Inhibiting heme-piracy by pathogenicEscherichia coliusingde novo-designed proteins

Inhibiting heme-piracy by pathogenic Escherichia coli using de novo-designed proteins | 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 Inhibiting heme-piracy by pathogenic Escherichia coli using de novo -designed proteins Daniel R. Fox , Kazem Asadollahi , Imogen Samuels , Bradley Spicer , Ashleigh Kropp , Chris Lupton , Kevin Lim , Chunxiao Wang , Hari Venugopal , Marija Dramicanin , View ORCID Profile Gavin Knott , View ORCID Profile Rhys Grinter doi: https://doi.org/10.1101/2024.12.05.626953 Daniel R. Fox 1 Department of Microbiology, Biomedicine Discovery Institute, Monash University , Clayton 3800, Australia 2 Centre for Electron Microscopy of Membrane Proteins, Monash Institute of Pharmaceutical Sciences , Parkville, 3052, Victoria, Australia 3 Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne , Parkville, Victoria 3010, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kazem Asadollahi 3 Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne , Parkville, Victoria 3010, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Imogen Samuels 3 Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne , Parkville, Victoria 3010, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Bradley Spicer 4 Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University , Clayton 3800, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ashleigh Kropp 1 Department of Microbiology, Biomedicine Discovery Institute, Monash University , Clayton 3800, Australia 2 Centre for Electron Microscopy of Membrane Proteins, Monash Institute of Pharmaceutical Sciences , Parkville, 3052, Victoria, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Chris Lupton 4 Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University , Clayton 3800, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kevin Lim 5 The Walter and Eliza Hall Institute of Medical Research , Parkville, Victoria 3052, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Chunxiao Wang 3 Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne , Parkville, Victoria 3010, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Hari Venugopal 6 Ramaciotti Centre for Cryo-Electron Microscopy, Biomedicine Discovery Institute, Monash University , Clayton 3800, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Marija Dramicanin 5 The Walter and Eliza Hall Institute of Medical Research , Parkville, Victoria 3052, Australia 7 Department of Medical Biology, University of Melbourne , Parkville, Victoria 3010, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Gavin Knott 4 Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University , Clayton 3800, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Gavin Knott For correspondence: rhys.grinter{at}unimelb.edu.au gavin.knott{at}monash.edu.au Rhys Grinter 1 Department of Microbiology, Biomedicine Discovery Institute, Monash University , Clayton 3800, Australia 2 Centre for Electron Microscopy of Membrane Proteins, Monash Institute of Pharmaceutical Sciences , Parkville, 3052, Victoria, Australia 3 Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne , Parkville, Victoria 3010, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Rhys Grinter For correspondence: rhys.grinter{at}unimelb.edu.au gavin.knott{at}monash.edu.au Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Iron is an essential nutrient for most bacteria and is often growth-limiting during infection, due to the host sequestering free iron as part of the innate immune response. To obtain the iron required for growth, many bacterial pathogens encode transporters capable of extracting the iron-containing cofactor heme directly from host proteins. Pathogenic E. coli and Shigella spp . produce the outer membrane transporter ChuA, which binds host hemoglobin and extracts its heme cofactor, before importing heme into the cell. Heme extraction by ChuA is a dynamic process, with the transporter capable of rapidly extracting heme from hemoglobin in the absence of an external energy source, without forming a stable ChuA-hemoglobin complex. In this work, we utilise a combination of structural modelling, Cryo-EM, X-ray crystallography, mutagenesis, and phenotypic analysis to understand the mechanistic detail of this process. Based on this understanding we utilise artificial intelligence-based protein design to create binders capable of inhibiting E. coli growth by blocking hemoglobin binding to ChuA. By screening a limited number of these designs, we identify several binders that inhibit E. coli growth at low nanomolar concentrations, without experimental optimisation. We determine the structure of a subset of these binders, alone and in complex with ChuA, demonstrating that they closely match the computational design. This work demonstrates the utility of de novo -designed proteins for inhibiting bacterial nutrient uptake and uses a workflow that could be applied to integral membrane proteins in other organisms. Introduction Escherichia coli and the closely related genus Shigella (containing the species S. boydii , S. dysenteriae, S. flexneri , and S. sonnei ) are important pathogens of humans and animals 1 – 4 . E. coli and Shigella are genetically diverse with different strains sharing as little as 40% of their protein-encoding genes 5 , 6 . This diversity allows these bacteria to adopt a range of lifestyles from harmless commensal to intestinal and extra-intestinal pathogen 2 . E. coli strains that cause intestinal infection are divided into six subtypes (DAEC, EAEC, EHEC, EIEC, EPEC, and ETEC) based on their phylogenetic lineage and the disease symptoms they elicit 2 , 7 . EIEC strains are closely related to Shigella, with Shigella spp. emerging several times from independent E. coli ancestors, with only a limited number of genetic markers distinguishing the species 8 . Enteric infections caused by pathogenic E. coli and Shigella are a leading cause of infant and childhood diarrhoeal mortality in the developing world 9 – 11 and a significant cause of food- related infections globally 4 . In 2016, there were an estimated 270 million Shigella cases leading to 212,000 deaths, 222 million ETEC cases leading to 51,000 deaths, and 14 million EPEC cases leading to 12,000 deaths globally 10 , 11 . Extra-intestinal pathogenic E. coli (ExPEC) predominantly cause urinary tract infections (UPEC) and neo-natal meningitis (NMEC). ExPEC E. coli is the most common cause of both these infections, leading to a significant disease burden 2 , 12 . Increasing antimicrobial resistance among pathogenic E. coli and Shigella is a major concern, with the World Health Organization ranking E. coli as third on the list of twelve antibiotic-resistant ‘priority pathogens’ and designating fluoroquinolone-resistant Shigella spp. high priority status in the Bacterial Priority Pathogens List 2024 13 , 14 . Like most bacterial pathogens, E. coli and Shigella require the essential nutrient iron to cause infection 15 – 18 . Most bacteria require iron as a cofactor for enzymes that perform diverse reactions key to their survival and persistence. For example, DNA biogenesis, gene regulation and respiratory ATP production all require enzymatic reactions dependent on iron 19 . While available sources of iron differ depending on the host context, it is a scarce resource due to sequestration by the host, via an innate immune process known as nutritional immunity 20 . E. coli and Shigella employ two general strategies to obtain iron during infection. In the first strategy, they secrete compounds known as siderophores, which sequester iron with high affinity, and then reimport and process the iron-siderophore complex 21 , 22 . In the second strategy, they import the iron-containing cofactor heme, either as free heme or by extraction from host heme-containing proteins like hemoglobin 23 . Both these strategies rely on a class of outer-membrane protein known as TonB-dependent transporters (TBDTs) 24 , 25 . TBDTs bind a target nutrient compound with high affinity and import it into the cell using energy provided by the inner-membrane TonB-ExbBD complex 26 . TBDTs consist of a 22-stranded transmembrane β-barrel with a pore occluded by a globular plug domain, which is transiently displaced during import 24 , 25 . While the structure of the TBDT barrel is highly conserved, different TBDTs have diverse extracellular loops that mediate a high level of substrate specificity, while the transient displacement of the plug domain prevents the ingress of noxious compounds 24 , 25 . While E. coli and Shigella strains encode many genes for the production and import of chemically diverse siderophores 21 , 22 , only two TBDTs that import heme have been identified: Hma which imports free heme, and ChuA (aka. ShuA) that imports either free heme or extracts heme directly from host hemoglobin 15 – 18 , 27 , 28 . Hma and ChuA are important for virulence, as mutants lacking hma and chuA were outcompeted by the WT parental strain in kidney during co-infection experiments in mice 28 , 29 . ChuA interacts transiently with host hemoglobin and rapidly extracts its heme cofactor 17 . It is also capable of extracting heme from myoglobin although at a much slower rate. Two histidine residues (His-86 and His-420) are essential for this process 17 . His-420 is located in extracellular loop 7, while His-86 is located in the plug domain 18 . The presence and location of these histidine residues imply a transition between distinct coordination states occurs during heme extraction and import. However, the structural basis for hemoglobin binding, heme extraction and import remained unresolved. In this work, we utilise a combination of structural modelling, Cryo-EM, X-ray crystallography, mutagenesis, and phenotypic analysis to determine the mechanistic detail of this process. Preventing bacterial growth by blocking access to essential nutrients remains an underexplored strategy, which could be exploited to develop novel treatments for infections caused by antibiotic-resistant bacterial pathogens. Leveraging our understanding of the molecular basis for ChuA function, we utilised RFdiffusion-based protein design to create binders that block hemoglobin binding to ChuA 30 . We screened a limited number of these designs, identifying several binders that inhibit E. coli growth at low nanomolar concentrations when hemoglobin or myoglobin is the sole available iron source. We characterised the affinity of a subset of these binders for ChuA, and determined representative structures, alone and in complex with ChuA, demonstrating that they closely match the computational design. This work demonstrates the utility of de novo -designed proteins for inhibiting the growth of bacterial pathogens by blocking the import of essential nutrients. Moreover, it demonstrates the utility of AI-based protein design to create binders capable of modulating the function of membrane transporters, using a workflow that could be applied to integral membrane proteins in other organisms. Main ChuA targets hemoglobin as its high-affinity substrate Previous phenotypic analysis indicates ChuA acts as a heme transporter, targeting either free hemin (ferric heme) or hemoglobin 15 – 18 , while biochemical analysis indicates that ChuA binds free hemin much more slowly than it extracts heme from hemoglobin, suggesting free hemin may not be the transporter’s preferred substrate 17 . In addition, ChuA also extracts heme from myoglobin, although at a much slower rate than from hemoglobin, suggesting this protein is also a ChuA substrate 17 . To reconcile these data, we sought to determine the substrate specificity of ChuA in bacterial cell culture. However, redundancy in TBDT-based iron-uptake systems makes it difficult to assess the contribution of a specific transporter to iron uptake. To solve this, we engineered an E. coli BW25113 strain that lacks all TBDTs involved in iron uptake 31 – 33 , so that it contains the Chu operon, which encodes ChuA and proteins required for cytoplasmic heme import and processing 16 , 34 ( Figure 1a ). This strain is impaired in its ability to grow under even mildly iron-limited conditions 31 – 33 , providing a clean background for assessing ChuA function. We tested the ability of this strain ( E. coli ΔTBDT:ChuOP ) to grow on agar containing free hemin or various human heme or iron-containing proteins as its iron source. In this assay, E. coli ΔTBDT:ChuOP was able to grow with hemoglobin (adult αβ or fetal αγ), myoglobin and hemin as a heme/iron source, but not cytoglobin, neuroglobin, or ferredoxin 1 ( Figure 1b ). The ability to grow on hemoglobin, myoglobin and hemin was abolished in a chuA knockout ( E. coli ΔTBDT:ChuOP:ΔchuA ), but restored by complementation with a plasmid- encoded copy of chuA , demonstrating the specificity of this effect ( Figure 1b , Figure S1a). Download figure Open in new tab Figure 1: ChuA targets hemoglobin, myoglobin and free hemin. (a) Schematic of the genetic engineering strategy for the generation of the ChuA reporter strain used in this study. (b) The growth phenotype of the strain described in (a), grown on LB agar (top, representative images shown) in the presence of iron-limited LB agar supplemented with either 5 µM αβHb, αγHb, hemin, myoglobin, neuroglobin, cytoglobin or human ferredoxin 1. (below) EC 50 values of E. coli ΔTBDT:ChuOP cultured in LB liquid medium in the presence of 200 µM 2,2’-bipyridine, supplemented with serially diluted αβHb, αγHb, hemin, myoglobin, neuroglobin, cytoglobin or ferredoxin 1. Next, we assessed the growth of E. coli ΔTBDT:ChuOP in liquid culture across a range of heme- containing substrate concentrations. While neither the parental E. coli ΔTBDT strain nor E. coli ΔTBDT:ChuOP:ΔchuA were able to grow at any substrate concentration, E. coli ΔTBDT:ChuOP exhibited growth in the presence of hemoglobin (adult αβHb or fetal αγHb), myoglobin (Mb), hemin, and to a lesser extent neuroglobin (Figure1b, Figure S1b). The affinity of ChuA for these substrates varied considerably, with αβHb and αγHb representing the highest affinity substrates (EC 50 values of 53 and 44 nM respectively), followed by hemin (EC 50 value of 350 nM), and myoglobin (EC 50 value of 1.3 µM) ( Figure 1b ). These data indicate that while ChuA can import heme derived from several sources, hemoglobin represents its highest affinity substrate. To determine the affinity of ChuA for αβHb and Mb, we performed bio-layer interferometry (BLI), with recombinant globins immobilised on the sensor. We measured a disassociation constant (K D ) for ChuA binding to αβHb of 71.5 nM, with relatively fast association (K on ) and dissociation rate (K off ) constants, consistent with the transient nature of the interaction (Figure S1c, Table S1). Some signal was detected for Mb but with insufficient response to measure binding kinetics, indicating that ChuA interacts more weakly with this substrate. ChuA binds heme and hemoglobin via extracellular loops Based on the observation that ChuA has the highest affinity for hemoglobin, we attempted to determine the structure of the ChuA-αβHb complex by crystallography. Crystallisation screening was performed with 2:1 molar ratio of αβHb to ChuA, with pink/brown crystals forming in several conditions. X-ray diffraction data was collected from these crystals and solved by molecular replacement (Table S2). However, only ChuA was observed in the crystal, with heme bound to His-420, located in extracellular loop 7 of the transporter ( Figure 2a ). Aside from bound heme, the overall structure was highly similar to the previously solved apo- structure of ShuA 18 (99% AA identity; RMSD = 0.430 Å out of 3626/4638 atoms). The presence of heme bound to ChuA is consistent with previous work showing that the transporter forms a transient interaction with αβHb, extracting heme, which binds at His-420 17 . We also attempted to determine the ChuA-αβHb complex by CryoEM, but only recovered class averages containing free αβHb and ChuA, further indicating that this interaction is transient (Figure S2). Download figure Open in new tab Figure 2: ChuA coordinates heme at His-420 and binds hemoglobin and myoglobin via extracellular loops 7 and 8. (a) Crystal structure of ChuA (light cyan) bound to hemin (orange) extracted from αβHb, showing the coordination of hemin by His-420 of loop 7. (b) The top- ranked AlphaFold2 multimer model of ChuA-αβHb dimer (αHb shaded light green, βHb shaded dark green). (c) The top-ranked AlphaFold2 multimer model of ChuA-αγHb dimer (αHb shaded pink, γHb shaded purple). (d) The top-ranked AlphaFold2 multimer model of ChuA-Mb (Mb shaded salmon). (e) Loop 7 and 8 of ChuA binding to βHb from the ChuA-αβHb dimer model, with hemin coordinated in βHb appended the crystal structure of Hb (PDB ID: 2HHB). (f) Loop 7 and 8 of ChuA bound to hemin, via interactions with His-420, from the ChuA-hemin crystal structure (g) Hemin coordinated in βHb by His-93 as in the ChuA-αβHb dimer model, overlayed with the coordination of hemin bound to ChuA by His-420 in the crystal structure. (h) Loop 7 and 8 of the ChuA-Mb model, showing hemin coordinated by Mb appended from the crystal structure of Mb (PDB ID: 3RGK). (i) Loop 7 and 8 of ChuA binding to γHb from the ChuA- αγHb dimer model, showing hemin coordinated in γHb appended from the crystal structure of αγHb (PDB ID: 4MQJ). (j) ChuA sidechain clashes with the hemin in βHb as shown in panel b. (k) Superimposition of αβHb-NbE11 (PDB ID: 8VYL) with the ChuA-αβHb dimer model, with NbE11 shown in surface view. (l) Superimposition of αβHb-Hp (PDB ID: 4WJG) with the ChuA- αβHb dimer model, with Hp shown in surface view. (m) Representative images of soft agar overlay assays (n=3) of E. coli ΔTBDT:ChuOP:ΔchuA complemented with WT ChuA on iron-limited agar spotted with serially-diluted αβHb, αβHb-NbE11 or αβHb-Hp. Minimal concentration supporting growth as indicated by red arrows. (n) Quantification of the minimal growth concentrations of αβHb, αβHb-NbE11 or αβHb-Hp across three biological replicates (n=3). To gain insight into the structure of the ChuA-hemoglobin complex we performed AlphaFold2 multimer modelling 35 , providing the sequence of ChuA and one or two copies of the α and β Hb subunits, corresponding to a hemoglobin dimer or tetramer. The oligomeric state of hemoglobin is pH and redox-state dependent (Figure S3). The dimeric form predominates in serum and thus is more likely to be relevant as an iron source for invading pathogens 17 , 23 , 36 , 37 . Consistent with this, while ChuA-αβHb tetramer modelling did not produce any realistic solutions, the top-ranked solution for the ChuA-αβHb dimer modelling placed the β-subunit of the Hb dimer in contact with extracellular loops 7 and 8 of ChuA, with reasonable confidence scores ( Figure 2b , Figure S4a). We also performed AlphaFold2 modelling with dimeric αγHb and monomeric Mb, with these substrates predicted to form a complex with ChuA in the same orientation to the βHb subunit, adding to our confidence in this prediction ( Figure 2c,d , Figure S4a). When hemin was appended to the βHb subunit in complex with ChuA, based on the crystal structure of hemoglobin (PDB ID: 2HHB, Figure 2e ) 38 , its iron centre is only 8.5 Å from that of bound heme in the ChuA crystal structure ( Figure 2f,g ). Given their similar binding modes, heme present in both the γHb and Mb subunits were in a similar position ( Figure 2h,i ). βHb in complex with heme at this position has several clashes with sidechains in loops 7 and 8 of ChuA ( Figure 2j ), which may be involved in destabilising bound heme, facilitating its transfer to the proximal heme binding residue His-420, providing a plausible mechanism for heme extraction by ChuA. To validate the ChuA-αβHb dimer AlphaFold model, we assessed the ability of ChuA to extract and import heme from αβHb in complex with dimeric human haptoglobin (αβHb-Hp) 39 and the hemoglobin binding nanobody NbE11 (αβHb-NbE11) 40 using an iron-limited agar plate growth assay. Based on the ChuA-αβHb dimer model and the crystal structures of αβHb-Hp and αβHb-NbE11 (PDB IDs = 4WJG, 8VYL) 39 , 40 , the binding of NbE11 should not affect heme extraction from ChuA, while clashes between haptoglobin and ChuA should have a negative effect on heme extraction from αβHb-Hp ( Figure 2k,l ). Consistent with this structural data, ChuA was equally effective at extracting heme from αβHb-NbE11 as αβHb, with a minimal growth concentration of 0.24 µM. Conversely, ChuA was significantly less effective at extracting heme from αβHb-Hp with a minimal growth αβHb concentration of 0.96 µM ( Figure 2m,n ). The ability of ChuA to extract heme from αβHb-Hp, despite the significant clashes observed in the static modelling of this complex, is likely due to the flexibility of binding loops 7 and 8, combined with the dynamic nature of the interaction. His-420 and His-86 are required for growth on hemoglobin and hemin To further validate the AlphaFold2 model of the ChuA-αβHb dimer and to gain insight into heme transfer, we complemented E. coli ΔTBDT:ChuOP:ΔchuA with either WT ChuA, a panel of ChuA mutants, or an empty vector control, and tested their ability to grow on αβHb. We utilised ChuA H420A , ChuA H86A , ChuA H68A , single mutants to probe the role of possible heme coordinating histidine residues, as well as ChuA I423A;N429A;F484A , ChuA H420A;I423A;N429A;F484A and ChuA V482F;D483E;A485E multiple mutants, based on the interaction interface of the αβHb binding site in our AlphaFold2 model ( Figure 3 ). We used iron-limited agar growth assays, spotted with serially diluted αβHb to determine the minimum concentration of αβHb that could support the growth of the complemented strains compared to WT ChuA ( Figure 3 ). E. coli ΔTBDT:ChuOP:ΔchuA complemented with wildtype ChuA has a minimal αβHb concentration of Download figure Open in new tab Figure 3: His-420 and His-86 are required for growth on Hb and heme extraction. Representative images of soft agar overlay assays (n=3) of E. coli ΔTBDT:ChuOP:ΔchuA complemented with either WT ChuA (a) , empty vector (b) , ChuA I423A,N429A,F484A (c) , ChuA V482F,D483E,A485E (d) , ChuA H420A,I423A,N429A,F484A (e) , ChuA H420A (f) , ChuA H86A (g) , or ChuA H68A (h) , grown on iron-limited LB agar spotted with serially-diluted αβHb or hemin. Minimal concentration supporting growth as indicated by red arrows. Inset displays WT and mutated residues as shown as sticks in the ChuA-αβHb dimer AlphaFold2 multimer model, with βHb shown in an overlaid cartoon-surface view. Quantification of the mutant to WT growth ratio of the minimal αβHb (i) and hemin (j) concentration supporting growth. (k) AlphaFold3 models of WT ChuA in complex with heme, suggesting that heme is coordinated by both His- 420 and His-86, facilitated by the flexibility of ChuA loops 7 and 8. (I) AlphaFold3 models of ChuA H420A and H86A mutants in complex with heme, with all models predicting heme coordination by the remaining histidine residue. 0.24 µM, with no growth observed for the empty vector control ( Figure 3a,b ). The ChuA I423A;N429A;F484A mutant, designed to stabilise interactions with αβHb ( Figure 3c ), grew at a minimal Hb concentration of 0.48 µM αβHb, whereas the ChuA V482F;D483E;A485E mutant, designed to disrupt αβHb interactions by steric or electrostatic hindrance, grew at a minimal concentration of 0.24 µM, the same as wildtype ( Figure 3d ). It is unclear why these mutants had a relatively minor or no effect on αβHb utilisation, given they cause changes at the predicted ChuA-αβHb interface. However, this may result from the dynamic nature of this interaction. Both the single and multiple mutants containing the H420A substitution (ChuA H420A, ChuA H420A;I423A;N429A;F484A ) , did not grow at any αβHb concentration, consistent with previous reports that this residue is critical for heme extraction ( Figure 3e,f ) 17 . The ChuA H86A mutant also had a significantly abrogated growth phenotype, with growth only observed at a αβHb concentration of 7.69 µM, confirming previous reports that this residue is also important for heme extraction ( Figure 3g ) 17 . ChuA H68A exhibited similar growth to wildtype, with a 0.24 µM minimal αβHb concentration ( Figure 3h ), indicating it is not likely to be involved in heme extraction or coordination. To assess differences in the importance of ChuA binding site residues for heme binding and import, rather than heme extraction from hemoglobin ( Figure 3i ), we repeated the growth experiments with free hemin ( Figure 3j ). ChuA I423A;N429A;F484A , and ChuA H68A mutants grew at minimal hemin concentrations equivalent to wildtype (6.1µM), indicating these substitutions do not affect hemin binding or import ( Figure 3a,c ,h). ChuA V482F;D483E;A485E exhibited a two-fold increase in the minimum hemin concentration for growth (12.2 µM) ( Figure 3d ). Interestingly, at higher hemin concentrations (>191.7 µM) we observed growth inhibition due to excessive hemin import, likely due to the production of toxic hydroxyl radicals via Fenton chemistry or metalloporphyrin toxicity ( Figure 3a,c ,d,h) 41 Analogous to growth on αβHb, the ChuA H86A mutant was functional, but impaired in its ability to utilise hemin, with growth observed to 96.6 µM ( Figure 3g ). The ChuA H420A mutant was barely functional for hemin import with weak growth observed at the highest hemin concentration (773 µM) ( Figure 3f ). Intriguingly, the additional substitutions in the ChuA H420A;I423A;N429A;F484A mutant partially restored ChuA function with a minimal hemin growth concentration of 48.3 µM ( Figure 3e ). These additional mutations replace bulky residues surrounding His-420 with alanine, possibly relieving steric hindrance and allowing hemin binding to His-86, facilitating import. To gain further insight into heme coordination by ChuA, we performed AlphaFold3 modelling of the ChuA-heme complex 42 . Heme modelling varied across the five ranked models produced by AlphaFold3, ranging from coordination analogous to the ChuA-heme crystal structure with Fe coordination only by His-420, to bidentate coordination by both His-420 and His-86. This His-420 and His-86 coordination was facilitated by significant movement of ChuA loops 7 and 8 towards the plug domain ( Figure 3k ). When the modelling was performed with either the H420A or H86A variant ChuA, heme was coordinated by the remaining histidine in a single binding mode ( Figure 3l ). While the confidence scores for heme in these models were modest (Figure S4b,c), given the experimental evidence supporting the role of both His-420 and His- 86 hemin coordination, it is likely that this modelling provides a realistic picture of heme coordination and dynamics by ChuA. De novo -designed ChuA binders block heme uptake from hemoglobin Considering the importance of heme acquisition by pathogenic E. coli during infection 28 , 29 , 43 , we sought to leverage our understanding of ChuA function to design de novo protein binders to block heme acquisition from hemoglobin. Successful binder design validates both our model for hemoglobin binding by ChuA and provides a proof of concept for inhibiting E. coli infection by denying access to heme from host hemoglobin. Using an AlphaFold2 model of ChuA as a target, we utilised RFdiffusion and proteinMPNN to design binders targeting extracellular loops 7 and 8 of ChuA, which our model indicated were responsible for hemoglobin binding ( Figure 2 ) 30 , 44 , 45 . We generated ∼20,000 ChuA binders in silico and selected 96 designs for wet lab screening using AlphaFold2 filtering and manual curation 35 , 46 . We expressed and partially purified these ChuA binders in parallel on a small scale, with most binders expressing at similar levels (Figure S5a). We screened these binders for their ability to inhibit the growth of E. coli ΔTBDT:ChuOP , by spotting them on hemoglobin agar overlaid with the bacteria. We observed weak zones of growth inhibition for many of the binders, suggesting a relatively high level of success at generating lower-affinity binders, with eight binders displaying more prominent zones of inhibition ( Figure 4a ). Zones of inhibition were more prominent at a lower hemoglobin concentration, suggesting the binders act competitively (Figure S5b). Interestingly, no inhibition was observed on agar containing free hemin suggesting binders block heme-extraction from hemoglobin but not heme transport by ChuA (Figure S5c). Download figure Open in new tab Figure 4: De novo- designed ChuA binders bind with high affinity, inhibiting heme extraction from hemoglobin. (a) A representative image of a soft agar overlay assay (n=3) of E. coli ΔTBDT:ChuOP , grown on iron-limited LB agar containing 2 µM αβHb, spotted with 2 µl of 96 purified the de novo ChuA binders. (b) A soft agar overlay assay of E. coli ΔTBDT:ChuOP , grown on iron-limited LB agar containing 2 µM αβHb, spotted with 2-fold serial dilution of de novo binders A10, C8, G7 and H3 (8 µM – 62 nM). (c) As in panel b but supplemented with 8 µM Mb instead of αβHb. (d) The top-ranked AlphaFold2 multimer model of ChuA bound to binders A10, C8, G7 or H3. (e) Binder IC 50 of E. coli ΔTBDT:ChuOP grown iron-limited LB broth containing 0.1 µM αβHb, with 2-fold serially diluted de novo binders A10, C8, G7 or H3. IC 50 values were calculated as a % relative to the growth of the untreated control. Data (n=3) displayed as mean ± s.e.m. (f) Representative BLI sensorgram traces (top) and associated steady-state binding kinetics (bottom) of A10, G7 and H3 binding to ChuA. We expressed and purified four binders with the most prominent inhibition zones (A10, C8, G7 and H3) ( Figure 4d , Figure S5d). All binders inhibited αβHb and Mb-dependent growth of E. coli ΔTBDT:ChuOP when spotted on agar, to a concentration of 250-500 nM for αβHb and 125- 250 nM for Mb ( Figure 4b,c ). There was no inhibition when hemin or Fe(II)SO 4 was provided as the iron source (Figure S5e,f). The higher potency of binders with Mb as the heme source is likely due to the lower affinity of ChuA for this protein. To more robustly quantify binder potency, we determined binder IC 50 for E. coli ΔTBDT:ChuOP in liquid culture supplemented with either 50 nM or 100 nM αβHb. At 100 nM αβHb, IC 50 values ranged from 3.3 µM for A10 to 42.5 nM for G7 ( Figure 4e ). Lower IC 50 values were obtained at 50nM αβHb consistent with competitive inhibition for the ChuA binding site (Figure S5g). Next, we determined the affinity of binders A10, G7, and H3 for purified ChuA using BLI, with average disassociation constants of 127, 84.9 and 71.4 nM recorded for A10, G7, and H3 respectively ( Figure 4f , Table S1), which are broadly consistent with growth inhibition values. The association (K on ) and dissociation rate (K off ) constants varied considerably between the binders (Table S1, Figure S5h), with A10 forming a more transient interaction with ChuA, which may explain its higher IC 50 value, despite its roughly comparable K D to G7 and H3. ChuA-binder complex structures closely match the computational design Our functional and biochemical analysis of the de novo -designed ChuA binders definitively demonstrates they are high-affinity inhibitors of heme extraction from hemoglobin. To resolve how closely their computational design matches reality, we attempted to determine the crystal structures of A10, C8, G7 and H3 in isolation. Diffracting crystals were only obtained for binder C8, and the structure was solved at 2.46 Å (Table S2). The crystal contained eight molecules per asymmetric unit, which have a pairwise similar RMSD of ∼0.6 Å (Figure S6a,b). This was comparable to the computational design of C8, which also had an RMSD of ∼0.6 Å to the C8 molecules in the crystal structure (Figure S6c, Table S3), indicating that the predicted model closely matches the X-ray structure, once flexibility of the protein and experimental error are accounted for. To determine if the similarity between the designed and experimental structure extends to the binders in complex with ChuA, we determined the CryoEM structures of G7 and H3 in complex with the transporter (Figure S2, Table S4). The structures of both binders in complex with ChuA were resolved to <3 Å, allowing us to model these complexes with high confidence ( Figure 5a,b ). These experimental structures were very similar to the predicted models, with the RMSD between the binders of ∼0.6 Å ( Figure 5c,d ). The position of H3 in the full complex differed slightly between the designed and experimental structures, due to a change in the conformation of ChuA loops 7 and 8 ( Figure 5d ). However, as these loops are flexible both the design and experimental structure likely represent possible conformations in solution. These structures provide compelling evidence that the de novo design of ChuA binding proteins closely matches experimental reality. Download figure Open in new tab Figure 5: The cryoEM structures of ChuA-G7 and ChuA-H3 closely match the computational design and block hemoglobin binding via steric hindrance. The final cryoEM density maps of the ChuA-G7 complex (a) or ChuA-H3 complex (b) (ChuA shaded cyan, G7 shaded dark pink, H3 shaded dark blue) (left) and the refined structures of the ChuA-G7 complex or the ChuA- H3 shown as a cartoon representation (right). Zoomed view of G7 (c) or H3 (d) bound to loops 7 and 8 of ChuA, with the cryoEM structure overlaid (G7 shaded dark pink, H3 shaded dark blue) with the AlphaFold2 model (G7 shaded light pink, H3 shaded light blue), showing the structures of ChuA-G7 and ChuA-H3 closely match the computation designs. The structure and model are depicted as cartoon representation, with sidechains shown as thin sticks. Overlay of the ChuA-G7 (e) or ChuA-H3 (f) complex structures with the ChuA-αβHb dimer AlphaFold2 multimer model, showing binders block the predicted ChuA αβHb binding site, preventing heme extraction. Overlay of the ChuA-G7 (g) or the ChuA-H3 complex (h) structures with the ChuA-heme crystal structure, showing only minor clashes with heme bound H420, and G7 or H3, and the area surrounding is H86 unaffected. An overlay of the ChuA-binder structures, with the predicted ChuA-αβHb complex, shows that both G7 and H3 occupy the majority of the predicted αβHb binding surface of ChuA loops 7 and 8 ( Figure 5e,f ), providing a clear mechanism for the observed inhibition of heme extraction by ChuA. Conversely, the same binder overlay with the ChuA-heme experimental structure, shows that only minor clashes occur between the heme bound at His-420 and the binding proteins ( Figure 5g,h ). Given that ChuA coordinates heme via both His-420 and His- 86, in a process mediated by the flexibility of loops 7 and 8, and the fact that His-420 is conditionally dispensable for heme uptake, these structures are consistent with ChuA remaining capable of heme import even in complex with these binding proteins. Conclusions In this work, we show that ChuA is a heme transporter that targets dimeric hemoglobin (αβHb or αγHb) as its high affinity substrate and is also capable of importing free hemin or heme from myoglobin. Considering most heme in the human body is contained within αβHb, and free heme is rapidly sequestered by hemopexin or serum albumin 23 , 47 , 48 , these findings indicate that αβHb is likely the physiological substrate for ChuA. Further, considering αβHb in serum is rapidly and tightly bound by haptoglobin, and ChuA can to some extent utilise the αβHb-Hp, it is likely this complex is also a physiological substrate for ChuA 49 . We also clarify the role of His-86 and His-420 in ChuA function, showing that while these histidines are important for heme extraction from αβHb, both are conditionally dispensable for the import of free hemin. This observation combined with our structural data indicating both His-86 and His-420 directly coordinate the hemin Fe centre, hints that heme extraction and import by ChuA is a dynamic multistage process. We leverage this understanding of ChuA function to generate de novo designed binding proteins, which selectively block heme extraction from αβHb by ChuA. Two of these binders achieved sub-100 nM affinity, without experimental optimisation, validating AI-guided protein design for binder generation against flexible, integral membrane protein targets. The success of our binder design supports our model for heme extraction and binding by ChuA, providing insight into this dynamic and difficult-to- study process, and demonstrating the utility of de novo-designed binders as research tools. Our structural analysis of the ChuA-G7 and Chu-H3 complexes by CryoEM shows that RFdiffusion-based computational design closely matches experimental reality and allows for a definitive explanation of the effect of these binders on ChuA function. Finally, the ability of these binders to inhibit E. coli growth provides a strong proof of concept of the use of de novo- designed binding proteins as antimicrobials, which block the uptake of essential nutrients at the cell surface. Methods Protein expression and purification ChuA WT ChuA lacking the signal peptide sequence was amplified from E. coli CFT073 by PCR, with primers containing NcoI and XhoI restriction sites to clone ChuA into a modified pET20b vector, carrying an N-terminal 10x His tag followed by a TEV protease cleavage site. pET20bChuA was then transformed into E. coli BL21 (DE3) C41 cells and plated onto LB agar supplemented with 100 µg/ml ampicillin and incubated at 37°C overnight. An overnight starter culture was prepared in the same medium, before being sub-cultured the next day in terrific broth supplemented with 100 µg/ml ampicillin at 37°C. 8L cultures were grown until an OD 600 of 0.8-1.0 was reached, and protein expression was then induced by the addition of 0.3 mM isopropyl 1-thio-b-D-galactopyranoside (IPTG), and were then grown for a further 16 hours at 24°C. Cells were harvested via centrifugation and pellets were homogenised in lysis buffer (50 mM Tris, 200 mM NaCl, pH 7.8, supplemented with 1 mM MgCl2, 0.1 mg/ml lysozyme, 0.05 mg/ml DNase 1 and x1 Complete protease inhibitor tablet (Roche)/8L culture) and placed in an ice bath slurry for 30 minutes before being lysed with a cell disruptor (Emulseflex, one pass-through). Lysate was clarified via centrifugation at 15,000 rpm for 10 minutes at 4°C and the supernatant was ultracentrifuged at 100,000 g for 1 hour at 4°C to isolate the membrane fraction. The membrane pellet was then solubilised in lysis buffer using a tight-fit dounce homogeniser before the addition of 5% Elugent (Santa Cruz Biotechnology) and incubated at room temperature with gentle rocking for 20 minutes. A 5 ml HisTrap nickel column was prepared via washing with 10-15 column volumes of MilliQ H2O followed by 10- 15 column volumes of binding buffer (50 mM Tris, 500 mM NaCl, 20 mM imidazole, 0.03% dodecyl maltoside (DDM), pH 7.8) before binding loaded with solubilised membranes. The resin was washed with a further 10-15 column volumes of binding buffer before elution of protein fractions via a stepwise gradient (10%, 25%, 50%, 75% and 100%) in elution buffer (50 mM Tris, 500 mM NaCl, 1 M imidazole, 0.03% DDM, pH 7.8). Fractions were run via SDS-PAGE and analysed via Coomassie gel staining. Fractions containing ChuA were pooled and concentrated using a 100 kDa spin filter column (Millipore) before loading onto a S200 26/600 Superdex size exclusion chromatography (SEC) column (Cytiva) preequilibrated in SEC buffer (50 mM Tris, 200 mM NaCl, 0.03% DDM, pH 7.8) and were run using the AKTA Pure system (Cytiva). Fractions were then run via SDS-PAGE and analysed via Coomassie staining. Fractions containing ChuA were pooled and concentrated with a separate 100 kDa spin filter column before being stored at -80°C. Recombinant globins Sequences for the expression of recombinant human αβ and αγ hemoglobin, myoglobin, neuroglobin, cytoglobin and ferredoxin 1 were ordered as gene fragments from Twist Biosciences and cloned into either pETDuet-1 (for hemoglobin), pET29a or pET22b, carrying an N-terminal 6x His tag. For αβ and αγ hemoglobin, a 6x His tag was added to either the N- terminal of the alpha subunit or the C-terminal of the β or γ subunit in the event presence of the tag interfered with tertiary structure formation. Expression vectors were transformed into E. coli BL21 (DE3) C41 cells and plated onto LB agar supplemented with 100 µg/ml ampicillin and incubated at 37°C overnight. An overnight starter culture was prepared in the same medium, before being sub-cultured the next day in terrific broth supplemented with 100 µg/ml ampicillin at 37°C. 3-4L cultures were grown until an OD 600 of 0.8-1.0 was reached, and protein expression was then induced by the addition of 0.3 mM IPTG and were then grown for a further 16 hours at 22°C. Cells were harvested and lysed in lysis buffer (50 mM Tris, 200 mM NaCl, pH 7.4, supplemented with 1 mM MgCl 2 , 0.1 mg/ml lysozyme, 0.05 mg/ml DNase 1 and x1 Complete protease inhibitor tablet (Roche)/8 L culture) and placed in an ice bath slurry for 30 minutes before being lysed with a cell disruptor (Emulseflex, one pass- through). Lysate was clarified via centrifugation at 15,000 rpm for 10 minutes at 4°C before loading into a HisTrap nickel column, pre-equilibrated in binding buffer (50 mM Tris, 500 mM NaCl, pH 7.4). Fractions were run via SDS-PAGE and analysed via Coomassie gel staining. Fractions containing purified globins were pooled and loaded onto a S75 26/600 Superdex SEC column (Cytiva) using a 50 ml Superloop (Cytiva) preequilibrated in SEC buffer (50 mM Tris, 200 mM NaCl, pH 7.4) and were run using the AKTA Pure system (Cytiva). Fractions were then run via SDS-PAGE and analysed via Coomassie staining. Fractions containing purified recombinant globins were pooled and concentrated with a separate 3 or 10 kDa spin filter column before being stored at -80°C. Hemoglobin nanobody NbE11 The human hemoglobin nanobody NbE11 was expressed and purified as described previously 40 . Purification of hemoglobin from erythrocytes Hemoglobin was purified from human blood collected from a consenting, healthy adult volunteer in accordance with 2022-30658-70864 approved by the Monash University Human Research Ethics Committee. Erythrocytes were isolated via centrifugation and cell pellets were diluted in 50 mM Tris, 200 mM NaCl, pH 7.4. To isolate hemoglobin, Erythrocytes were lysed with a tight-fit dounce homogeniser and clarified by centrifugation at 18,000 rpm. Cell lysate was further purified using a Superdex 200 10/300 column (Cytiva) preequilibrated in 50 mM Tris, 200 mM NaCl, pH 7.4. Fractions were collected and pooled and concentrated to ∼10 mg/ml using a 30 kDa spin filter column (Millipore). Generation of different hemoglobin oligomeric and redox states To generate different oligomeric states of hemoglobin, 500ul of hemoglobin purified from human erythrocytes was diluted to 1.5 ml in 50 mM BisTris, 100 mM NaCl, at either pH 6.0, 6.5, 7.0, or 8.0. The hemoglobin samples were run through pre-equilibrated a HiTrap desalting column (Cytiva) and the flow through containing hemoglobin was collected. The flow-through was applied to the HiTrap desalting column three times to ensure full buffer exchange. Hemoglobin samples were left to incubate at room temperature in buffer for 30 minutes before loading 5 µM onto a pre-equilibrated S200 10/300 column for analytical SEC analysis. The mass of hemoglobin in buffer at either pH 6.0 or 8.0 was further confirmed using a Refeyn 2MP Mass Photometer. To generate methemoglobin (metHb) from oxyhemoglobin (oxyHb), oxyHb was incubated with NaNO 2 at a 1:5 MR at room temperature shaking for 2 hours, as described previously 36 . The samples were then buffer exchanged into 50 mM Tris, 200 mM NaCl, pH 7.4 to remove excess NaNO 2 . The absorbance spectra of NaNO 2 -treated and untreated samples were measured using a Tecan Infinite 200 PRO microplate reader between 500-750 nm to identify the presence or absence of distinctive peaks corresponding to each of the Hb redox states 50 . Genetic engineering of ChuA mutants Mutant genotypes of E. coli BW25113 were generated using the λ-red system as described previously 51 . In brief, the E. coli ΔTBDT strain lacking all native TonB-dependent transporters; deleted in the following sequence: fhuA, fecA, cirA, fepA, fhuE, fiu, yncD, and yddB, was used as the base strain 31 – 33 .The E. coli ΔTBDT strain was then recombineered with the entire Chu operon ( Figure 1a ) to generate E. coli ΔTBDT:ChuOP . DNA encoding the Chu operon, +500 bp up- and downstream, was amplified by PCR from the uropathogenic E. coli strain CFT073. 100 µg of this PCR product was electroporated into E. coli ΔTBDT containing the λ-recombinase containing plasmid pKD46 induced with arabinose. Cells were plated onto LB-agar containing 150 µM 2’2-bipyridine and 1 µM αβHb. Emergent colonies were screened by PCR to confirm integration of the Chu operon into the E. coli ΔTBDT genome. The chuA gene was then deleted from E. coli ΔTBDT:ChuOP strain using the λ-red system to generate E. coli ΔTBDT:ChuOP:ΔchuA . E. coli ΔTBDT and all derived strains required additional iron for growth, which during passage was supplied supplement LB agar supplemented with 2.5 nM Fe(II)SO 4 at 37°C. Bacterial agar growth assays To determine whether the ChuA strains could utilise different sources of heme or iron, each strain was streaked onto different LB agar plates containing 150 µM 2’2-bipyridine (BP; an iron chelator, to control for any iron present in the LB), supplemented with 5 µM sterile human αβ or αγ hemoglobin, myoglobin, cytoglobin, neuroglobin or ferredoxin 1. Plates were then incubated at 37°C overnight. Complementation of E. coli ΔTBDT:ChuOP:ΔchuA with WT or mutant ChuA Sequences for the 6 ChuA mutants, in addition to WT ChuA, were ordered from Twist Bioscience and were amplified and cloned into pTET (originally from Ben Adler at UC Berkeley) using GoldenGate cloning using the BbsI restriction sites. The resultant vectors (pTET WT or mutant ChuA) were sequenced at Primordium Inc. and were confirmed to carry the correct sequences. pTET WT or mutant ChuA were electroporated into E. coli ΔTBDT:ChuOP:ΔchuA and were maintained on LB agar supplemented with 2.5 nM Fe(II)SO 4 and 34 µg/ml chloramphenicol at 37°C. To test for complementation E. coli ΔTBDT:ChuOP:ΔchuA :pTET WT or mutant ChuA were inoculated in soft agar overlay assays (base: 1.5% agar, supplemented with 100 µM BP, 200 ng/ml anhydrous tetracyline and 34 µg/ml chloramphenicol; soft agar overlay: 0.6% agar, supplemented with 100 µM BP, 200ng/ml anhydrous tetracyline and 34 µg/ml chloramphenicol) at an OD 600 of 0.1. 1:2 Serially-diluted sterile hemoglobin, hemin, NbE11- Hb (MR 2:1 NbE11:Hb tetramer ) or Hp-Hb (MR 1:2 Hp:Hb dimer ), was spotted over the dry plates (with a top concentration of 0.5 mg/ml) and were subsequently cultured at 30°C for two days. Serial dilution E. coli growth experiments Globins To determine if ChuA preferentially acquires heme from different heme sources, E. coli ΔTBDT , E. coli ΔTBDT:ChuOP and E. coli ΔTBDT:ChuOP:ΔChuA were grown in 5 mL of 2.5 nM Fe(II)SO 4 - supplemented LB broth and incubated overnight at 37°C. The next day, 10 ml of LB supplemented with 150 µM BP was inoculated with each genotype at OD 600 of 0.01. 100 µL of inoculated medium was then added to each well in a 96-well plate. 12 µM of αβ or αγ hemoglobin or 48 µM of either hemin, myoglobin, cytoglobin, neuroglobin or ferredoxin 1 was then added to 200 µL of the inoculated medium in an Eppendorf tube, and 100 µL of this medium was then added to the first well and then serially diluted by a factor of 2. Plates were then incubated at 37°C overnight and the OD 600 was measured using a Tecan Infinite 200 PRO microplate reader. De novo binders To generate IC50s, 10 ml of LB was supplemented with 200 µM BP and 0.2 µM hemoglobin, and was inoculated with the E. coli ΔTBDT:ChuOP strain to an initial OD 600 of 0.01. In a 96-well plate, binders were serially diluted 1:2 for 24 dilutions, with a top concentration of 400 µM, and the plates were wrapped in parafilm before incubation at 37°C at 100 rpm. The OD 600 was read 24 hours later using a ClarioStar microplate reader. ChuA binders were also serially diluted onto soft agar overlay assays (base: 1.5% agar, supplemented with 100 µM BP; soft agar overlay: 0.6% agar, supplemented with 100 µM BP, inoculated with E. coli ΔTBDT:ChuOP at a starting OD 600 of 0.1, with either 2 µM hemoglobin, 8 µM myoglobin, 8 µM hemin or 2.5nM Fe(II)SO 4 , with the latter without any BP added to either the base or soft overlay). A 1:2-fold dilution series of ChuA binders A10, C8, G7 and H3 (with a top concentration of 8 µM) were spotted over the dry plates and were subsequently cultured at 37°C overnight. AlphaFold2/3 modelling AlphaFold2 multimer modelling predictions were run using AlphaFold version 2.1.1 35 through the Monash MASSIVE M3 computing cluster. Modelling of ChuA with heme using AlphaFold3 was conducted using the AlphaFold Server 42 . The top five ranked models were interrogated, and the top-ranked model was visualised using PyMOL (Schrödinger). The following structures were used as overlay in the models for the appropriate placement of heme in hemoglobin (PDB ID: 2HHB) 52 and myoglobin (PDB ID: 3RGK) 53 , or for superimposition of the αβHb-Hp complex (PDB ID: 4WJG) 39 . Design of de novo binders by RFdiffusion-ProteinMPNN-AF2 initial guess A search model was prepared for binder backbone generation in RFdiffusion 30 , using an AlphaFold2 model of ChuA, defining the outer loops of the barrel and plug domain and excluding the remainder of the model from the search (ChuA search amino acids 51-61, 66- 70, 81-89, 175-193, 218-246, 272-291, 321-340, 366-381, 406-442, 474-499, 523-539, 564-579, 609-621). This model was provided to RFdiffusion as a target, with amino acids 572, 594, 563, and 568 defined as hotspots. Binder size was specified as 70-90 amino acids. The denoiser noise scale and scale frame were 0, and standard model weights were used. A total of 5,000 models generated in RFdiffusion, were provided to the DL binder design pipeline 45 , for sequence assignment using proteinMPNN 44 and quality screening using AlphaFold2 initial guess. Binders were screened for in silico success, using an AlphaFold2 initial guess pAE interact score cutoff of <10. The RFdiffusion output for successful designs (∼200 models) where then recycled through the DL binder design pipeline five times, to generate a final pool of models for selection (∼550 models) with a pAE interact score cutoff of <10. These models were further screened by complex prediction with full-length ChuA using AlphaFold2 multimer 2.3.2 35 , 54 , and manually curated to select 96 designs for synthesis and testing. Expression and purification of de novo binders Binder gene sequences were synthesized by Twist Bioscience and inserted in a pET29b expression vector between NdeI and XhoI binding sites. A N or C-terminal 6xHis tag was utilised depending on which of the binder termini was not involved in interactions with ChuA. Initial binder expression was performed in parallel. Binder expression plasmids were transformed into E. coli C41 DE3 cells by heat shock in a 2 ml 96 well plate (0.5 µl plasmid DNA at 10-50 µg/ml, 10 µl chemically competent E. coli cells). 1 ml of LB broth was added to cells before recovery at 37 ° C for 1 hour, with shaking at 400 rpm. Kanamycin selection was then added (50 µg/ml), and cells were incubated overnight at 37 °C. The binder construct transformed E. coli C41 DE3 cells were used to inoculate 4 ml of overnight express terrific broth (Merck) with 50 µg/ml Kanamycin in 24 well plate format. Cultures were grown for 18 hours at 30 ° C before the cells were harvested via centrifugation. The supernatant was eluted and cells were lysed using B-PER reagent (ThermoFisher Scientific) following the manufacturer’s instructions. Lysed cells were clarified by centrifugation and the supernatant was transferred to a 2 ml 96 well plate, and 50µl of Ni-agarose resin slurry was added, before incubation with shaking at 200 rpm for 1.5 hours. The cell lysate was then added to a 96-well filter plate (30-40 µM cutoff), and lysate was removed with a vacuum manifold retaining the resin on the filter. Wells were washed with 3 x 1.5 ml of wash buffer (50 mM Tris, 500 mM NaCl, 20 mM imidazole, pH 7.8), before binders were eluted by adding 2 x 200 µl of elution buffer (50 mM Tris, 500 mM NaCl, 500 mM imidazole, pH 7.8), into a separate 500 µl 96 well plate. Binder expression and purity were assessed by SDS-PAGE. Larger scale production of binders A10, C8, G7, and H3 was performed as described above for the recombinant globins. Screening of de novo binders For screening of the putative de novo binders, the E. coli ΔTBDT:ChuOP strain in a soft agar overlay assay was used. The base agar (1.5%) was supplemented with 100 µM BP and was left to set. To this, a soft agar (0.6%) overlay was supplemented with 100 µM BP, 0.125 mg/ml (1.94 µM) hemoglobin and inoculated with the E. coli ΔTBDT:ChuOP strain at an initial OD 600 of 0.1 and left to set. 2 µl of each of the 96 binders was spotted onto the soft agar overlay and plates were then incubated at 37°C. X-Ray crystallography, data processing, refinement and analysis ChuA-heme Purified ChuA (10 mg/ml final concentration) in 0.8% β-octylglucoside was combined with purified human αβHb (∼20 mg/ml final concentration) in a 1:2 ratio, and the complex was screened for crystallisation conditions (sitting drop, 100 nl protein + 100 nl crystallisation solution) using commercially available crystallisation screens (Index, JCSG+, MIDAS, PACT, ShotGunSG1 and PEG Ion) (Hampton Research, Molecular Dimensions). Pink-brown crystals formed in the PACT crystal screen condition containing: 0.1 M MIB buffer, 25% (w/v) PEG 1500, pH 6. Crystals for data collection were prepared from an optimisation grid screen ranging from 24-28 (w/v) PEG 1500, and pH 4-7, with the MIB buffer concentration constant. Excess mother liquor was removed by wicking, before being cryocooled in liquid N 2 at 100 K. Data was collected at the Australian Synchrotron, with crystals diffracting anisotropically to 2.8 Å (resolution ranged from 2.8 Å along the k-axis to 3.43 along the l-axis, judged by I/σ(I) = 1.5) in the P2 1 2 1 2 1 space group. The crystal structure of ShuA from Shigella dysenteriae (PDB ID = 3FHH) 38 was used for phasing via molecular replacement using Phaser 55 . Based on this solution we found that the crystals only contain ChuA in complex with heme that had been extracted from αβHb. The resulting structure was rebuilt manually and refined using PHENIX 56 and Coot 57 . De novo ChuA binder C8 Purified binder C8 (8 mg/ml) was screened for crystallisation conditions (sitting drop, 100 nl protein + 100 nl crystallisation solution) using commercially available crystallisation screens (Index, JCSG+, MIDAS, PACT, ShotGunSG1 and PEG Ion) (Hampton Research, Molecular Dimensions). Crystals with different morphologies formed in a range of conditions (Sheet/rectangular crystals formed in 0.2 M Na Acet, 0.1 M Tris pH 8.5, 30% w/v PEG 4K; Needle-like crystals formed in 0.2 M (NH 4 ) 2 SO 4 , 0.1M Na Acet pH 4.6, 30% w/v PEG MME 2K; Hexagonal crystals formed in 0.2 M K Na Tart, 2 M (NH 4 ) 2 SO 4 , 0.1 M Na 3 Citrate pH 5.6). Crystals were looped directly from screening trays, and the excess mother liquor was removed before cryocooling in liquid N 2 at 100 K. Data was collected at the Australian Synchrotron. Most crystals diffracted poorly (highest resolution diffraction = 6-8 Å). However, a single crystal from the 0.2 M Na Acet, 0.1 M Tris pH 8.5, 30% w/v PEG 4K condition diffracted to ∼2.5 Å. Data was collected on this crystal and the structure was solved by molecular replacement using an AlphaFold2 model of C8. The crystal contained 8 molecules per asymmetric unit, consisting of two groups of four molecules related by strong translational non-crystallographic symmetry (tNCS). The structure was rebuilt manually and refined using PHENIX 56 and Coot 57 . While the majority of all 8 molecules could be built into the available electron density, the refinement R-factors remained high due to the tNCS. However, the maps were predictive and of sufficient quality to compare the predicted and experimental structure of binding protein C8. CryoEM imaging of ChuA-G7 and ChuA-H3 complexes ChuA (72.3 µM final concentration; 5 mg/ml) was mixed with binder G7 or H3 (72.3 µM final concentration) 2 hours before grid preparation and 3.5 μl of the complex was applied onto a glow-discharged UltrAuFoil grid (Quantifoil GmbH) and were flash frozen in liquid ethane using the Vitrobot mark IV (ThermoFisher Scientific) set at 95% humidity and 4 °C for the prep chamber. Data were collected on a G1 Titan Krios microscope (ThermoFisher Scientific) with S-FEG as electron source operated at an accelerating voltage of 300 kV. A C2 aperture of 50 μm was used and no objective aperture was used. Data was collected at a nominal magnification of 105 K in nanoprobe EFTEM mode. Gatan K3 direct electron detector positioned post a Gatan BioQuantum energy filter was operated in a zero-energy-loss mode using a slit width of 10 eV to acquire dose fractionated images of the ChuA-G7 and ChuA-H3 complexes. One dataset was collected, composed of ∼7,000 movies. Movies were recorded in hardware-binned mode yielding a physical pixel size of 0.82 Å pixel −1 with a dose rate of 8.5 e− pixel −1 s −1 . An exposure time of 7 s yielded a total dose of 70.0 e− Å −2 , which was further fractionated into 70 subframes. Automated data collection was performed using EPU (ThermoFisher Scientific) with periodic centring of zero loss peak. A defocus range was set between −1.4 and −0.5 μm. CryoEM data processing and analysis Micrographs from all datasets were motion-corrected using MotionCor 3.0 (Chan Zuckerberg Institute) and dose-weighted averages had their CTF parameters estimated using CTFFIND 4.1.8, implemented using Relion 5.0 58 . Particle coordinates were determined by crYOLO 1.7.6 using a general model 59 . 4x binned particles were extracted from micrographs using Relion 5.0, before being imported into cryoSPARC 4.4.1 for initial 2D classification to remove bad particles, followed by ab initio model generation and 3D refinement 60 . Subsequently, the data from each dataset was processed as described in Figure S2b,c with a final resolution of 2.97 Å for ChuA-G7 and 2.51Å for ChuA-H3, respectively (FSC = 0.143, gold standard). An AlphaFold2 model of the ChuA-G7 and ChuA-H3 complexes were fitted into the CryoEM density maps before model improvement and refinement using PHENIX 56 and Coot 57 . Biolayer interferometry analysis BLI experiments were conducted using the Gator Plus Label-Free Bioanalysis System (Gator Bio). Ni-NTA probes were hydrated in binding buffer (50 mM Tris, 200 mM NaCl, 0.03% DDM, pH 7.8) for at least 10 minutes before use, and all experiments were performed at 25°C with an orbital shaking speed of 1000 rpm. Samples were diluted in binding buffer and probes were then loaded with either binder, αβHb dimer or myoglobin to a shift of 0.2-0.4 nm, to avoid crowding on the probe surface. An unloaded reference probe was included as a control to define any non-specific binding. After loading probes were dipped in buffer and then subsequently exposed to a 2-fold increasing ChuA concentration series, starting at 10nM, to determine association signals. Between concentrations, probes were also dipped in binding buffer to determine dissociation signals. Each association-dissociation step lasted for five minutes to ensure enough binding and dissociation occurred before starting the next cycle. Analysis was conducted using the integrated Gator® GatorOne software (Gator Bio). Data was reference-subtracted, inter-step corrected (to association) and Savitzky-Golay filtered (for experiments with strong binding signal). For binder experiments, binding data was calculated using a combined association-dissociation 1:1 model, with global fitting (association window: 0-300 seconds, dissociation window: 0-90 seconds). Each experiment was performed in triplicate, and the K D values were averaged. Data was plotted using GraphPad Prism 10 Software. Statistical analysis Data analysis was conducted using GraphPad Prism 10 software. Data are shown as mean ± s.e.m. For three or more groups statistical significance was determined by a one-way ANOVA with multiple comparisons tests. P < 0.05 was considered statistically significant. IC 50 and EC 50 values were calculated using a nonlinear regression (curve fit). BLI binding statistics were determined using the integrated Gator® GatorOne software (Gator Bio). Only full association and dissociation data with R 2 values > 0.98 and Χ 2 < 3 were included for analysis. Data Availability CryoEM maps and atomic models generated from this study have been deposited in the Protein Data Bank (PDB IDs: 9DHE, 9DIV, 9DIR, 9DIS) and the Electron Microscopy Data Bank (EMDB IDs: EMD-46916, EMD-46917). All other data to support the findings of the study are either available with this article or are available from the corresponding authors on request. Author Contributions Conceived and designed the experiments: D.R.F., K.A., G.N.L.J., M.D., G.K., and R.G.; performed the experiments: D.R.F., K.A., I.S., B.S., A.K., C.L., K.L., G.N.L.J., H.V., M.D., and R.G., analysed the data: D.R.F., K.A., I.S., B.S., G.N.L.J., H.V., M.D., and R.G.; contributed reagents/materials/analysis tools: G.N.L.J., H.V., M.D., G.K., and R.G.; wrote and edited the manuscript: D.R.F. and R.G.; acquired funding and provided project supervision: R.G. and G.K. All authors edited and approved the manuscript. Abbreviations BLI: biolayer interferometry, CryoEM: cryogenic electron microscopy, DAEC: diffusely adherent E. coli, EAEC: enteroaggregative E. coli, EHEC: enterohaemorrhagic E. coli , EIEC: enteroinvasive E. coli , EPEC: enteropathogenic E. coli, ETEC: enterotoxigenic E. coli , ExPEC: extra-intestinal pathogenic E. coli, Hb: hemoglobin, Mb: Myoglobin, NMEC: neo-natal meningitis, RMSD: root mean square deviation, SEC: size exclusion chromatography, TBDTs: TonB-dependent transporters, UPEC: uropathogenic E. coli Download figure Open in new tab Figure S1 Capacity of ChuA to target different heme-containing substrates. (a) A hemoglobin agar plate streaked with E. coli ΔTBDT:ChuOP:ΔchuA or E. coli ΔTBDT:ChuOP:ΔchuA pChuA, showing ChuA is required in this strain for hemoglobin-dependent growth. (b) Liquid culture growth assays with different heme-containing substrates, with E. coli ΔTBDT , E. coli ΔTBDT:ChuOP, or E. coli ΔTBDT:ChuOP:ΔchuA , showing the minimum substrate concentrations able to support ChuA dependent growth. (n=3, biological replicates, statistical significance was determined through a one-way ANOVA with multiple comparisons tests. P < 0.05 was considered statistically significant.). (c) Representative BLI sensorgram trace (left) and associated steady-state binding kinetics (right) of αβHb binding to ChuA. Download figure Open in new tab Figure S2. CryoEM processing data for ChuA-Hb and binder complexes. (a) 2d class averages from grids prepared with a 1:1 ratio of ChuA to αβHb, showing only ChuA-alone class averages were obtained. (b) Data processing workflow for the ChuA-H3 binder complex. (c) ChuA-H3 binder final map coloured by local resolution, and plots of resolution, B-factor and particle angle distribution. (d) Data processing workflow for the ChuA-G7 binder complex. (e) ChuA- G7 binder final map coloured by local resolution, and plots of resolution, B-factor and particle angle distribution. Download figure Open in new tab Figure S3. The effect of pH and redox state on hemoglobin oligomeric state. Analytical SEC of oxy-αβHb (a) and met-αβHb (b) , at pH 6 to 8, showing that αβHb is predominantly tetrameric at pH 8.0, and largely dimeric at pH 6.0. (c) The change in absorbance of oxy-αβHb between 500-600 nm, after treatment with O 2 or NaNO 2 to generate met-αβHb. (d) Mass- photometry data confirming that met-αβHb is predominantly tetrameric at pH 8.0, and largely dimeric at pH 6.0. (e) SEC profile of recombinant αβHb pH7.4, 6xhis-tagged at either the n-term of the α-subunit or at the c-term of the β-subunit, showing that tagging the α- subunit induces dimerization. Download figure Open in new tab Figure S4. AlphaFold modelling quality indicators for ChuA-substrate complexes. (a) AlphaFold2 models of the ChuA-αβHb, ChuA-αγHb, and ChuA-Mb complexes coloured by pLDDT confidence score. (b) AlphaFold3 models of ChuA-hemin complexes coloured by pLDDT confidence score. (c) pAE scores for the AlphaFold3 ChuA-hemin models. Download figure Open in new tab Figure S5. Screening and validation of de novo designed ChuA binders. (a) SDS-PAGE analysis of initial purification of binders for functional screening. The ability of binder to inhibit an overlay of E. coli ΔTBDT:ChuOP on agar containing different concentrations of αβHb (b) or hemin (c) . (d) SDS-PAGE analysis of large-scale purification of ChuA binders selected for further analysis. (e) A soft agar overlay assay of E. coli ΔTBDT:ChuOP , grown on iron-limited LB agar containing 8 µM hemin, spotted with 2-fold serial dilution of de novo ChuA binders A10, C8, G7 and H3 (8 µM – 62 nM). (f) A soft agar overlay assay of E. coli ΔTBDT:ChuOP , grown on LB agar containing 2.5 nM FeSO 4 , spotted with de novo as in panel d. (g) Binder IC 50 of E. coli ΔTBDT:ChuOP grown iron limited LB broth containing 0.05 µM αβHb, with 2-fold serially diluted de novo binders A10, C8, G7 or H3. IC 50 values were calculated as a % relative to the growth of the untreated control. Data (n=3) displayed as mean ± s.e.m. (h) a plot of K on and K off rates for ChuA to binding proteins measured by BLI. Download figure Open in new tab Figure S6. The ChuA binder C8 computational model closely matches the crystal structure. (a) A cartoon representation of the eight C8 molecules in the asymmetric unit of the crystal structure of C8 crystal structure. (b) A superimposition of the eight C8 molecules from the C8 crystal structure, showing they exhibit some variation in both sidechain and backbone conformation. (c) A superimposition of the C8 AlphaFold2 model (tan) and a representative C8 molecule from the crystal structure (dark blue), show experiment and prediction match closely. Supplemental Tables Table S1. BLI statistical parameters for globins and binding proteins. Table S2. Crystallographic data collection and refinement statistics Table S3. Comparison of RMSD of C8 molecules in the crystal and RFdiffusion model Table S4. CryoEM data collection and model statistics Acknowledgements The authors acknowledge the use of electron microscopy and cryo-sample preparation facilities at the Ian Holmes Imaging Centre of the Bio21 Molecular Science & Biotechnology Institute, the University of Melbourne; in particular Dr. Hamish Brown for training and assistance, and at the Ramaciotti Centre for Cryo-Electron Microscopy of the Biomedicine Discovery Institute, Monash University. This research was supported by ARC LIEF grants (LE200100045, LE120100090) for the Titan Krios Gatan K3 Camera and the Titan Krios, and an ARC discovery project grant awarded to R.G. and G.K. (DP230102150). This research was undertaken on the MX2 beamlines at the Australian Synchrotron, part of ANSTO (CAP20894). R.G. and D.R.F. are members of the Australian Research Council Industrial Transformation Training Centre for Cryo-Electron Microscopy of Membrane Proteins for Drug Discovery (IC200100052). R.G. was funded by an NHMRC EL1 investigator grant (APP1197376). D.R.F. was supported by an Australian Government Research Training Program (RTP) Scholarship. The authors thank Dr. Roxanne Smith at the Bio21-WEHI Crystallisation Facility, at the University of Melbourne, for her assistance with sample characterisation, crystallographic screening, and optimisation. 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Nat Methods 14 , 290 – 296 ( 2017 ). doi: 10.1038/nmeth.4169 OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted December 06, 2024. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Inhibiting heme-piracy by pathogenic Escherichia coli using de novo-designed proteins Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Inhibiting heme-piracy by pathogenic Escherichia coli using de novo -designed proteins Daniel R. Fox , Kazem Asadollahi , Imogen Samuels , Bradley Spicer , Ashleigh Kropp , Chris Lupton , Kevin Lim , Chunxiao Wang , Hari Venugopal , Marija Dramicanin , Gavin Knott , Rhys Grinter bioRxiv 2024.12.05.626953; doi: https://doi.org/10.1101/2024.12.05.626953 Share This Article: Copy Citation Tools Inhibiting heme-piracy by pathogenic Escherichia coli using de novo -designed proteins Daniel R. Fox , Kazem Asadollahi , Imogen Samuels , Bradley Spicer , Ashleigh Kropp , Chris Lupton , Kevin Lim , Chunxiao Wang , Hari Venugopal , Marija Dramicanin , Gavin Knott , Rhys Grinter bioRxiv 2024.12.05.626953; doi: https://doi.org/10.1101/2024.12.05.626953 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 (7651) Biochemistry (17746) Bioengineering (13928) Bioinformatics (42064) Biophysics (21499) Cancer Biology (18650) Cell Biology (25579) Clinical Trials (138) Developmental Biology (13409) Ecology (19947) Epidemiology (2067) Evolutionary Biology (24373) Genetics (15633) Genomics (22557) Immunology (17774) Microbiology (40504) Molecular Biology (17217) Neuroscience (88793) Paleontology (667) Pathology (2845) Pharmacology and Toxicology (4836) Physiology (7664) Plant Biology (15178) Scientific Communication and Education (2047) Synthetic Biology (4304) Systems Biology (9839) Zoology (2272)