Structural basis of substrate recognition and membrane association by the bacterial lysyl-phosphatidylglycerol hydrolase AcvB

Structural basis of substrate recognition and membrane association by the bacterial lysyl-phosphatidylglycerol hydrolase AcvB | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (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];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Structural basis of substrate recognition and membrane association by the bacterial lysyl-phosphatidylglycerol hydrolase AcvB Mizuki Hoshi , View ORCID Profile Yasunori Watanabe doi: https://doi.org/10.1101/2025.11.10.687609 Mizuki Hoshi 1 Graduate School of Science and Engineering, Yamagata University , 1-4-12, Kojirakawa-machi, Yamagata 990-8560, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yasunori Watanabe 2 Faculty of Science, Yamagata University , 1-4-12 Kojirakawa-machi, Yamagata 990-8560, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Yasunori Watanabe For correspondence: yasunori{at}sci.kj.yamagata-u.ac.jp Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Bacteria adapt to environmental stresses via membrane phospholipid remodeling; however, the underlying molecular mechanism remains largely elusive. In Agrobacterium tumefaciens , the lysyl-phosphatidylglycerol (Lys-PG) synthase LpiA and periplasmic hydrolase AcvB genes form an operon that controls Lys-PG levels. We determined the crystal structures of mature AcvB and its C-terminal catalytic domain at 3.1 Å and 1.8 Å resolution, respectively. The catalytic domain forms a negatively charged cavity that recognizes the positively charged Lys-PG head group through multiple acidic residues, including Asp271, Asp340, and Asp370. A hydrophobic protruding loop containing Trp378 and Leu379 mediates transient membrane association and contributes to Lys-PG acyl-chain recognition. Further, AcvB interacts with LpiA via its C-terminal domain, suggesting a cooperative module for Lys-PG turnover. These findings reveal the structural basis of Lys-PG hydrolysis and provide mechanistic insight into adaptive lipid modification at the bacterial membrane interface, and may guide future development of antibacterial agents against plant-pathogenic bacteria. Introduction Biological membranes are composed of diverse phospholipids that not only provide structural integrity but also play critical roles in cellular adaptation to environmental stresses. For example, in response to environmental temperature changes, the level of unsaturated fatty acids in membrane phospholipids is modulated to maintain appropriate membrane fluidity 1 , 2 . Bacterial cells can adapt in response to harmful compounds by modifying their membrane phospholipids 3 . Thus, dynamic membrane phospholipid remodeling represents a fundamental mechanism by which bacteria and other organisms adapt to environmental challenges; however, the molecular basis of these adaptive lipid modifications remains largely elusive. Bacterial membranes mainly consist of phosphatidylethanolamine (PE) and the anionic phospholipids phosphatidylglycerol (PG) and cardiolipin and therefore have a net negative surface charge 3 , 4 . Cationic antimicrobial peptides (CAMPs) bind to the negatively charged bacterial membrane, inducing membrane disruption and antibacterial activity 5 – 7 . To resist CAMPs, many bacteria reduce the net negative surface charge by adding aminoacyl groups to PG, forming aminoacyl-PG. Aminoacyl-PGs exist in various forms, of which lysyl-PG (Lys-PG) and alanyl-PG (Ala-PG) are the most common 8 – 10 . Many Gram-positive bacteria such as Staphylococcus aureus , and certain Gram-negative bacteria, including the plant pathogen Agrobacterium tumefaciens , have Lys-PG modification, decreasingin their membrane, decreasing the net negative charge of the membrane and conferring resistance to CAMPs 11 , 12 . Ala-PG is found in certain Gram-positive as well as Gram-negative bacteria, such as Enterococcus faecalis and Pseudomonas aeruginosa , respectively 8 , 12 , 13 . Aminoacyl-PGs are synthesized by multiple peptide resistance factor (MprF), which was originally identified in S. aureus 10 . MprF is a bifunctional protein; its cytoplasmic domain functions as an aminoacyl-PG synthase that uses PG and aminoacyl-tRNA as substrates, whereas its membrane-embedded domain acts as a flippase that translocates aminoacyl-PG to the outer leaflet of the membrane 14 . In A. tumefaciens , Lys-PG is synthesized by the Lys-PG synthase LpiA, an MprF homolog 12 , 15 . In addition, A. tumefaciens has a soluble periplasmic Lys-PG hydrolase, Agrobacterium chromosomal virulence protein B (AcvB), which hydrolyzes Lys-PG into PG and lysine and is encoded in an operon along with lpiA 15 , 16 . The physiological importance of AcvB has been demonstrated by a genetic study in A. tumefaciens 16 ; deletion of acvB resulted in elevated Lys-PG levels, a severe growth defect at low pH, and impaired tumor formation in host cells. Further, the elevated Lys-PG levels inhibited T-DNA transfer to host cells via the type IV secretion system, thereby impairing the virulence of A. tumefaciens . These observations indicated that AcvB plays a crucial role in the physiological adaptation to acidic conditions and maintenance of appropriate Lys-PG levels to ensure membrane homeostasis and optimal virulence in A. tumefaciens . Structural studies of LpiA/MprF have provided insights into the molecular mechanisms of substrate recognition and aminoacyl-PG flipping 17 – 19 . Recently, the crystal structure of the N-terminal D1 domain of the AcvB homolog VirJ from Brucella abortus , which is distinct from the catalytic domain, has been reported 20 . However, the precise molecular mechanisms involved in Lys-PG hydrolysis, substrate recognition, and membrane binding remain unclear. In this study, we determined the crystal structures of the mature form of A. tumefaciens AcvB (lacking the signal sequence) and its C-terminal catalytic domain at 3.1 Å and 1.8 Å resolution, respectively. The C-terminal catalytic domain of AcvB forms a negatively charged cavity surrounding the active site, which is responsible for the recognition of the positively charged head group of Lys-PG. Furthermore, we showed that the protruding loop region near the active site contributes to membrane binding and the recognition of Lys-PG acyl chains. These findings provide fundamental insights into the molecular mechanism of Lys-PG hydrolysis and establish a structural framework for understanding adaptive lipid modifications at the bacterial membrane interface. Results Crystal structure of the mature form of AcvB To obtain structural insights into the mechanism of Lys-PG hydrolysis by AcvB, we attempted to determine the crystal structure of AcvB. AcvB (456 amino acids) contains an N-terminal signal sequence for periplasmic localization, with a predicted cleavage site after residue 24 according to SignalP 6.0 21 . As the mature form of AcvB, we expressed and purified AcvB(25– 456), lacking the N-terminal 24 residues, using the E. coli expression system, and confirmed that the recombinant protein retained the Lys-PG hydrolase activity (Supplementary Fig. 1). Ser336 has been proposed to be a conserved catalytic residue in AcvB homologs 16 , 22 . We substituted this residue with alanine to generate the AcvB(25–456)(S336A) mutant and assessed its Lys-PG hydrolase activity. The S336A mutation abolished the enzymatic activity, confirming the proposed role of Ser336 (Supplementary Fig. 1). Therefore, we used AcvB(25– 456) for crystallization and successfully obtained well-diffracting crystals. The crystal structure of AcvB(25–456) was determined via the molecular replacement method, using the structure predicted by ColabFold as the search model 23 , 24 , and subsequently refined to 3.1 Å resolution ( Table 1 ). The asymmetric unit of the crystal contained two AcvB(25–456) molecules with a similar conformation, with a root mean square deviation (r.m.s.d.) of 0.38 Å over 386 Cα atoms. In the crystal structure, AcvB consisted of two domains: the N-terminal (residues 29–248) domain and the C-terminal catalytic (residues 249–456) domain. Both adopted α/β hydrolase folds with a similar architecture ( Fig. 1A ). The N-terminal domain formed a six-stranded parallel β-sheet (β1–β6) surrounded by six α-helices (α1–α6), and the C-terminal domain formed a seven-stranded parallel β-sheet (β7–β13) surrounded by eight α-helices (α7–α14). Although the N-terminal and C-terminal domains of AcvB were highly similar to the AlphaFold3-predicted domain structures, with r.m.s.d. values of 0.39 Å over 174 Cα atoms and 0.37 Å over 173 Cα atoms, respectively, the overall domain orientation differed from the prediction (Supplementary Fig. 2). Ser336, a catalytic residue, was located between β10 and α10 of the C-terminal domain. In addition, the C-terminal domain harbored a protruding loop region between β11 and α12, which is located near Ser336, suggesting that its proximity to the catalytic residue may contribute to substrate recognition. Electrostatic surface analysis revealed that the region surrounding Ser336 formed a negatively charged cavity, which could accommodate the positively charged head group of Lys-PG ( Fig. 1B ). Download figure Open in new tab Fig. 1: Structure of mature AcvB. (A) Ribbon diagram of the AcvB(25–456) structure. The N-terminal and C-terminal domains are colored blue and pink, respectively. Secondary structural elements are labeled. The catalytic residue Ser336 is shown in stick representation. (B) Electrostatic surface potential of AcvB(25–456) calculated using PyMOL. Positive and negative potentials are shown in blue and red, respectively. View this table: View inline View popup Download powerpoint Table 1. Data collection and refinement statistics Crystal structure of the C-terminal domain of AcvB The C-terminal domain of AcvB has been shown to be necessary and sufficient for the Lys-PG hydrolase activity 16 . To gain a more detailed understanding of its catalytic mechanism, we next attempted to determine the high-resolution crystal structure of the C-terminal domain of AcvB (residues 249–456). The domain was expressed and purified using the E. coli expression system, and well-diffracting crystals were successfully obtained. Its crystal structure, determined at 1.8 Å resolution, closely resembled the corresponding region in mature AcvB, with an r.m.s.d. of 0.34 Å over 178 Cα atoms ( Fig. 2A ). His433 reportedly is a catalytic residue, along with Ser336 16 . In the crystal structure, His433 was positioned in close proximity to Ser336, suggesting that Ser336 and His433 likely function as a catalytic dyad ( Fig. 2B ). Notably, six acidic residues (Asp271, Asp340, Glu368, Asp370, Glu410, and Glu413) were also located near Ser336, suggesting a role in recognizing the positively charged head group of Lys-PG ( Fig. 2B ). To investigate their roles in catalytic activity, we substituted these residues with alanine (D271A, D340A, E368A, D370A, E410A, and E413A). The Lys-PG hydrolase activity of the AcvB mutants was assessed by co-expressing each mutant with LpiA, the Lys-PG synthase of A. tumefaciens , in E. coli cells. Protein expression was confirmed using immunoblotting ( Fig. 2C ). Lipid extracts from E. coli cells co-expressing AcvB and LpiA were analyzed using thin-layer chromatography (TLC) followed by ninhydrin staining of the TLC plate to assess the levels of Lys-PG ( Fig. 2D ). The results revealed that the membranes of E. coli cells harboring empty vectors contained only PE, whereas those of cells expressing LpiA contained both PE and Lys-PG. Co-expression of wild-type AcvB with LpiA in E. coli cells resulted in a marked decrease in the amount of Lys-PG compared with that in cells expressing LpiA alone, indicating that wild-type AcvB catalyzed Lys-PG hydrolysis. Consistent with its essential role in catalysis, the S336A mutation abolished the hydrolase activity of AcvB, resulting in the accumulation of Lys-PG in the membranes ( Fig. 2D, E ). Similarly, the D271A mutation caused strong Lys-PG accumulation, whereas D340A and D370A resulted in moderate accumulation. By contrast, the E368A, E410A, and E413A mutants did not accumulate Lys-PG; their Lys-PG levels were comparable to those in cells expressing wild-type AcvB. These results indicated that Asp271, Asp340, and Asp370 are critical for Lys-PG hydrolysis, whereas Glu368, Glu410, and Glu413 are not essential for the catalytic activity. Download figure Open in new tab Fig. 2: Structure of the C-terminal domain of AcvB. (A) Ribbon diagram of the C-terminal domain of AcvB. (B) Magnified view of the region around the active site. Asp271, Ser336, Asp340, Glu368, Asp370, Glu410, Glu413, and His433 are shown in stick representation. (C) Western blot showing relative expression levels of C-terminally His 10 -tagged LpiA and C-terminally 3xFLAG-tagged AcvB mutants in E. coli cells. (D) Total phospholipids were extracted from E. coli cells expressing C-terminally His 10 -tagged LpiA and C-terminally 3xFLAG-tagged AcvB mutants, separated by TLC, and visualized by ninhydrin staining. (E) Quantification of Lys-PG based on the data in (D), using the ImageJ software. Values represent means ± SDs (n = 3). Structural basis of substrate Lys-PG recognition To explore the structural basis of substrate recognition, we attempted to co-crystallize AcvB with Lys-PG. Despite numerous crystallization trials, crystals of the complex could not be obtained. Therefore, we instead carried out docking simulations using AutoDock Vina 25 to model the binding of a lysyl-glycerol head-group fragment, representing the polar head group of Lys-PG, to the C-terminal domain of AcvB. The lysyl-glycerol fragment was docked into a cavity corresponding to the putative substrate-binding pocket of AcvB ( Fig. 3A ). In the docking model, the carbonyl carbon of the ester group was positioned in close proximity to the catalytic residue S336 (4.1 Å), consistent with a plausible model for substrate binding and catalysis. The lysine moiety of the fragment was positioned near Asp271, Ser336, Phe337, Asp340, Glu368, Asp370, and Glu384. The side chains of Asp271 and Ser336 were within a distance that allowed hydrogen bonding with the α-amino group of lysine, whereas the side chains of Asp370 and Glu384 were able to form hydrogen bonds with its ε-amino group. The glycerol moiety of the fragment lied adjacent to Leu365, Ser366, Glu410, Glu413, Glu414, and His433, with the side chain of Glu410 capable of forming hydrogen bonds with the hydroxyl groups of glycerol. Given that substitution of Asp271, Asp340, and Asp370 reduced Lys-PG hydrolysis ( Fig. 2D ), the docking model was consistent with the notion that recognition of the lysine moiety by these acidic residues is critical for the catalytic activity of AcvB. Download figure Open in new tab Fig. 3: Residues responsible for Lys-PG head-group recognition. (A) Magnified view of the area around the lysyl-glycerol molecule in the docking model of the C-terminal domain of AcvB with a docked lysyl-glycerol. The lysyl-glycerol molecule is shown as a ball-and-stick model in yellow. Residues located near the lysyl-glycerol are shown in stick representation. The distance between the carbonyl carbon of the ester group of the lysyl-glycerol and the hydroxyl oxygen of Ser336 is indicated by a solid line. Dashed lines indicate possible hydrogen bonds. (B) Schematic representation of the hydrolysis reaction of Lys-EE catalyzed by AcvB. AcvB hydrolyzes Lys-EE into lysine and ethanol. (C) Purified AcvB(25–456) mutants were analyzed using SDS-PAGE followed by CBB staining. (D) Lys-EE and purified AcvB(25–456) mutants were incubated at 37 °C for 30 min. Proteins were separated using TLC and visualized using ninhydrin staining. (E) Quantification of lysine based on the data in (D), using ImageJ. Values represent means ± SDs (n = 3). To assess the specific contribution of the acidic residues to lysine recognition, we examined the hydrolysis of lysine ethyl ester (Lys-EE), a simplified substrate that mimics the lysine head group without a PG moiety. Lys-EE was expected to be hydrolyzed by AcvB, yielding lysine and ethanol ( Fig. 3B ). N-terminally His 6 -tagged AcvB(25–456) mutants (S336A, D271A, D340A, and E410A) were prepared and used in Lys-EE hydrolysis assays ( Fig. 3C ). Lys-EE was incubated with the mutants, and the reaction products were analyzed using TLC. Wild-type AcvB efficiently generated lysine when compared with the S336A mutant, demonstrating that the hydrolysis of Lys-EE was catalyzed by AcvB ( Fig. 3D, E ). Both the D271A and D340A mutations reduced Lys-EE hydrolase activity, whereas the E410A mutant retained activity comparable to that of the wild-type. These results supported that Asp271 and Asp340 play a critical role in lysine recognition during Lys-PG hydrolysis. A protruding loop region contributes to membrane association AcvB has been described as a soluble periplasmic protein 16 ; however, its substrate Lys-PG is a membrane phospholipid, implying that AcvB likely transiently associates with the membrane during substrate recognition. In the crystal structure, one AcvB molecule interacted with the N-terminal domain of a neighboring AcvB molecule via a protruding loop in the C-terminal domain ( Fig. 4A ). Trp378 and Leu379 in the protruding loop were buried in the hydrophobic pocket of the N-terminal domain formed by hydrophobic residues such as Leu136, His206, and Leu210 ( Fig. 4B ). Although analysis using the PISA server 26 indicated that this interaction was a crystal packing artifact, the protruding loop was in contact with a hydrophobic region of a neighboring AcvB molecule in the crystal, suggesting that it may bind to hydrophobic regions such as the membrane. To investigate how AcvB interacts with the membrane, we predicted its membrane-binding orientation using the Positioning of Proteins in Membrane (PPM) server 27 . Consistent with our hypothesis, the protruding loop, particularly Trp378 and Leu379, was predicted to mediate membrane association ( Fig. 4C ). Taken together, these findings supported the notion that membrane association mediated by Trp378 and Leu379 provides a plausible mechanism for Lys-PG recognition, as the loop region is positioned near the catalytic residue Ser336. Download figure Open in new tab Fig. 4: A protruding loop region mediates the membrane association of AcvB. (A) Crystal packing interface of AcvB showing the protruding loop in the C-terminal domain interacting with the N-terminal domain of a neighboring molecule. Trp378 and Leu379 in the protruding loop, as well as the catalytic residue Ser336, are shown in stick representation. The neighboring molecule is shown as a surface representation. (B) Magnified view of the crystal contact shown in (A). Trp378 and Leu379 in the protruding loop of one molecule interact with a hydrophobic pocket in the N-terminal domain of the neighboring molecule. Residues forming the hydrophobic pocket are shown in stick representation. (C) Predicted membrane-binding orientation of AcvB calculated using the PPM server. Trp378 and Leu379 in the protruding loop are positioned to interact with the membrane surface. The protruding loop region is important for recognition of Lys-PG acyl chains To investigate the role of the protruding loop in catalytic activity, we constructed an AcvB mutant lacking the loop region (residues 373–382; ΔLoop) ( Fig. 5A ). The Lys-PG hydrolase activity of the ΔLoop mutant was assessed by co-expressing it with LpiA in E. coli cells ( Fig. 5B ). Similar to S336A mutation, deletion of the loop region caused Lys-PG accumulation, indicating that the loop region is important for the Lys-PG hydrolase activity ( Fig. 5B,C ). Trp378 and Leu379 in the protruding loop region were likely to be involved in membrane binding ( Fig. 4C ). To assess the role of these residues in catalytic activity, we generated two double mutants in which both residues were substituted with either hydrophilic asparagine (W378N/L379N) or hydrophobic phenylalanine (W378F/L379F). The W378N/L379N mutant showed Lys-PG accumulation, whereas the W378F/L379F mutant did, similar to wild-type AcvB ( Fig. 5B, C ). These results indicated that the hydrophobic property of Trp378 and Leu379 plays a critical role in Lys-PG hydrolase activity. Download figure Open in new tab Fig. 5: Trp378 and Leu379 are involved in Lys-PG acyl-chain recognition. (A) Magnified view of the area around the protruding loop region of the C-terminal domain of AcvB. The loop region (residues 373–382) is colored yellow. Trp378 and Leu379 in the protruding loop, as well as the catalytic residue Ser336, are shown in stick representation. (B) E. coli cells harboring expression plasmids encoding C-terminally His 10 -tagged LpiA and C-terminally 3xFLAG-tagged AcvB mutants were cultured at 37 °C. When the OD 600 reached ∼0.5, IPTG was added to a final concentration of 0.5 mM and the cells were incubated at 37 °C for 3 h. Cell lysates were subjected to SDS-PAGE, followed by immunoblotting with anti-6x His antibody and anti-FLAG antibody to detect LpiA (top panel) and AcvB (middle panel), respectively. Total phospholipids were extracted from E. coli cells expressing C-terminally His 10 -tagged LpiA and C-terminally 3xFLAG-tagged AcvB mutants, separated through TLC, and visualized using ninhydrin staining (bottom panel). (C) Quantification of Lys-PG based on the data in (B), using ImageJ. Values represent means ± SDs (n = 3). (D) Soluble and membrane fractions were separated from E. coli cells expressing N-terminally His 6 -tagged AcvB(25–456) mutants and subjected to SDS-PAGE and immunoblotting with anti-6x His antibody. (E) Quantification of AcvB in the membrane fraction based on the data in (D), using ImageJ. Values represent means ± SDs (n = 3). (F) Purified AcvB(25–456) mutants were analyzed using SDS-PAGE followed by CBB staining. (G) Lys-EE and purified AcvB(25–456) mutants were incubated at 37 °C for 30 min. Proteins were separated through TLC and visualized using ninhydrin staining. (H) Quantification of lysine based on the data in (G), using ImageJ. Values represent means ± SDs (n = 3). We next examined whether Trp378 and Leu379 are required for substrate recognition or membrane association during Lys-PG hydrolysis. To this end, we first tested the membrane binding of AcvB mutants. N-terminally His 6 -tagged AcvB(25–456) W378N/L379N and W378F/L379F mutants were expressed in E. coli cells, and soluble and membrane fractions were isolated and analyzed using immunoblotting ( Fig. 5D ). Wild-type AcvB and the W378F/L379F mutant showed comparable levels of membrane association, whereas the W378N/L379N mutant was present at only ∼5% in the membrane fraction ( Fig. 5E ). Consistent with the predicted membrane-binding orientation ( Fig. 4C ), these findings demonstrated that the hydrophobic nature of Trp378 and Leu379 is crucial for membrane association. To specifically assess the contributions of Trp378 and Leu379 to substrate recognition, the W378N/L379N and W378F/L379F mutants were subjected to Lys-EE hydrolysis assays ( Fig. 5F ). Interestingly, both mutants generated lysine from Lys-EE at levels comparable to that of wild-type AcvB, suggesting that Trp378 and Leu379 are not essential for Lys-PG head-group recognition ( Fig. 5G, H ). Collectively, these results indicated that Trp378 and Leu379 in the protruding loop are dispensable for recognition of the polar head group of Lys-PG but are essential for membrane association and contribute to the recognition of the acyl chains of Lys-PG through their hydrophobic side chains. AcvB interacts with LpiA via the C-terminal domain In various Gram-negative bacteria, such as A. tumefaciens C58, lpiA and acvB are organized in an operon 15 . Given this genomic arrangement, we hypothesized that AcvB and LpiA may functionally cooperate through direct interaction. To test this hypothesis, we examined whether AcvB physically associates with LpiA. N-terminal glutathione S-transferase (GST)-fused AcvB and LpiA were purified and subjected to an in-vitro GST pull-down assay, which revealed that AcvB interacted with LpiA ( Fig. 6A ). We next investigated whether the N- terminal or C-terminal domain of AcvB is responsible for this interaction. GST pull-down assays revealed that the C-terminal domain interacted with LpiA, whereas the N-terminal domain did not ( Fig. 6A ). These results indicated that the C-terminal domain of AcvB mediates the interaction with LpiA, suggesting that AcvB and LpiA function cooperatively in regulating Lys-PG metabolism. Download figure Open in new tab Fig. 6: AcvB interacts with LpiA via the C-terminal domain. (A) GST pull-down assay showing the interaction between AcvB and LpiA. GST–AcvB(25– 456), GST–AcvB(25–248), and GST–AcvB(249–456) bound to GST-accept resin were incubated with LpiA–His 10 . Proteins bound to the resin were eluted with glutathione and analyzed using SDS-PAGE followed by CBB staining (upper panels) and immunoblotting with anti-6x His antibody (lower panels). (B) Schematic model illustrating the cooperation between LpiA and AcvB in regulating Lys-PG metabolism in A. tumefaciens . LpiA, located in the inner membrane, catalyzes Lys-PG synthesis from PG and lysyl-tRNA and flips Lys-PG to the periplasmic leaflet. AcvB, localized in the periplasm, interacts with LpiA via its C-terminal domain. Multiple acidic residues around the active site recognize the positively charged head group of Lys-PG, whereas a protruding hydrophobic loop recognizes its acyl chains. AcvB subsequently hydrolyzes Lys-PG to PG and lysine. Discussion In A. tumefaciens , AcvB catalyzes the hydrolysis of Lys-PG at the periplasmic side of the inner membrane, thereby maintaining an optimal balance between Lys-PG synthesis and degradation. In this study, we determined the crystal structures of the mature form of AcvB and its C-terminal catalytic domain and assessed how the enzyme recognizes its lipid substrate and interacts with the membrane. The C-terminal domain forms a negatively charged cavity that accommodates the positively charged Lys-PG head group through several acidic residues, while a hydrophobic protruding loop contributes to transient membrane association ( Figs. 4 and 5 ). This loop contains two hydrophobic residues (Trp378 and Leu379) that are essential for membrane association and Lys-PG hydrolysis. Notably, the relative orientation of the N- and C-terminal domains in the crystal structure differed from that predicted by AlphaFold3 28 . In the predicted model, the protruding loop contacted the N-terminal domain within the same molecule, whereas in the crystal structure, it interacted with the hydrophobic region of the N-terminal domain from a neighboring molecule ( Fig. 4A, B ). This intermolecular contact likely mimics the interaction between AcvB and the membrane surface, suggesting that the domain arrangement observed in the crystal represents a membrane-bound conformation. Several peripheral enzymes act on membrane phospholipids without having transmembrane segments. The E. coli phosphatidylserine synthase (PssA), which catalyzes phosphatidylserine synthesis, and the phospholipid N -methyltransferases (PmtA) from Rhodothermus thermophilus and A. tumefaciens , which catalyze phosphatidylcholine synthesis, associate with membranes via amphipathic helices 29 – 31 . The E. coli phosphatidylserine decarboxylase (Psd), responsible for PE synthesis, binds to membranes via three hydrophobic helices comprising approximately 60 residues 32 , 33 . In contrast, AcvB lacks amphipathic or hydrophobic helices and instead associates weakly with the membrane via a short loop region. Notably, we found that the C-terminal domain of AcvB directly interacts with LpiA ( Fig. 6A ). Because LpiA possesses an integral membrane flippase domain that translocates Lys-PG from the cytoplasmic to the periplasmic leaflet, the interaction likely anchors AcvB to the membrane and facilitates efficient substrate recognition. Therefore, we propose that the LpiA–AcvB complex constitutes a cooperative module in which LpiA enhances the localization and catalytic efficiency of AcvB, thereby maintaining an balanced amount of Lys-PG in the membrane. Mutational and docking analyses revealed a potential recognition mechanism for the lysyl head group of Lys-PG by AcvB. Asp271, Asp340, and Asp370 are responsible for Lys-PG hydrolase activity. In the docking model, Asp271 and Asp340 were located close to the α-amino group of the lysine moiety of the substrate, whereas Asp370 was positioned near its ε-amino group. Sequence alignment revealed that Asp271 and Asp340 are conserved in AcvB homologs, including A. tumefaciens VirJ, Rhizobium tropici AtvA, and the Pseudomonas homologs (PA0919 from P. aeruginosa and PP_1201 from P. putida ) (Supplementary Fig. 3). Only in the P. aeruginosa and P. putida homologs, Asp370 is replaced with serine. Given that A. tumefaciens and R. tropici produce Lys-PG, whereas P. aeruginosa and P. putida produce Ala-PG, Asp370 likely contributes to the specific recognition of the lysine head group of Lys-PG. Thus, AcvB homologs from bacteria that synthesize Lys-PG may have evolved a more acidic environment around the active site to facilitate electrostatic complementarity and selective lysine-containing substrate recognition. Based on the results of structural and biochemical analyses, we propose a working model for Lys-PG hydrolysis by AcvB at the periplasmic membrane surface ( Fig. 6B ). AcvB transiently associates with the membrane via the hydrophobic loop containing Trp378 and Leu379, orienting the catalytic site containing Ser336 toward the membrane surface, where Trp378 and Leu379 are involved in the recognition of the acyl chain moiety of Lys-PG. The lysyl head group of Lys-PG is recognized electrostatically by multiple acidic residues, including Asp271, Asp340, and Asp370, enabling cleavage of the ester linkage. Direct interaction with LpiA via the C-terminal domain of AcvB likely stabilizes the membrane-bound state of AcvB and facilitates efficient substrate transfer between the two enzymes. Through this coupled activity, the LpiA–AcvB module functions to maintain an optimal Lys-PG level and membrane homeostasis in A. tumefaciens . Future structural studies of the LpiA– AcvB complex will provide mechanistic insights into substrate transfer between the two enzymes. In conclusion, our findings establish a structural basis for understanding the molecular mechanism of Lys-PG hydrolysis by AcvB and may guide future development of antibacterial agents against plant-pathogenic bacteria. Methods Construction of expression plasmids acvB and lpiA were amplified from the genome of A. tumefaciens NBRC 15193. AcvB(25– 456), AcvB(25–248), and AcvB(249–456) were cloned into the pGEX-6p-1 vector (GE Healthcare) to construct N-terminal GST-fusion protein expression plasmids. AcvB(25–456) was also cloned into the pETDuet-1 vector (Novagen) to construct the N-terminally His 6 -tagged protein expression plasmid. Full-length AcvB was cloned into a modified pACYCDuet-1 vector (Novagen) to add a C-terminal 3xFLAG tag to the expressed protein. LpiA was cloned into a modified pET21a vector (Novagen) to add a C-terminal His 10 -tag to the expressed protein. Mutations for amino-acid substitutions were introduced using PCR-based site-directed mutagenesis. All constructs were sequenced to confirm their identities. Protein expression and purification All constructs were expressed in E. coli C43 (DE3) cells (Lucigen) cultured in Luria–Bertani (LB) medium at 37 °C. When the culture reached an optical density at 600 nm (OD 600 ) of approximately 0.8, protein expression was induced with 0.1 mM isopropyl β-D-thiogalactopyranoside. After growth at 25 °C for 18 h, the cells were harvested. For GST-tagged AcvB protein purification, cells were resuspended in buffer A (20 mM Tris-HCl [pH 8.0] and 150 mM NaCl) supplemented with 5 mM dithiothreitol, disrupted by sonication, and centrifuged at 20,000 × g for 40 min to remove the insoluble debris. The supernatant was loaded onto a GST-Accept column (Nacalai Tesque) equilibrated with buffer A. The column was washed with buffer A, and GST-tagged proteins were eluted using buffer B (50 mM Tris-HCl [pH 8.0] and 10 mM reduced glutathione). The GST-tag was cleaved using human rhinovirus 3C protease at 4 °C overnight and removed by reloading the sample onto the GST-Accept column. For N-terminally His 6 -tagged AcvB protein purification, cells were resuspended in buffer C (50 mM Tris-HCl [pH 8.0], 500 mM NaCl, and 20 mM imidazole). After cell disruption by sonication, the supernatant was loaded onto a Ni-NTA column (Qiagen) equilibrated with buffer C. The column was washed with buffer C, and His 6 -tagged proteins were eluted using buffer D (50 mM Tris-HCl [pH 8.0], 100 mM NaCl, and 250 mM imidazole). All recombinant AcvB proteins, regardless of the affinity tag used, were further purified through size-exclusion chromatography using a Superdex 200 Increase column (GE Healthcare) with buffer A. GST-tag purified AcvB(25–456) and AcvB(249–456) proteins were used for crystallization trials after cleavage of the GST-tag. For C-terminally His 10 -tagged LpiA purification, cells were resuspended in buffer C. After cell disruption by sonication, cell debris was removed by centrifugation at 10,000 × g for 10 min and the membrane fraction was collected by ultracentrifugation at 100,000 × g for 90 min. The membrane fraction was solubilized in buffer C supplemented with 1.5% dodecyl-β-D-maltopyranoside (DDM) at 4 °C for 60 min. Insoluble components were removed by ultracentrifugation at 100,000 × g for 30 min and the supernatant was loaded onto a Ni-NTA column equilibrated with buffer C supplemented with 0.03% DDM. The column was washed with buffer C supplemented with 0.03% DDM, and the protein was eluted using buffer D supplemented with 0.03% DDM. LpiA was further purified through size-exclusion chromatography using a Superdex 200 Increase column with buffer A supplemented with 0.03% DDM. Crystallization and X-ray crystallography Crystallization trials were performed at 20 °C using the sitting drop vapor diffusion method. For crystallization of AcvB(25–456), 0.2-μL drops of approximately 27 mg/mL AcvB(25-456) in 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl were mixed with an equal amount of reservoir solution consisting of 2.4 M sodium malonate (pH 7.0) and equilibrated against 70 μL of the same reservoir solution through vapor diffusion. For crystallization of AcvB(249–456), 0.2-μL drops of approximately 6.8 mg/mL AcvB(249–456) in 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl were mixed with an equal amount of reservoir solution consisting of 0.1 M ammonium iodide, 0.1 M sodium acetate (pH 6.0), and 20% w/v polyethylene glycol 3,350, and equilibrated against 70 μL of the same reservoir solution through vapor diffusion. The crystals were soaked in a reservoir solution supplemented with 15% ethylene glycol, flash-cooled, and maintained in a stream of nitrogen (N 2 ) gas at 100 K during data collection. X-ray diffraction data were collected at the SPring-8 beamline BL32XU, with a 10 × 15-μm (width × height) microbeam using the helical data collection method. The diffraction data were collected using the ZOO automated data collection system 34 . The data were processed using the KAMO 35 and XDS 36 software. Structures were determined through molecular replacement with PHASER 37 , using the structure predicted by ColabFold 23 , 24 as a search model. Further model building was performed manually using COOT 38 , and crystallographic refinement was performed using PHENIX 39 . MolProbity 40 was used to assess the quality and geometry of structural models. Detailed data collection and processing statistics are shown in Table 1 . Ramachandran analysis showed that 94.36% and 97.88% of AcvB(25–456) and AcvB(249–456) residues were located in favored regions, and 5.64% and 2.12% in allowed regions, respectively, whereas no outliers were observed. Molecular docking Molecular docking of a lysyl-glycerol molecule into the crystal structure of AcvB(249–456) was examined using AutoDock Vina 25 . Docking was performed using SwissDock 41 , with a sampling exhaustivity of 8. The AcvB(249–456) structure was set as rigid during docking, and a 30 × 30 × 30 Å grid box was placed around the S336 residue. Separation of soluble and membrane fractions Soluble and membrane fractions of E. coli lysates were separated as previously described 31 , 32 , with several modifications. E. coli C43(DE3) cells carrying expression plasmids for C-terminally His 10 -tagged LpiA and C-terminally 3xFLAG-tagged AcvB variants were cultured in 80 mL of LB medium at 37 °C. When the OD 600 reached approximately 0.5, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 500 μM, and the cultures were incubated at 37 °C for 3 h to induce protein expression. Next, the cells were harvested, resuspended in lysis buffer (20 mM Tris-HCl [pH 8.0] and 150 mM NaCl), disrupted through sonication, and centrifuged at 10,000 × g for 10 min to pellet the insoluble debris. The samples were ultracentrifuged at 150,000 × g for 1 h. The supernatant and pellet fractions were defined as soluble and membrane fractions, respectively, and analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting. TLC analysis To analyze the hydrolysis of Lys-PG by AcvB variants expressed in E. coli cells, membrane fractions were prepared from 80-mL E. coli cultures and resuspended in 100 μL of lysis buffer. Fifty microliters of the membrane suspension was mixed with 750 μL of chloroform/methanol (2:1, v/v ) and vortexed for 10 min. Then, 100 μL of water was added, and the samples were vortexed again for 10 min. The organic phase was separated by centrifugation at 1,000 × g for 2 min, collected, and dried under a stream of N 2 gas. The resulting lipid films were dissolved in 40 μL of chloroform. Twenty-microliter aliquots of each sample were spotted onto an HPTLC Silica gel 60 plate (Merck Millipore) and analyzed through TLC using chloroform/methanol/water (65:25:4, v/v/v ). To analyze the hydrolysis of Lys-EE by purified AcvB variants, 40 mM Lys-EE was incubated with 40 μM purified AcvB variants in 50 μL of assay buffer A (8 mM Tris-HCl [pH 8.0] and 60 mM NaCl) at 37 °C for 30 min. The reaction was stopped by adding 5 μL of 10% SDS solution. One-microliter aliquots of each sample were spotted onto an HPTLC Silica gel 60 plate and analyzed through TLC using 1-butanol/acetic acid/water (3:1:1, v/v/v ). To analyze Lys-PG hydrolysis by purified AcvB variants, 8 mg of 18:1-18:1 Lys-PG (Avanti Polar Lipids) was incubated with 20 μM purified AcvB variants in 50 μL of assay buffer B (20 mM HEPES [pH 7.0], 150 mM NaCl, and 0.2% Triton X-100) at 37 °C. After incubation for the indicated time periods, 750 μL of chloroform/methanol (2:1, v/v ) was added, and the samples were vortexed for 10 min. After the addition of 100 μL of water, the samples were vortexed again for 10 min. The organic phase was separated by centrifugation at 1,000 × g for 2 min, collected, and dried under a stream of N 2 gas. The resulting lipid films were dissolved in 40 μL of chloroform. Twenty-microliter aliquots of each sample were spotted onto a TLC Silica gel 60 plate and analyzed through TLC using chloroform/methanol/water (65:25:4, v/v/v ). Sample spots were visualized through staining with ninhydrin using Ninhydrin-ethanol TS Spray (FUJIFILM Wako). In-vitro pull-down assay GST-tagged AcvB variants (20 μM) and C-terminally His 10 -tagged LpiA (20 μM) were incubated with 50 μL of GST-Accept resin in buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.03% DDM at 4 °C for 60 min. The resin was washed three times with 500 μL of the same buffer, and bound proteins were eluted with 150 μL of 10 mM glutathione in 50 mM Tris-HCl (pH 8.0) and 0.03% DDM. The eluted proteins were analyzed using SDS-PAGE followed by Coomassie brilliant blue (CBB) staining and immunoblotting with an anti-6x His antibody. Immunoblotting For immunoblotting, mouse monoclonal anti-6x His antibody (clone 9C11; FUJIFILM Wako) and mouse monoclonal anti-FLAG antibody (clone M2; Sigma-Aldrich) were used at dilutions of 1:2,000 and 1:3,000, respectively. A fluorophore-conjugated secondary antibody, goat anti-mouse IgG (H + L) cross-absorbed secondary antibody, cyanine5 (A10524; Life Technologies), was used at a 1:2,000 dilution. Fluorescent signals were detected using a Typhoon FLA 9500 imaging system (GE Healthcare). Amino-acid sequence alignment The amino acid sequences of AcvB and its homologs were analyzed using Clustal Omega 42 and ESPript 3.0 43 . Statistics and reproducibility Data in Figs. 2E , 3E , and 5C, E , and H were obtained from three independent experiments and are presented as the mean ± standard deviation (SD) from three independent experiments. Quantification was performed using ImageJ software. Data availability The atomic coordinates and structure factor files were deposited in the Protein Data Bank under accession codes 9XHM (AcvB(25–456)) and 9XHN (AcvB(249–456)). Contributions Y.W. conceived the project. M.H. performed most of the experiments. M.H. and Y.W. conducted structural studies. Y.W. wrote the original draft, and both authors reviewed and edited the manuscript. Competing interests The authors declare no competing interests. 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Share Structural basis of substrate recognition and membrane association by the bacterial lysyl-phosphatidylglycerol hydrolase AcvB Mizuki Hoshi , Yasunori Watanabe bioRxiv 2025.11.10.687609; doi: https://doi.org/10.1101/2025.11.10.687609 Share This Article: Copy Citation Tools Structural basis of substrate recognition and membrane association by the bacterial lysyl-phosphatidylglycerol hydrolase AcvB Mizuki Hoshi , Yasunori Watanabe bioRxiv 2025.11.10.687609; doi: https://doi.org/10.1101/2025.11.10.687609 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Biochemistry Subject Areas All Articles Animal Behavior and Cognition (7635) Biochemistry (17697) Bioengineering (13894) Bioinformatics (41951) Biophysics (21455) Cancer Biology (18592) Cell Biology (25507) Clinical Trials (138) Developmental Biology (13380) Ecology (19903) Epidemiology (2067) Evolutionary Biology (24321) Genetics (15610) Genomics (22509) Immunology (17737) Microbiology (40398) Molecular Biology (17182) Neuroscience (88618) Paleontology (667) Pathology (2833) Pharmacology and Toxicology (4825) Physiology (7641) Plant Biology (15158) Scientific Communication and Education (2046) Synthetic Biology (4296) Systems Biology (9825) Zoology (2271)