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• W2989454912 abstract "Single-chain antibodies from camelids have served as powerful tools ranging from diagnostics and therapeutics to crystallization chaperones meant to study protein structure and function. In this study, we isolated a single-chain antibody from an Indian dromedary camel (ICab) immunized against a bacterial 14TM helix transporter, NorC, from Staphylococcus aureus. We identified this antibody in a yeast display screen built from mononuclear cells isolated from the immunized camel and purified the antibody from Escherichia coli after refolding it from inclusion bodies. The X-ray structure of the antibody at 2.15 Å resolution revealed a unique feature within its CDR3 loop, which harbors a Zn2+-binding site that substitutes for a loop-stabilizing disulfide bond. We performed mutagenesis to compromise the Zn2+-binding site and observed that this change severely hampered antibody stability and its ability to interact with the antigen. The lack of bound Zn2+ also made the CDR3 loop highly flexible, as observed in all-atom simulations. Using confocal imaging of NorC-expressing E. coli spheroplasts, we found that the ICab interacts with the extracellular surface of NorC. This suggests that the ICab could be a valuable tool for detecting methicillin-resistant S. aureus strains that express efflux transporters such as NorC in hospital and community settings. Single-chain antibodies from camelids have served as powerful tools ranging from diagnostics and therapeutics to crystallization chaperones meant to study protein structure and function. In this study, we isolated a single-chain antibody from an Indian dromedary camel (ICab) immunized against a bacterial 14TM helix transporter, NorC, from Staphylococcus aureus. We identified this antibody in a yeast display screen built from mononuclear cells isolated from the immunized camel and purified the antibody from Escherichia coli after refolding it from inclusion bodies. The X-ray structure of the antibody at 2.15 Å resolution revealed a unique feature within its CDR3 loop, which harbors a Zn2+-binding site that substitutes for a loop-stabilizing disulfide bond. We performed mutagenesis to compromise the Zn2+-binding site and observed that this change severely hampered antibody stability and its ability to interact with the antigen. The lack of bound Zn2+ also made the CDR3 loop highly flexible, as observed in all-atom simulations. Using confocal imaging of NorC-expressing E. coli spheroplasts, we found that the ICab interacts with the extracellular surface of NorC. This suggests that the ICab could be a valuable tool for detecting methicillin-resistant S. aureus strains that express efflux transporters such as NorC in hospital and community settings. Multidrug efflux has emerged as a major strategy employed by drug-resistant pathogens to overcome antibiotic and biocide stress that they encounter in their environments (1Walsh C. Molecular mechanisms that confer antibacterial drug resistance.Nature. 2000; 406 (10963607): 775-78110.1038/35021219Crossref PubMed Scopus (1183) Google Scholar). Integral membrane transporters belonging to five major families, including the major facilitator superfamily (MFS), 6The abbreviations used are: MFSmajor facilitator superfamilyMATEmultidrug and toxic compound extrusionSMRsmall multidrug resistanceABCATP-binding cassetteRNDresistance nodulation divisionDHAdrug:H+ antiporter(s)TMtransmembraneMRSAmethicillin-resistant S. aureusVHHvariable heavy chain antibodyCDRcomplementarity-determining regionDDMdodecyl-β-d-maltopyranosideSECsize-exclusion chromatographyFSECfluorescence-detection size-exclusion chromatographyPBMCperipheral blood mononuclear cellFRframework regionITCisothermal titration calorimetrynano-DSFnano-differential scanning fluorimetryDAPI4′,6-diamidino-2-phenylindoleODoptical densityLBLuria brothESIelectrospray ionizationRMSDroot mean square deviation. multidrug and toxic compound extrusion (MATE) family, small multidrug resistance (SMR) family, ATP-binding cassette (ABC) transporters, and resistance nodulation division (RND) superfamily, are involved in the process of active efflux of antibacterial compounds (2Webber M.A. Piddock L.J. The importance of efflux pumps in bacterial antibiotic resistance.J. Antimicrob. Chemother. 2003; 51 (12493781): 9-1110.1093/jac/dkg050Crossref PubMed Scopus (495) Google Scholar). Among the numerous efflux pumps observed in bacterial species, the major facilitator superfamily comprises the largest class of secondary active transporters (3Reddy V.S. Shlykov M.A. Castillo R. Sun E.I. Saier Jr., M.H. The major facilitator superfamily (MFS) revisited.FEBS J. 2012; 279 (22458847): 2022-203510.1111/j.1742-4658.2012.08588.xCrossref PubMed Scopus (314) Google Scholar). Efflux transporters within this superfamily are grouped into drug:H+ antiporters (DHA) containing 12 or 14 transmembrane (TM) helices referred to as DHA1 and DHA2 families, respectively (3Reddy V.S. Shlykov M.A. Castillo R. Sun E.I. Saier Jr., M.H. The major facilitator superfamily (MFS) revisited.FEBS J. 2012; 279 (22458847): 2022-203510.1111/j.1742-4658.2012.08588.xCrossref PubMed Scopus (314) Google Scholar, 4Saier Jr., M.H. Reddy V.S. Tamang D.G. Västermark A. The transporter classification database.Nucleic Acids Res. 2014; 42 (24225317): D251-D25810.1093/nar/gkt1097Crossref PubMed Scopus (328) Google Scholar). The DHA2 family members are capable of transporting a wide array of substrates, including antibiotics, bile salts, and lipophilic cations (5Paulsen I.T. Brown M.H. Skurray R.A. Proton-dependent multidrug efflux systems.Microbiol. Rev. 1996; 60 (8987357): 575-608Crossref PubMed Google Scholar, 6Mallonee D.H. Hylemon P.B. Sequencing and expression of a gene encoding a bile acid transporter from Eubacterium sp. strain VPI 12708.J. Bacteriol. 1996; 178 (8955384): 7053-705810.1128/jb.178.24.7053-7058.1996Crossref PubMed Google Scholar). A large number of DHA2 efflux transporters exist within pathogenic bacteria, although, unlike the DHA1 members, there is no representative atomic structure available for any of them. Efflux pumps have gained increased attention in recent times as a potential target for efflux pump inhibitors that could serve as adjuvants enhancing the efficacy of existing antibiotics (7Wright G.D. Antibiotic adjuvants: rescuing antibiotics from resistance (Trends in Microbiology 24, 862–871; October 17, 2016).Trends Microbiol. 2016; 24 (27522372): 92810.1016/j.tim.2016.07.008Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 8Douafer H. Andrieu V. Phanstiel 4th, O. Brunel J.M. Antibiotic adjuvants: make antibiotics great again!.J. Med. Chem. 2019; 62 (31063379): 8665-868110.1021/acs.jmedchem.8b01781Crossref PubMed Scopus (110) Google Scholar). major facilitator superfamily multidrug and toxic compound extrusion small multidrug resistance ATP-binding cassette resistance nodulation division drug:H+ antiporter(s) transmembrane methicillin-resistant S. aureus variable heavy chain antibody complementarity-determining region dodecyl-β-d-maltopyranoside size-exclusion chromatography fluorescence-detection size-exclusion chromatography peripheral blood mononuclear cell framework region isothermal titration calorimetry nano-differential scanning fluorimetry 4′,6-diamidino-2-phenylindole optical density Luria broth electrospray ionization root mean square deviation. The role of DHA members acquires prominence in Gram-positive pathogens like methicillin-resistant S. aureus (MRSA), which lack RND family transporters due to the absence of an outer membrane (9Piddock L.J. Multidrug-resistance efflux pumps—not just for resistance.Nat. Rev. Microbiol. 2006; 4 (16845433): 629-63610.1038/nrmicro1464Crossref PubMed Scopus (1048) Google Scholar). Numerous DHA2 efflux transporters are observed in MRSA, including QacA, NorB, and NorC, that are proposed to efflux antibacterial compounds (10Paulsen I.T. Brown M.H. Littlejohn T.G. Mitchell B.A. Skurray R.A. Multidrug resistance proteins QacA and QacB from Staphylococcus aureus: membrane topology and identification of residues involved in substrate specificity.Proc. Natl. Acad. Sci. U.S.A. 1996; 93 (8622987): 3630-363510.1073/pnas.93.8.3630Crossref PubMed Scopus (236) Google Scholar) and quinolone antibiotics, respectively (11Costa S.S. Viveiros M. Amaral L. Couto I. Multidrug efflux pumps in Staphylococcus aureus: an update.Open Microbiol. J. 2013; 7 (23569469): 59-7110.2174/1874285801307010059Crossref PubMed Scopus (239) Google Scholar, 12Truong-Bolduc Q.C. Strahilevitz J. Hooper D.C. NorC, a new efflux pump regulated by MgrA of Staphylococcus aureus.Antimicrob. Agents Chemother. 2006; 50 (16495280): 1104-110710.1128/AAC.50.3.1104-1107.2006Crossref PubMed Scopus (103) Google Scholar). MRSA is known to cause infections ranging from minor skin infections like boils and abscesses to lethal infections like bacteremia, sepsis, and endocarditis (13Tong S.Y. Davis J.S. Eichenberger E. Holland T.L. Fowler Jr., V.G. Staphylococcus aureus infections: epidemiology, pathophysiology, clinical manifestations, and management.Clin. Microbiol. Rev. 2015; 28 (26016486): 603-66110.1128/CMR.00134-14Crossref PubMed Scopus (2409) Google Scholar). Also, detection of efflux pumps in the bacterial membrane would aid in ascertaining whether the superbug could efflux drugs of certain chemical classes. Any tool that either detects or blocks efflux in pathogens would serve as a diagnostic or therapeutic agent to test for the presence and treatment of superbugs, respectively. In this study, we employ NorC as a test case to generate single domain camelid antibodies from an Indian dromedary camel (Camelus dromedarius) for performing structural studies. Single-domain camelid antibodies, also known as variable heavy chain antibodies (VHHs) or nanobodies, consist of only a heavy chain in their structure with a single domain retaining the epitope-binding complementarity-determining regions (CDRs) instead of an antibody fragment that comprises a light chain and a heavy chain (14Muyldermans S. Nanobodies: natural single-domain antibodies.Annu. Rev. Biochem. 2013; 82 (23495938): 775-79710.1146/annurev-biochem-063011-092449Crossref PubMed Scopus (1228) Google Scholar). VHH domains were identified in dromedaries (15Hamers-Casterman C. Atarhouch T. Muyldermans S. Robinson G. Hamers C. Songa E.B. Bendahman N. Hamers R. Naturally occurring antibodies devoid of light chains.Nature. 1993; 363 (8502296): 446-44810.1038/363446a0Crossref PubMed Scopus (2168) Google Scholar) and in sharks (16Greenberg A.S. Avila D. Hughes M. Hughes A. McKinney E.C. Flajnik M.F. A new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification in sharks.Nature. 1995; 374 (7877689): 168-17310.1038/374168a0Crossref PubMed Scopus (538) Google Scholar) and have become powerful tools due to their small size (∼15 kDa), high stability, greater permeability and ease of heterologous expression compared with conventional antibodies (∼150 kDa) (17Beghein E. Gettemans J. Nanobody technology: a versatile toolkit for microscopic imaging, protein-protein interaction Analysis, and protein function exploration.Front. Immunol. 2017; 8 (28725224): 77110.3389/fimmu.2017.00771Crossref PubMed Scopus (117) Google Scholar, 18Baral T.N. MacKenzie R. Arbabi Ghahroudi M. Single-domain antibodies and their utility.Curr. Protoc. Immunol. 2013; 103 (Unit 2.17) (24510545)10.1002/0471142735.im0217s103Crossref PubMed Scopus (37) Google Scholar). The paratope in VHH domains comprises three hypervariable CDRs that link the framework regions of the single-domain antibody as opposed to conventional antibody or Fabs that have six CDRs, three from the heavy and three from the light chain that constitute the antigen-binding site. The three CDRs of VHH are also longer than those of conventional antibodies. In particular, the CDR3 of VHH domains between strands F and G displays a stretched twisted turn conformation to shield the FR2 region, which is involved in the formation of the heterodimer interface between VH and VL domains in conventional antibodies (14Muyldermans S. Nanobodies: natural single-domain antibodies.Annu. Rev. Biochem. 2013; 82 (23495938): 775-79710.1146/annurev-biochem-063011-092449Crossref PubMed Scopus (1228) Google Scholar). The CDR3 loop is typically stabilized by an additional disulfide between the CDR1 and -3 loops, particularly in camel VHH domains that have a longer CDR3 compared with that of llamas. The long CDRs in VHH domains compensate for the absence of the VL domain and its CDRs that form the large antigen-binding site in conventional antibodies. This property gives the VHH domains their characteristic prolate ellipsoid shape that allows them to target cavities deep within their target antigens (19De Genst E. Silence K. Decanniere K. Conrath K. Loris R. Kinne J. Muyldermans S. Wyns L. Molecular basis for the preferential cleft recognition by dromedary heavy-chain antibodies.Proc. Natl. Acad. Sci. U.S.A. 2006; 103 (16537393): 4586-459110.1073/pnas.0505379103Crossref PubMed Scopus (462) Google Scholar). VHH domains are frequently observed to target active sites of enzymes (20Kromann-Hansen T. Oldenburg E. Yung K.W. Ghassabeh G.H. Muyldermans S. Declerck P.J. Huang M. Andreasen P.A. Ngo J.C. A camelid-derived antibody fragment targeting the active site of a serine protease balances between inhibitor and substrate behavior.J. Biol. Chem. 2016; 291 (27226628): 15156-1516810.1074/jbc.M116.732503Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) and specific conformations of receptors (21Rasmussen S.G. Choi H.J. Fung J.J. Pardon E. Casarosa P. Chae P.S. Devree B.T. Rosenbaum D.M. Thian F.S. Kobilka T.S. Schnapp A. Konetzki I. Sunahara R.K. Gellman S.H. Pautsch A. Steyaert J. Weis W.I. Kobilka B.K. Structure of a nanobody-stabilized active state of the β(2) adrenoceptor.Nature. 2011; 469 (21228869): 175-18010.1038/nature09648Crossref PubMed Scopus (1314) Google Scholar) and transporters (22Jiang X. Smirnova I. Kasho V. Wu J. Hirata K. Ke M. Pardon E. Steyaert J. Yan N. Kaback H.R. Crystal structure of a LacY-nanobody complex in a periplasmic-open conformation.Proc. Natl. Acad. Sci. U.S.A. 2016; 113 (27791182): 12420-1242510.1073/pnas.1615414113Crossref PubMed Scopus (31) Google Scholar) and interact with structured epitopes of their targets (23Mitchell L.S. Colwell L.J. Comparative analysis of nanobody sequence and structure data.Proteins. 2018; 86 (29569425): 697-70610.1002/prot.25497Crossref PubMed Scopus (91) Google Scholar). Owing to their unique properties, VHH domains have become increasingly popular for applications in medicine, biotechnology, and research as therapeutics, diagnostics, and research tools (24Wesolowski J. Alzogaray V. Reyelt J. Unger M. Juarez K. Urrutia M. Cauerhff A. Danquah W. Rissiek B. Scheuplein F. Schwarz N. Adriouch S. Boyer O. Seman M. Licea A. et al.Single domain antibodies: promising experimental and therapeutic tools in infection and immunity.Med. Microbiol. Immunol. 2009; 198 (19529959): 157-17410.1007/s00430-009-0116-7Crossref PubMed Scopus (378) Google Scholar, 25Harmsen M.M. De Haard H.J. Properties, production, and applications of camelid single-domain antibody fragments.Appl. Microbiol. Biotechnol. 2007; 77 (17704915): 13-2210.1007/s00253-007-1142-2Crossref PubMed Scopus (591) Google Scholar, 26Hassanzadeh-Ghassabeh G. Devoogdt N. De Pauw P. Vincke C. Muyldermans S. Nanobodies and their potential applications.Nanomedicine (Lond.). 2013; 8 (23730699): 1013-102610.2217/nnm.13.86Crossref PubMed Scopus (213) Google Scholar). They have also been used successfully as crystallization chaperones to obtain diffraction quality crystals of protein targets that are otherwise difficult to crystallize like integral membrane proteins (27Desmyter A. Spinelli S. Roussel A. Cambillau C. Camelid nanobodies: killing two birds with one stone.Curr. Opin. Struct. Biol. 2015; 32 (25614146): 1-810.1016/j.sbi.2015.01.001Crossref PubMed Scopus (75) Google Scholar, 28Mujić-Delić A. de Wit R.H. Verkaar F. Smit M.J. GPCR-targeting nanobodies: attractive research tools, diagnostics, and therapeutics.Trends Pharmacol. Sci. 2014; 35 (24690241): 247-25510.1016/j.tips.2014.03.003Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). As a result, many of the transporters (22Jiang X. Smirnova I. Kasho V. Wu J. Hirata K. Ke M. Pardon E. Steyaert J. Yan N. Kaback H.R. Crystal structure of a LacY-nanobody complex in a periplasmic-open conformation.Proc. Natl. Acad. Sci. U.S.A. 2016; 113 (27791182): 12420-1242510.1073/pnas.1615414113Crossref PubMed Scopus (31) Google Scholar, 29Ehrnstorfer I.A. Geertsma E.R. Pardon E. Steyaert J. Dutzler R. Crystal structure of a SLC11 (NRAMP) transporter reveals the basis for transition-metal ion transport.Nat. Struct. Mol. Biol. 2014; 21 (25326704): 990-99610.1038/nsmb.2904Crossref PubMed Scopus (130) Google Scholar, 30Kumar H. Finer-Moore J.S. Jiang X. Smirnova I. Kasho V. Pardon E. Steyaert J. Kaback H.R. Stroud R.M. Crystal structure of a ligand-bound LacY-nanobody complex.Proc. Natl. Acad. Sci. U.S.A. 2018; 115 (30108145): 8769-877410.1073/pnas.1801774115Crossref PubMed Scopus (24) Google Scholar), channels (31Löw C. Yau Y.H. Pardon E. Jegerschöld C. Wåhlin L. Quistgaard E.M. Moberg P. Geifman-Shochat S. Steyaert J. Nordlund P. Nanobody mediated crystallization of an archeal mechanosensitive channel.PLoS One. 2013; 8 (24205053)e7798410.1371/journal.pone.0077984Crossref PubMed Scopus (18) Google Scholar, 32Hassaine G. Deluz C. Grasso L. Wyss R. Tol M.B. Hovius R. Graff A. Stahlberg H. Tomizaki T. Desmyter A. Moreau C. Li X.D. Poitevin F. Vogel H. Nury H. X-ray structure of the mouse serotonin 5-HT3 receptor.Nature. 2014; 512 (25119048): 276-28110.1038/nature13552Crossref PubMed Scopus (291) Google Scholar), and G protein–coupled receptors (33Manglik A. Kobilka B.K. Steyaert J. Nanobodies to study G protein-coupled receptor structure and function.Annu. Rev. Pharmacol. Toxicol. 2017; 57 (27959623): 19-3710.1146/annurev-pharmtox-010716-104710Crossref PubMed Scopus (147) Google Scholar) that are considered recalcitrant targets could be successfully crystallized and their structures determined with the use of VHHs. Isolation of VHH domains against a target antigen was primarily done through antigen immunization of llamas or camels followed by construction of phage or yeast display libraries for screening of high-affinity binders against the target antigen (21Rasmussen S.G. Choi H.J. Fung J.J. Pardon E. Casarosa P. Chae P.S. Devree B.T. Rosenbaum D.M. Thian F.S. Kobilka T.S. Schnapp A. Konetzki I. Sunahara R.K. Gellman S.H. Pautsch A. Steyaert J. Weis W.I. Kobilka B.K. Structure of a nanobody-stabilized active state of the β(2) adrenoceptor.Nature. 2011; 469 (21228869): 175-18010.1038/nature09648Crossref PubMed Scopus (1314) Google Scholar, 34Uchański T. Zögg T. Yin J. Yuan D. Wohlkönig A. Fischer B. Rosenbaum D.M. Kobilka B.K. Pardon E. Steyaert J. An improved yeast surface display platform for the screening of nanobody immune libraries.Sci. Rep. 2019; 9 (30674983): 38210.1038/s41598-018-37212-3Crossref PubMed Scopus (41) Google Scholar, 35Pardon E. Laeremans T. Triest S. Rasmussen S.G. Wohlkönig A. Ruf A. Muyldermans S. Hol W.G. Kobilka B.K. Steyaert J. A general protocol for the generation of Nanobodies for structural biology.Nat. Protoc. 2014; 9 (24577359): 674-69310.1038/nprot.2014.039Crossref PubMed Scopus (404) Google Scholar). More recently, naive synthetic nanobody yeast libraries have been constructed and used to isolate high-affinity binders against antigens (36McMahon C. Baier A.S. Pascolutti R. Wegrecki M. Zheng S. Ong J.X. Erlandson S.C. Hilger D. Rasmussen S.G.F. Ring A.M. Manglik A. Kruse A.C. Yeast surface display platform for rapid discovery of conformationally selective nanobodies.Nat. Struct. Mol. Biol. 2018; 25 (29434346): 289-29610.1038/s41594-018-0028-6Crossref PubMed Scopus (211) Google Scholar). In this study, we followed the conventional route of immunizing an Indian camel against a putative multidrug efflux transporter, NorC, and built a yeast display library using Saccharomyces cerevisiae to isolate single-domain VHH antibodies that we refer to as ICabs (Indian camelid antibodies). We isolated a high-affinity ICab against NorC that displays a rather interesting Zn2+-binding site in the CDR3 region to compensate for the absence of the disulfide. We observe that this Zn2+-binding site plays a vital role in determining the stability of the antibody and its affinity to NorC. All atom simulations clearly reveal enhanced flexibility in the CDR3 region upon removal of the Zn2+ ion. The ICab also demonstrates an ability to bind NorC at the extracellular surface that could serve to develop efflux pump detection assays in pathogenic organisms. A 4-year-old male Indian Camel (C. dromedarius) was immunized with purified WT NorC isolated through heterologous expression in Escherichia coli membranes. The transporter was extracted from crude membranes using dodecyl-β-d-maltopyranoside (DDM) followed by exchange into lauryl maltose neopentlyglycol during size-exclusion chromatography (Fig. S1). Purified NorC was reconstituted into proteoliposomes prior to immunization using GERBU-FAMA as adjuvant. Sera from the immunized animal were used to check for peak shifts of NorC-CGFP in fluorescence-detection size-exclusion chromatography (FSEC) (Fig. S2B). Repeated immunization was carried out to increase the titer against NorC (Fig. S2A). The pre-immune serum displayed weak binding against NorC-CGFP, evident in the shift of the NorC-GFP peak to a higher hydrodynamic size as compared to the control. However, the binding was enhanced by the sixth week of immunization, with the serum having an enriched antibody titer compared with the pre-immune serum (Fig. S2, C and D). After six rounds of immunization with the antigen, total mRNA from peripheral blood mononuclear cells (PBMCs) were isolated, and cDNA was prepared using RT-PCR. The VHH regions were amplified from the cDNA using standard methods (35Pardon E. Laeremans T. Triest S. Rasmussen S.G. Wohlkönig A. Ruf A. Muyldermans S. Hol W.G. Kobilka B.K. Steyaert J. A general protocol for the generation of Nanobodies for structural biology.Nat. Protoc. 2014; 9 (24577359): 674-69310.1038/nprot.2014.039Crossref PubMed Scopus (404) Google Scholar). The amplified VHH sequences were cloned into pPNL6 vector (a kind gift from Prof. Rajan Dighe, IISc) followed by electroporation into S. cerevisiae (EBY100) cells to build the yeast display library (Fig. 1A). The VHH fragment was cloned 3′ to the aga2 gene in between hemagglutinin and c-Myc tags. Expression of VHH on yeast surface was followed using c-Myc tag at the C terminus. The yeast display library was constructed to screen binders against NorC using fluorescently labeled protein that was cross-linked with fluorescein maleimide after creating a substitution (cysteine) at the N terminus (T4C). FACS was performed to screen and isolate yeast cells displaying binders against NorC with high library expression and showing upward shifts in the mean fluorescence intensity when incubated with the labeled antigen (Fig. S3, A–D). About 28 of the 60 clones used for screening displayed promising FACS profiles. Sanger sequencing yielded four unique clones (ICab1 to -4) (Fig. S3). We could overexpress three of the four ICabs in E. coli Rosetta cells that yielded protein in inclusion bodies. We were successful at refolding one of the three proteins, ICab3 (Fig. 1B), using urea denaturation and refolding by gradually reducing urea concentration in a stepwise dialysis at 4 °C. Interestingly, ICab3 lacks a Cys residue in the CDR3 region, unlike the remaining ICabs identified in this study (Fig. 2F and Fig. S3E). The refolded protein was homogeneous (Fig. S6A) and pure (Fig. S6B) and gave a typical β-sheet secondary structure profile in CD suggesting proper folding (Fig. S6C). The leader sequence cleaved post-expression as the estimated mass of ICab3 correlated to protein without the pelB signal sequence in MALDI-TOF. The pure protein behaved as a monomer in solution when analyzed by FSEC and displayed shifts in the FSEC when incubated with NorC-CGFP, suggesting formation of a stable NorC ICab3 complex (Fig. S6D). ICab3 was crystallized by hanging-drop vapor diffusion method at 4 °C, and the structure was determined through molecular replacement using a related VHH structure (PDB code 3JBE) as a template to a resolution of 2.15 Å (Table 1).Table 1Crystallographic data and refinement statisticsNativeZn2+ anomalousData collection statistics X-ray sourceCuKαESRF ID29 Temperature100 K100 K Space groupP32 2 1P32 2 1 Cell dimensionsa, b, c (Å)106.9, 106.9, 70.6106.7, 106.7, 71.4α, β, γ (degrees)90, 90, 12090, 90, 120 Wavelength (Å)1.54171.2814 Resolution (Å)53.44–2.15 (2.22–2.15)71.40–2.10 (2.16–2.10) Total number of observations225,745 (17,407)231,995 (15,128) Unique reflections25,502 (2171)27,730 (2218) Multiplicity8.9 (8.0)8.4 (6.8) Data completeness99.6 (98.7)99.9 (99.1) I/σI11.1 (1.9)17.0 (3.0) Rpim (%)4.5 (42.2)2.8 (21.7) CC½0.997 (0.624)0.998 (0.862) Anomalous completeness (%)99.7 (98.2) Anomalous multiplicity4.1 (3.4) DelAnom CC½0.625 (0.044)Refinement statistics Resolution (Å)46.28–2.15 Unique reflections25,479 Rwork (%)/Rfree (%)19.32/23.24 Non-hydrogen atoms/moleculesProtein2008Ethylene glycol18Water121Sodium1Zinc2 Average B-factor (Å2)Protein59.4Ethylene glycol67.4Zinc39.2Sodium49.12Water60.9 RMSD, bond lengths (Å)0.006 RMSD, bond angles (°)0.914 Ramachandran plotFavored (%)98.47Allowed (%)1.53Outliers (%)0.00Values in parentheses indicate the highest-resolution shell. Open table in a new tab Values in parentheses indicate the highest-resolution shell. The refined X-ray structure of ICab3 displays a canonical immunoglobulin fold of VHH with nine β-strands organized into two β-sheets (Fig. 2A). The β-sandwich comprises five and four β-strands in each of the two β-sheets. The conserved disulfide bond, a defining feature of immunoglobulin structure, is present between Cys26 and Cys103 connecting the two β-sheets. The uniqueness of any VHH is determined by its hypervariable loops or CDRs because they constitute the structural determinants forming the paratope that defines the unique binding propensities with the cognate epitope. The length and sequence variability of CDR loops determine the binding affinity and specificity. The regions between the CDRs, referred to as “framework regions” (FRs), are made up of highly conserved sequences. A unique feature of VHHs that differentiates them from conventional antibodies is the presence of long CDR loops 1 and 3. Interestingly, in the ICab3 structure, CDR1 is unusually long compared with most of the VHH structures available in the Protein Data Bank. CDR1 to -3 comprise 13 residues (Gly30–Gly42), 6 residues (Lys59–Ser64), and 19 residues (Asp106–Tyr124), respectively. A close inspection of the loop sequence reveals that the additional length of CDR1 loop is due to the repetition of the motif STY (Fig. 2F), making the loop polar and rich in aromatic residues. Tyrosines were found to have a higher representation in the CDRs of many VHHs and are known to mediate strong interactions with the antigen in many of the known VHH-antigen complexes (37Zavrtanik U. Lukan J. Loris R. Lah J. Hadži S. Structural basis of epitope recognition by heavy-chain camelid antibodies.J. Mol. Biol. 2018; 430 (30205092): 4369-438610.1016/j.jmb.2018.09.002Crossref PubMed Scopus (63) Google Scholar). Despite having a longer length, the B-factors of atoms in the additional residues in CDR1 are comparable with the rest of the framework regions, likely due to their close interactions with symmetry-related partners. As mentioned earlier, VHHs bind to their antigen with high shape surface complementarity, and the long protruding CDR1 suggests the likelihood that it could closely interact with crevices or the vestibular region in NorC. Interestingly, CDR1 and -2 of ICab3 are not only enriched in Tyr but also have several Ser and His residues. Thus, the paratope of ICab3 has a more hydrophilic character in contrast to the paratope of other VHHs characterized so far, which have a more hydrophobic surface. The long CDR3 loop is a hallmark feature of camel VHHs and is usually shorter in the case of llama VHHs. The long loop folds back over the FR2 region, assuming a typical stretched-twist conformation (38Sircar A. Sanni K.A. Shi J. Gray J.J. Analysis and modeling of the variable region of camelid single-domain antibodies.J. Immunol. 2011; 186 (21525384): 6357-636710.4049/jimmunol.1100116Crossref PubMed Scopus (61) Google Scholar) covering a part of the FR2 region. In conventional antibody, FR2 of the VH domain has residues Val42, Gly49, Leu50, and Trp52 that make the surface hydrophobic, thus readily allowing VL and VH domains to dimerize and exist as a stable dimer in solution (39Chothia C. Novotný J. Bruccoleri R. Karplus M. Domain association in immunoglobulin molecules. The packing of variable domains.J. Mol. Biol. 1985; 186 (4093982): 651-66310.1016/0022-2836(85)90137-8Crossref PubMed Scopus (332) Google Scholar). The corresponding residues of FR2 of ICab3 are Phe44, Glu51, Arg52, and Gly54, allowing it to exist as a stable monomer. These amino acid substitutions are a characteristic feature of VHHs and are highly conserved (40Muyldermans S. Atarhouch T. Saldanha J. Barbosa J.A. Hamers R. Sequence and structure of VH domain from naturally occurring camel heavy chain immunoglobulins lacking light chains.Protein Eng. 1994; 7 (7831284): 1129-113510.1093/protein/7.9.1129Crossref PubMed Scopus (394) Google Scholar, 41Vu K.B. Ghahroudi M.A. Wyns L. Muyldermans S. Comparison of llama VH sequences from conventional and heavy chain antibod" @default.
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• W2989454912 title "Isolation and structural characterization of a Zn2+-bound single-domain antibody against NorC, a putative multidrug efflux transporter in bacteria" @default.
• W2989454912 cites W1538833285 @default.
• W2989454912 cites W1598136901 @default.
• W2989454912 cites W1608855442 @default.
• W2989454912 cites W1667902581 @default.
• W2989454912 cites W1878404532 @default.
• W2989454912 cites W1927502485 @default.
• W2989454912 cites W1964538987 @default.
• W2989454912 cites W1988729093 @default.
• W2989454912 cites W1992032455 @default.
• W2989454912 cites W1998292458 @default.
• W2989454912 cites W2000106654 @default.
• W2989454912 cites W2011075430 @default.
• W2989454912 cites W2014327275 @default.
• W2989454912 cites W2016123211 @default.
• W2989454912 cites W2019486739 @default.
• W2989454912 cites W2020601776 @default.
• W2989454912 cites W2027285268 @default.
• W2989454912 cites W2032054300 @default.
• W2989454912 cites W2033079214 @default.
• W2989454912 cites W2035266068 @default.
• W2989454912 cites W2037042031 @default.
• W2989454912 cites W2037302035 @default.
• W2989454912 cites W2038652838 @default.
• W2989454912 cites W2040751953 @default.
• W2989454912 cites W2054597378 @default.
• W2989454912 cites W2067174909 @default.
• W2989454912 cites W2071251528 @default.
• W2989454912 cites W2081693079 @default.
• W2989454912 cites W2095553811 @default.
• W2989454912 cites W2100355920 @default.
• W2989454912 cites W2101272434 @default.
• W2989454912 cites W2106231619 @default.
• W2989454912 cites W2108921801 @default.
• W2989454912 cites W2120929471 @default.
• W2989454912 cites W2124759299 @default.
• W2989454912 cites W2128572087 @default.
• W2989454912 cites W2129026291 @default.
• W2989454912 cites W2129906420 @default.
• W2989454912 cites W2134668367 @default.
• W2989454912 cites W2134836136 @default.
• W2989454912 cites W2144081223 @default.
• W2989454912 cites W2147451568 @default.
• W2989454912 cites W2160544821 @default.
• W2989454912 cites W2163341755 @default.
• W2989454912 cites W2169610187 @default.
• W2989454912 cites W2180229411 @default.
• W2989454912 cites W2403564902 @default.
• W2989454912 cites W2477491193 @default.
• W2989454912 cites W2523100511 @default.
• W2989454912 cites W2532595958 @default.
• W2989454912 cites W2555870966 @default.
• W2989454912 cites W2621074137 @default.
• W2989454912 cites W2647441530 @default.
• W2989454912 cites W2724786424 @default.
• W2989454912 cites W2765322245 @default.
• W2989454912 cites W2786832567 @default.
• W2989454912 cites W2793899290 @default.
• W2989454912 cites W2887979370 @default.
• W2989454912 cites W2891129333 @default.
• W2989454912 cites W2913432304 @default.
• W2989454912 cites W2943885243 @default.
• W2989454912 cites W2950797462 @default.
• W2989454912 cites W4210675630 @default.
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