of AmVg, both for the full-length protein and the cleavage product were also analyzed in cryoSPARC 4.1\u003csup\u003e71\u003c/sup\u003e. 3D classification and 3D variability jobs did not provide obvious clues into alternative conformations, while flexible refinement did not produce final densities of better quality than local and non-uniform refinement.\u003c/p\u003e \u003cp\u003eThe AlphaFold 2 structure prediction for AmVg (Uniprot ID: Q868N5) was docked in the cryo-EM map in UCSF Chimera\u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. From there, the model was manually fitted and rebuilt in Coot\u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e (see supplementary information for details on map quality, model building and regions not included in the final models). The smaller cleavage product was modelled manually from the full-length AmVg model. Iterative cycles of real-space refinement were done in Phenix\u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e with further model rebuilding in Coot. Final geometries were assessed with Molprobity\u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e. Glycan geometries were assessed with Privateer\u003csup\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e. Structural analysis, comparison and figures were done in PyMOL (Schr\u0026ouml;dinger, LLC). The volumes of the lipid binding cavities were calculated with CASTp\u003csup\u003e\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e using a probe of 2.5 \u0026Aring; radius optimized to exclude areas outside of the lipid binding cavities.\u003c/p\u003e \u003cp\u003eThe cryo-EM maps were deposited in the Electron Microscopy Data Bank with accession codes EMD-19842 and EMD-19843 for full-length AmVg and the 150 kDa cleavage product of AmVg, respectively. The atomic coordinates of the models were deposited in the Protein Data Bank with accession numbers 9ENR for full-length AmVg and 9ENS for the cleavage product of AmVg.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eAlphafold 3 model generation\u003c/h2\u003e \u003cp\u003eThe Alphafold 3 model was generated using release 23/6/2024 of the AlphaFold server (alphafoldserver.com). The sequence for AmVg (Uniprot ID: Q868N5) was used as input, together with three Zn\u003csup\u003e2+\u003c/sup\u003e ions and one Ca\u003csup\u003e2+\u003c/sup\u003e ion. Additionally, the glycosylation observed in our cryo-EM density was input to occur at residue N298.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAmVg\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;honey bee vitellogenin\u003c/p\u003e\n\u003cp\u003eapoB\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;mammalian apolipoprotein B\u003c/p\u003e\n\u003cp\u003eapoLp-II/I \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;insect apolipophorins II/I\u003c/p\u003e\n\u003cp\u003eCG\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;chorionic gonadotropin\u003c/p\u003e\n\u003cp\u003ecryo-EM\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;cryogenic electron microscopy\u003c/p\u003e\n\u003cp\u003eCTCK domain\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;C-terminal cystine knot domain\u003c/p\u003e\n\u003cp\u003efbVg\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;fat-body vitellogenin\u003c/p\u003e\n\u003cp\u003eGlcNAc\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;N-acetyl glucosamine\u003c/p\u003e\n\u003cp\u003eHsCG\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;human chorionic gonadotropin\u003c/p\u003e\n\u003cp\u003eHsMTP\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;human microsomal triglyceride transfer protein\u003c/p\u003e\n\u003cp\u003eHsvWF\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;human von Willebrand factor\u003c/p\u003e\n\u003cp\u003eIuVg\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;silver lamprey vitellogenin\u003c/p\u003e\n\u003cp\u003eIuLv\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;silver lamprey lipovitellin\u003c/p\u003e\n\u003cp\u003eLC-MS/MS\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;liquid chromatography coupled to tandem mass spectrometry\u003c/p\u003e\n\u003cp\u003eLLTP superfamily \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;large lipid transfer protein superfamily\u003c/p\u003e\n\u003cp\u003eLv\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;lipovitellin\u003c/p\u003e\n\u003cp\u003eMTP\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;microsomal triglyceride transfer protein\u003c/p\u003e\n\u003cp\u003eNMR\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;nuclear magnetic resonance\u003c/p\u003e\n\u003cp\u003ePAMP\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;pathogen-associated molecular pattern\u003c/p\u003e\n\u003cp\u003epolyS region\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;polyserine region\u003c/p\u003e\n\u003cp\u003eTIL domain\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;trypsin inhibitor-like domain\u003c/p\u003e\n\u003cp\u003eVg\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;vitellogenin\u003c/p\u003e\n\u003cp\u003evWF\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;von Willebrand factor\u003c/p\u003e\n\u003cp\u003evWD domain\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;von Willebrand factor type D domain\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCryo-EM data were collected at the Cryo-EM Swedish National Facility funded by the Knut and Alice Wallenberg, Family Erling Persson and Kempe Foundations, SciLifeLab, Stockholm University and Ume\u0026aring; University. We thank Marta Carroni and Dustin Morado for facilitating grid freezing, remote data collection and initial assistance with data processing. We thank Gabriele Cordara for his help during model building and Marta Sanz-Gaitero for her work during the initial negative-staining EM studies of AmVg. We acknowledge The Research Council of Norway grant number 262137 and 335244 for funding toward running costs and positions. This project received funding from the European Union\u0026apos;s Horizon Europe Research and Innovation Programme under grant agreement number 101087571 (H.L). Arne Moeller was supported by SFB 944 and SFB 1557 and the DFG INST190/196-1 FUGG.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.M.-C. performed cryo-EM data processing, built the structural models and performed structural analysis under the supervision of H.L. and E.C. E.C. also performed initial cryo-EM data processing. K.S. processed the cryo-EM data, obtained the final reconstructions and contributed to manuscript writing under the supervision of A.M. V.L. purified AmVg supervised by \u0026Oslash;.H. and G.A. The manuscript draft was written by M.M.-C. with contributions from all authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSmolenaars MMW, Madsen O, Rodenburg KW, Van Der Horst DJ (2007) Molecular diversity and evolution of the large lipid transfer protein superfamily. J Lipid Res 48:489\u0026ndash;502\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRakhshandehroo M et al (2014) CD1d-mediated presentation of endogenous lipid antigens by adipocytes requires microsomal triglyceride transfer protein. 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Protein Sci 31:1\u0026ndash;18\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4768326/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4768326/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eVitellogenin (Vg) is the main yolk precursor lipoprotein in almost all egg-laying animals. In addition, along its evolutionary history, Vg has developed a range of new functions in different taxa. In the honey bee, Vg has functions related to immunity, antioxidant protection, social behavior and longevity. However, the molecular mechanisms underlying Vg functionalities are still poorly understood. Here, we report the cryo-EM structure of full-length honey bee Vg, onestep purified directly from hemolymph. The structure provides structural insights into the overall domain architecture, including the lipid binding cavity and the previously uncharacterized von Willebrand factor type D domain. A domain of unknown function has been identified as a C-terminal cystine knot domain based on structural homology. Information about post-translational modifications, cleavage products, metal and lipid binding allow an improved understanding of the mechanisms underlying the range of Vg functionalities. The findings have numerous implications for the structure-function relationship of vitellogenins of other species as well as members of the same protein superfamily, which share the same structural elements.\u003c/p\u003e","manuscriptTitle":"Cryo-EM structure of native honey bee vitellogenin","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-07 10:17:56","doi":"10.21203/rs.3.rs-4768326/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f405d683-bbc1-420e-ba79-e2c453effc02","owner":[],"postedDate":"August 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":35625505,"name":"Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy"},{"id":35625506,"name":"Biological sciences/Molecular biology/Post-translational modifications/Glycosylation"},{"id":35625507,"name":"Biological sciences/Zoology/Entomology"},{"id":35625508,"name":"Biological sciences/Biochemistry/Proteins/Lipoproteins"}],"tags":[],"updatedAt":"2025-07-02T07:35:14+00:00","versionOfRecord":{"articleIdentity":"rs-4768326","link":"https://doi.org/10.1038/s41467-025-58575-y","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-07-01 04:00:00","publishedOnDateReadable":"July 1st, 2025"},"versionCreatedAt":"2024-08-07 10:17:56","video":"","vorDoi":"10.1038/s41467-025-58575-y","vorDoiUrl":"https://doi.org/10.1038/s41467-025-58575-y","workflowStages":[]},"version":"v1","identity":"rs-4768326","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4768326","identity":"rs-4768326","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}