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Antigen Specificity and High Affinity Binding Provided by One Single Loop of a Camel Single-domain Antibody

2001; Elsevier BV; Volume: 276; Issue: 28 Linguagem: Inglês

10.1074/jbc.m102107200

ISSN

1083-351X

Autores

Aline Desmyter, Klaas Decanniere, Serge Muyldermans, Lode Wyns,

Tópico(s)

Protein purification and stability

Resumo

Detailed knowledge on antibody-antigen recognition is scarce given the unlimited antibody specificities of which only few have been investigated at an atomic level. We report the crystal structures of an antibody fragment derived from a camel heavy chain antibody against carbonic anhydrase, free and in complex with antigen. Surprisingly, this single-domain antibody interacts with nanomolar affinity with the antigen through its third hypervariable loop (19 amino acids long), providing a flat interacting surface of 620 Å2. For the first time, a single-domain antibody is observed with its first hypervariable loop adopting a type-1 canonical structure. The second hypervariable loop, of unique size due to a somatic mutation, reveals a regular β-turn. The third hypervariable loop covers the remaining hypervariable loops and the side of the domain that normally interacts with the variable domain of the light chain. Specific amino acid substitutions and reoriented side chains reshape this side of the domain and increase its hydrophilicity. Of interest is the substitution of the conserved Trp-103 by Arg because it opens new perspectives to 'humanize' a camel variable domain of heavy chain of heavy chain antibody (VHH) or to 'camelize' a human or a mouse variable domain of heavy chain of conventional antibody (VH). Detailed knowledge on antibody-antigen recognition is scarce given the unlimited antibody specificities of which only few have been investigated at an atomic level. We report the crystal structures of an antibody fragment derived from a camel heavy chain antibody against carbonic anhydrase, free and in complex with antigen. Surprisingly, this single-domain antibody interacts with nanomolar affinity with the antigen through its third hypervariable loop (19 amino acids long), providing a flat interacting surface of 620 Å2. For the first time, a single-domain antibody is observed with its first hypervariable loop adopting a type-1 canonical structure. The second hypervariable loop, of unique size due to a somatic mutation, reveals a regular β-turn. The third hypervariable loop covers the remaining hypervariable loops and the side of the domain that normally interacts with the variable domain of the light chain. Specific amino acid substitutions and reoriented side chains reshape this side of the domain and increase its hydrophilicity. Of interest is the substitution of the conserved Trp-103 by Arg because it opens new perspectives to 'humanize' a camel variable domain of heavy chain of heavy chain antibody (VHH) or to 'camelize' a human or a mouse variable domain of heavy chain of conventional antibody (VH). variable domain of heavy chain of heavy chain antibody variable domain of heavy chain of conventional antibody variable domain of light chain complementarity determining region camel single-domain antibody fragment root-mean-square deviations human chorionic gonadotropin structural loop around, respectively, the first and second antigen-binding region of a VH Conventional antibody IgG molecules consist of two light chains folded in two domains and two heavy chains folded in four domains (1Padlan E.A. Mol. Immunol. 1994; 31: 169-217Crossref PubMed Scopus (790) Google Scholar). Surprisingly, the serum of Camelidae contains in addition a large proportion (∼50%) of functional antibodies devoid of light chains and heavy chains possessing only three domains since the equivalent of the first constant domain is missing (2Hamers-Casterman C. Atarhouch T. Muyldermans S. Robinson G. Hamers C. Bajyana Songa E. Bendahman N. Hamers R. Nature. 1993; 363: 446-448Crossref PubMed Scopus (2177) Google Scholar). The two C-terminal domains of the heavy chain homodimers within camelids and conventional IgG molecules share large sequence identities and are responsible for the effector functions. Also, the N-terminal variable domain of the heavy chain antibodies (referred to as VHH)1 (3Muyldermans S. Lauwereys M. J. Mol. Recognit. 1999; 12: 1-10Crossref PubMed Scopus (116) Google Scholar) has an overall sequence and structure that is homologous to the variable domain (VH) of the heavy chain of a classical human antibody (4Muyldermans S. Atarhouch T. Saldanha J. Barbosa J.A.R.G. Hamers R. Protein Eng. 1994; 7: 1129-1135Crossref PubMed Scopus (394) Google Scholar, 5Decanniere K. Desmyter A. Lauwereys M. Ghahroudi M.A. Muyldermans S. Wyns L. Structure. 1999; 7: 361-370Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 6Desmyter A. Transue T.R. Arbabi Ghahroudi M. Dao-Thi M.-H. Poortmans F. Hamers R. Muyldermans S. Wyns L. Nat. Struct. Biol. 1996; 3: 803-811Crossref PubMed Scopus (415) Google Scholar, 7Spinelli S. Frenken L. Bourgeois D. de Ron L. Bos W. Verrips T. Anguille C. Cambillau C. Tegoni M. Nature Struc. Biol. 1996; 3: 752-757Crossref PubMed Scopus (130) Google Scholar, 8Spinelli S. Frenken L. Hermans P.W.J.J. Verrips T. Brown K. Tegoni M. Cambillau C. Biochem. 2000; 39: 1217-1222Crossref PubMed Scopus (141) Google Scholar). Important amino acid differences occur between the VH and VHH in their framework 2 region. This region is hydrophilic in VHHs rendering the domain soluble in aqueous solution, whereas the region is hydrophobic in the VH, and its amino acids associate with the VL. The VHH domain represents the smallest naturally occurring, intact antigen-binding site (9Sheriff S. Constantine K.L. Nature Struct. Biol. 1996; 3: 733-736Crossref PubMed Scopus (59) Google Scholar), comprising only one single immunoglobulin domain with three antigen-binding loops (or complementarity determining regions, CDRs). Heavy chain antibodies with high specificity and affinity can be generated against a wide variety of antigens (10van der Linden R. de Geus B. Stok W. Bos W. van Wassenaar D. Verrips T. Frenken L. J. Immunol. Methods. 2000; 240: 185-195Crossref PubMed Scopus (121) Google Scholar). Their VHHs are readily cloned (11Ghahroudi M.A. Desmyter A. Wyns L. Hamers R. Muyldermans S. FEBS Lett. 1997; 414: 521-526Crossref PubMed Scopus (583) Google Scholar, 12Lauwereys M. Ghahroudi M.A. Desmyter A. Kinne J. Hölzer W. De Genst E. Wyns L. Muyldermans S. EMBO J. 1998; 17: 3512-3520Crossref PubMed Scopus (397) Google Scholar) and expressed in bacteria and yeast (13Frenken L. van der Linden R.H.J. Hermans P.W.J.J. Bos W. Ruuls R.C. de Geus B. Verrips T. J. Biotechnol. 2000; 78: 11-21Crossref PubMed Scopus (227) Google Scholar) and are extremely stable (14van der Linden R.H.J. Frenken L. de Geus B. Harmsen M.M. Ruuls R.C. Stok W. de Ron L. Wilson S. Davis P. Verrips T. Biochim. Biophys. Acta. 1999; 1431: 37-46Crossref PubMed Scopus (353) Google Scholar). In human and mouse, the first two antigen-binding loops of a VH domain, CDR1 and CDR2, can be assigned to a limited number of possible conformations referred to as canonical structures (15Al-Lazikani B. Lesk A.M. Chothia C. J. Mol. Biol. 1997; 273: 927-948Crossref PubMed Scopus (587) Google Scholar, 16Chothia C. Lesk A.M. Gherardi E. Tomlinson I.M. Walter G. Marks J.D. Llewelyn M.B. Winter G. J. Mol. Biol. 1992; 227: 799-817Crossref PubMed Scopus (365) Google Scholar, 17Chothia C. Lesk A.M. J. Mol. Biol. 1987; 196: 901-917Crossref PubMed Scopus (1184) Google Scholar, 18Chothia C. Lesk A.M. Tramontano A. Levitt M. Smith-Gill S.J. Air G. Sheriff S. Padlan E.A. Davies A.H. Tulip W. Colman P.M. Spinelli S. Alzari P.M. Poljak R. Nature. 1989; 342: 877-883Crossref PubMed Scopus (1083) Google Scholar). The conformation of these loops depends both on their length and on the presence of specific residues at key positions. In contrast, the x-ray structure analysis of four VHH domains showed that their CDR1 and CDR2 deviate significantly from the canonical loop structures observed in human or mouse VHs (19Decanniere K. Muyldermans S. Wyns L. J. Mol. Biol. 2000; 300: 83-91Crossref PubMed Scopus (81) Google Scholar). The third antigen-binding loop (CDR3) of the VHH fragments is often constrained by an interloop disulfide bond and is, on average, longer than a human or mouse VH-CDR3 loop (4Muyldermans S. Atarhouch T. Saldanha J. Barbosa J.A.R.G. Hamers R. Protein Eng. 1994; 7: 1129-1135Crossref PubMed Scopus (394) Google Scholar). This allows for a potentially larger antigen-binding surface (20Vu K.B. Ghahroudi M.A. Wyns L. Muyldermans S. Mol. Immunol. 1997; 34: 1121-1131Crossref PubMed Scopus (248) Google Scholar). About half of the dromedary single-domain binders to enzymes are potent inhibitors (12Lauwereys M. Ghahroudi M.A. Desmyter A. Kinne J. Hölzer W. De Genst E. Wyns L. Muyldermans S. EMBO J. 1998; 17: 3512-3520Crossref PubMed Scopus (397) Google Scholar). This can be explained by their long CDR3 loop inserting into the active site cleft on the enzyme surface, as illustrated by the lysozyme binder cAb-Lys3. In this case, the N-terminal part of the 24-amino acid-long CDR3 loop protrudes from the remaining antigen-binding surface, penetrates deeply into the active site of the enzyme (6Desmyter A. Transue T.R. Arbabi Ghahroudi M. Dao-Thi M.-H. Poortmans F. Hamers R. Muyldermans S. Wyns L. Nat. Struct. Biol. 1996; 3: 803-811Crossref PubMed Scopus (415) Google Scholar), and mimics the lysozyme natural substrate (21Transue T.R. De Genst E. Ghahroudi M.A. Wyns L. Muyldermans S. Proteins: Structure, Function, and Genetics. 1998; 32: 515-522Crossref PubMed Scopus (95) Google Scholar). However, several non-inhibiting antibody fragments with a long CDR3 loop were also isolated (12Lauwereys M. Ghahroudi M.A. Desmyter A. Kinne J. Hölzer W. De Genst E. Wyns L. Muyldermans S. EMBO J. 1998; 17: 3512-3520Crossref PubMed Scopus (397) Google Scholar), and these fragments are not expected to interact with the active site of their enzymes. Therefore, it was hypothesized that these non-inhibiting VHH molecules would interact with other clefts present on the protein surface. Here, we present the crystal structures of a camel VHH fragment, cAb-CA05, both as free antibody and in complex with its antigen. This specific non-inhibiting enzyme binder recognizes the bovine erythrocyte carbonic anhydrase with an affinity of 72 nm(K d), which is in the same range as other VHHs or single chain variable fragment (11Ghahroudi M.A. Desmyter A. Wyns L. Hamers R. Muyldermans S. FEBS Lett. 1997; 414: 521-526Crossref PubMed Scopus (583) Google Scholar, 12Lauwereys M. Ghahroudi M.A. Desmyter A. Kinne J. Hölzer W. De Genst E. Wyns L. Muyldermans S. EMBO J. 1998; 17: 3512-3520Crossref PubMed Scopus (397) Google Scholar). Surprisingly, the structural data reveal for the first time an antibody using only one single loop, its CDR3, to interact directly with the antigen. The cAb-CA05 was extracted from Escherichia coli periplasm and purified by chromatography on Ni-NTA (Qiagen) and Superdex 75 (Amersham Pharmacia Biotech) and gel filtration (12Lauwereys M. Ghahroudi M.A. Desmyter A. Kinne J. Hölzer W. De Genst E. Wyns L. Muyldermans S. EMBO J. 1998; 17: 3512-3520Crossref PubMed Scopus (397) Google Scholar). The cAb-CA05-carbonic anhydrase complex was prepared by mixing cAb-CA05 in phosphate buffered saline with bovine erythrocyte carbonic anhydrase (Sigma) in a molar ratio of 1.2: 1 and applied on Superdex 75 (Amersham Pharmacia Biotech). Crystals from the antigen-free cAb-CA05 (1.7 mg/ml) and from the complex cAb-CA05·carbonic anhydrase (3.5 mg/ml) were grown in 25% (w/v) PEG8000 (Hampton), 0.1 m sodium citrate, pH 5.6, using the hanging drop vapor diffusion method. A data set to 2.1 Å for cAb-CA05 was collected using a Rigaku RU-H2R rotating anode generator (Kobe, Japan) and a MarResearch image plate (MarResearch, Hamburg, Germany). Data for the antigen-antibody crystal were collected on beam line BW7A at EMBL-Hamburg using a MarResearch image plate. Primary data processing was done with DENZO (22Otwinowski Z. Swayer L. Isaacs N. Bailey S. Proceedings of the CCP4 Study Weekend. CLRC Daresbury Laboratory, Warrington, England1993: 107-113Google Scholar), scaling was done with SCALA, and further processing was done with the CCP4 program suite (23Collaborative Computational Project, Number 4 Acta Crystallogr. D. 1994; 50: 760-763Crossref PubMed Scopus (19769) Google Scholar). The structure of the free antigen was solved by molecular replacement as implemented in AMORE (24Navaza J. Acta Crystallogr. A. 1994; 50: 157-163Crossref Scopus (5029) Google Scholar), using the VHH cAb-RN05 (Protein Data Bank entry code 1BZQ) as a search model. The structure was refined with X-PLOR (25Brünger A.T. A system for X-ray Crystallography and NMR, X-PLOR Version 3.1. Yale University Press, New Haven, CT1992Google Scholar) and REFMAC (26Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. D. 1997; 53: 240-255Crossref PubMed Scopus (13870) Google Scholar). The CDR loops were deleted from the search model and rebuilt from scratch. Possible water positions were identified with ARP (27Lamzin V.S. Wilson K.S. Acta Crystallogr. D. 1993; 49: 129-147Crossref PubMed Google Scholar) and checked manually. Model and structure factors are deposited in the Protein Data Bank, entry 1F2X. The antigen-antibody complex structure was solved by molecular replacement with the antigen-free cAb-CA05 structure and human carbonic anhydrase structure (1CA2) as search models. For refinement, we used the CNS program (28Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.-S. Kuszewskie J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren L. Acta Crystallogr. D. 1998; 54: 905-921Crossref PubMed Scopus (16967) Google Scholar), and included a simulated-annealing step to reduce possible model bias. As the high resolution limit of the data set is 3.5 Å, a grouped B factor refinement scheme was used (only one B factor for main chain atoms and one B factor for side chain atoms/residue). Model and structure factors are accessible through the Protein Data Bank, entry 1G6V. Superposition of structures or structure fragments and calculation of rmsd were done with LSQKAB (23Collaborative Computational Project, Number 4 Acta Crystallogr. D. 1994; 50: 760-763Crossref PubMed Scopus (19769) Google Scholar). Inter-residue and inter-atom distances were calculated with CONTACT (23Collaborative Computational Project, Number 4 Acta Crystallogr. D. 1994; 50: 760-763Crossref PubMed Scopus (19769) Google Scholar), and accessible surface areas were calculated with NACCESS (29Hubbard S.J. Thornton J.M. NACCESS Computer Program, Version 2.1.1. Dept. of Biochemistry and Molecular Biology, University College London, UK1993Google Scholar). Figures were produced with MOLSCRIPT 2.1 (30Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar) and rendered with RASTER3D (31Merrit E.A. Murphy M.E. Acta Crystallogr. D. 1994; 50: 869-873Crossref PubMed Scopus (2857) Google Scholar). cAb-CA05 is a VHH antibody fragment of 135 residues (M r 15,000) that binds specifically to bovine erythrocyte carbonic anhydrase (K d = 72 nm) but does not inhibit the enzymatic activity of the antigen. It was selected by panning from a VHH library of an immunized dromedary (12Lauwereys M. Ghahroudi M.A. Desmyter A. Kinne J. Hölzer W. De Genst E. Wyns L. Muyldermans S. EMBO J. 1998; 17: 3512-3520Crossref PubMed Scopus (397) Google Scholar). The cAb-CA05 shares a high sequence identity with other VHHs or human VHs of family III (Fig.1). Nevertheless, the VHH characteristic amino acids in framework 2 (Phe-37, Glu-44, Arg-45, and Gly-47) (4Muyldermans S. Atarhouch T. Saldanha J. Barbosa J.A.R.G. Hamers R. Protein Eng. 1994; 7: 1129-1135Crossref PubMed Scopus (394) Google Scholar, 20Vu K.B. Ghahroudi M.A. Wyns L. Muyldermans S. Mol. Immunol. 1997; 34: 1121-1131Crossref PubMed Scopus (248) Google Scholar) are all present (Kabat amino acid numbering (32Kabat E. Wu T.T. Perry H.M. Gottesman K.S. Foeller C. Sequence of Proteins of Immunological Interest, Publication 91-3242. U.S. Public Health Services, National Institutes of Health, Bethesda, MD1991Google Scholar)). In addition, residue Trp-103, constitutively conserved in all VHs and interacting with the VL, is substituted by Arg in cAb-CA05. Like most other VHHs from camelids, the cAb-CA05 contains a long CDR3 (19 amino acids) with a cysteine at position 100c expected to form a disulfide bond with Cys-33 located in the CDR1. The CDR2 of cAb-CA05 is unusually short as it contains 15 amino acids instead of a standard length of 16, 17, or 19 amino acids (32Kabat E. Wu T.T. Perry H.M. Gottesman K.S. Foeller C. Sequence of Proteins of Immunological Interest, Publication 91-3242. U.S. Public Health Services, National Institutes of Health, Bethesda, MD1991Google Scholar). The cAb-CA05 was crystallized both as free antibody and in complex with its antigen. Antigen-free cAb-CA05 crystallizes in space group P 21 with cell dimensions of a = 29.98 Å,b = 43.86 Å, c = 87.95 Å, β = 93.23 ° and two VHH molecules in the asymmetric unit. The antigen-antibody complexes crystallize in space group P 41212 with cell dimensions of a= 83.86 Å and c = 224.05 Å and one antigen-antibody complex in the asymmetric unit. The structure of the antigen-free cAb-CA05 was refined to 2.1 Å resolution (Table I). It adopts the standard fold of an immunoglobulin variable domain with nine conserved anti-parallel β-strands (Fig. 2) and three hypervariable regions clustering at one end of the domain (1Padlan E.A. Mol. Immunol. 1994; 31: 169-217Crossref PubMed Scopus (790) Google Scholar, 33Chothia C. Gelfand I.M. Kister A.E. J. Mol. Biol. 1998; 278: 457-479Crossref PubMed Scopus (157) Google Scholar,34Chothia C. Boswell D.R. Lesk A.M. EMBO J. 1988; 7: 3745-3755Crossref PubMed Scopus (631) Google Scholar). The Cys-22 and Cys-92 are oxidized into an intradomain disulfide bond, conserved in all immunoglobulin domains. Its general structure superimposes very well with a human VH reference structure (1igm) and with all available VHH structures of camel (1mel, 1bzq) and llama (1hcv, 1qdo). Root-mean-square deviations for the main chain atoms of the framework residues (residues 2–24, 32–52, 55–72, 77–92, and 103–112) ranged between 0.58 and 0.88 Å.Table ICrystallographic data and refinement statistics for antigen-free and antigen-complexed cAb-CA05Data statistics (highest shell)Antigen-freeAntigen-complexedResolution limits (Å)31.0–2.126.5–3.5(2.21–2.1)(3.69–3.5)R factor (%)10.3 (22.7)15.9 (34.5)Completeness99.8 (99.8)99 (99)I/ς4.9 (2.7)4.4 (2.1)Multiplicity5.8 (5.3)5.6 (5.8)Refinement statistics# of reflections1278610533# of reflections free666502R (%)19.021.0Rfree (%)27.027.6GeometryRmsd bond lengths (Å)0.009 (refmac)0.008 (cns)Rmsd bond angles (°)0.027 (refmac)1.360 (cns)Rmsd planarity (Å)0.026/ESU1-aESU, estimated standard uncertainty. Rfree (Å)0.23/ESU ML1-bESU ML, estimated standard uncertainty maximum likelihood. residual (Å)0.15/1-a ESU, estimated standard uncertainty.1-b ESU ML, estimated standard uncertainty maximum likelihood. Open table in a new tab Structural adaptations, however, are expected to occur in the side of the domain that corresponds to the VL-interacting side of a VH domain. This area is hydrophobic in all VHs by the presence of Val-37, Leu-45, Trp-47, and Trp-103 side chains, conserved in sequence and structural position (Fig. 3 A) (35Chothia C. Novotny J. Bruccoleri R. Karplus M. J. Mol. Biol. 1985; 186: 651-663Crossref PubMed Scopus (332) Google Scholar). The L45R and W47G substitutions and the Trp-103 rotated over its Cβ-Cγ bond in cAb-Lys3 (Fig. 3 C) make this VHH region more hydrophilic. The V37F mutation fills a hydrophobic pocket created by the side chains of the Trp-103, Tyr-91, and the CDR3, where the conserved Tyr (three amino acids upstream of Trp-103) plays a central role (6Desmyter A. Transue T.R. Arbabi Ghahroudi M. Dao-Thi M.-H. Poortmans F. Hamers R. Muyldermans S. Wyns L. Nat. Struct. Biol. 1996; 3: 803-811Crossref PubMed Scopus (415) Google Scholar). The W103R substitution found here in cAb-CA05 renders this 'former VL side' of the VHH even more hydrophilic. It also allows a shift of the Phe-37 side chain toward the Tyr-91 and Arg-103 (Fig.3 D). As a result, the backbone of the long CDR3 approaches the former VL-side even more closely (Fig.4 D). All these modifications occur in the absence of distortions of the framework structure. In contrast, the partial camelization of a human VH in this area by L45R and W47I substitutions makes the isolated domain more soluble but induces backbone deformations at positions 37–38 and 45–47 (36Riechmann L. J. Mol. Biol. 1996; 259: 957-969Crossref PubMed Scopus (60) Google Scholar). In addition, the side chain of Trp-103 takes a completely new position (Fig. 3 B).Figure 4Structure of the antigen-binding loops in cAb-CA05. A, stereo presentation of superposition of the H1 loop of the human Pot VH (1igm) adopting a type-1 canonical structure (brown) and the cAb-CA05 (blue) including the structure-determining side chains forming a hydrophobic core. B, the peptide backbone of the H2 loop of cAb-CA05 and C, of a canonical structure type-1 human VH (7fab) shown in ball-and-stick representation. Thedashed lines indicate the interloop hydrogen bonds.D, stereo picture of the superposition of the H1 and H3 loops of cAb-Lys3 (1mel) (brown) and cAb-CA05 (blue and red, respectively). The interloop disulfide bond is shown as well as the conserved disulfide bond between the scaffold Cys-22 and Cys-92. The Kabat numbering of a few amino acids is given for reference.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The conformation of the H1 loop (residues 26–32, the solvent-exposed loop around the CDR1) of cAb-CA05 fits with the canonical structure type-1, a conformation observed in all VH structures containing a 7-amino acid H1 loop (15Al-Lazikani B. Lesk A.M. Chothia C. J. Mol. Biol. 1997; 273: 927-948Crossref PubMed Scopus (587) Google Scholar, 16Chothia C. Lesk A.M. Gherardi E. Tomlinson I.M. Walter G. Marks J.D. Llewelyn M.B. Winter G. J. Mol. Biol. 1992; 227: 799-817Crossref PubMed Scopus (365) Google Scholar). This canonical loop structure is shaped by a sharp turn at Gly-26, clustering of the hydrophobic side chains of Ala-24, Phe-27, Phe-29, and Met-34 (Fig. 4 A), and the hydrophobic part of the Arg-94 side chain (Cδ-Cε of Lys-94 in Pot VH) (16Chothia C. Lesk A.M. Gherardi E. Tomlinson I.M. Walter G. Marks J.D. Llewelyn M.B. Winter G. J. Mol. Biol. 1992; 227: 799-817Crossref PubMed Scopus (365) Google Scholar). The sequence composition of the cAb-CA05 H1 loop harbors the key elements for a type-1 structure except for the R94G and conservative F27Y and F29V substitutions (Fig. 1). These substitutions lead to a slightly different organization of the side chains forming the hydrophobic core of the loop but do not influence the main chain conformation of the loop (Fig. 4 A). In contrast, all previously solved camel or llama VHH structures had their H1 loop folded into completely different main chain architectures. Residues 52–56 form a hairpin loop (denoted H2) that constitutes the antigen-binding region of the second hypervariable region (15Al-Lazikani B. Lesk A.M. Chothia C. J. Mol. Biol. 1997; 273: 927-948Crossref PubMed Scopus (587) Google Scholar, 16Chothia C. Lesk A.M. Gherardi E. Tomlinson I.M. Walter G. Marks J.D. Llewelyn M.B. Winter G. J. Mol. Biol. 1992; 227: 799-817Crossref PubMed Scopus (365) Google Scholar). Canonical structures of the H2 loop are described for loops with sizes of five, six, or eight amino acids. We previously showed that VHH H2 loops of six amino acids adopt conformations not yet observed in VH structures (19Decanniere K. Muyldermans S. Wyns L. J. Mol. Biol. 2000; 300: 83-91Crossref PubMed Scopus (81) Google Scholar). Here, we are facing an H2 loop with only four amino acids (Fig. 1) that adopts a regular β-hairpin structure (Fig.4 B) known as type II′ (37Hutchinson E.G. Thornton J.M. Protein Sci. 1996; 5: 212-220Crossref PubMed Scopus (997) Google Scholar). A comparison of four- and five-amino acid-long H2 loops indicates that the addition of a fifth amino acid introduces a bulge at position 55 and converts the loop to an H2 canonical structure type-1 (Fig. 4, B andC). The CDR3 (residues 95–102) of a VHH is on average longer than that of a VH (17 versus 12 residues) (4Muyldermans S. Atarhouch T. Saldanha J. Barbosa J.A.R.G. Hamers R. Protein Eng. 1994; 7: 1129-1135Crossref PubMed Scopus (394) Google Scholar), although a notable fraction of llama VHHs were found with 'short' CDR3 loops (20Vu K.B. Ghahroudi M.A. Wyns L. Muyldermans S. Mol. Immunol. 1997; 34: 1121-1131Crossref PubMed Scopus (248) Google Scholar, 38Harmsen M.M. Ruuls R.C. Nijman I.J. Niewold T.A. Frenken L. de Geus B. Molec. Immunol. 2001; 37: 579-590Crossref Scopus (170) Google Scholar). Another remarkable feature of the CDR3 of VHHs is the frequent presence of a cysteine forming a disulfide bond with a cysteine in the CDR1 (4Muyldermans S. Atarhouch T. Saldanha J. Barbosa J.A.R.G. Hamers R. Protein Eng. 1994; 7: 1129-1135Crossref PubMed Scopus (394) Google Scholar,6Desmyter A. Transue T.R. Arbabi Ghahroudi M. Dao-Thi M.-H. Poortmans F. Hamers R. Muyldermans S. Wyns L. Nat. Struct. Biol. 1996; 3: 803-811Crossref PubMed Scopus (415) Google Scholar). In this respect, the cAb-CA05 with a 19-amino acid-long CDR3 loop and cysteines at positions 33 and 100c forming a disulfide bond is comparable with cAb-Lys3 (6Desmyter A. Transue T.R. Arbabi Ghahroudi M. Dao-Thi M.-H. Poortmans F. Hamers R. Muyldermans S. Wyns L. Nat. Struct. Biol. 1996; 3: 803-811Crossref PubMed Scopus (415) Google Scholar) (Figs. 1 and 4 D). The cysteine at position 100c can be considered to divide the CDR3 region into an N-terminal and a C-terminal part. The C-terminal part of the CDR3 loop folds back onto the side of the VHH domain corresponding to the side of the VH interacting with VL (1Padlan E.A. Mol. Immunol. 1994; 31: 169-217Crossref PubMed Scopus (790) Google Scholar) (see TableII for a list of contacting residues) (39Padlan E.A. Adv. Protein Chem. 1996; 49: 57-133Crossref PubMed Google Scholar). Large parts of the former VL-side are apparently shielded from the solvent by the C-end of the CDR3. A similar location and function has been observed for the C-terminal part of the cAb-Lys3 CDR3-loop (Fig. 4 D) (6Desmyter A. Transue T.R. Arbabi Ghahroudi M. Dao-Thi M.-H. Poortmans F. Hamers R. Muyldermans S. Wyns L. Nat. Struct. Biol. 1996; 3: 803-811Crossref PubMed Scopus (415) Google Scholar) for the entire, much shorter CDR3 loop of cAb-RN05 (5Decanniere K. Desmyter A. Lauwereys M. Ghahroudi M.A. Muyldermans S. Wyns L. Structure. 1999; 7: 361-370Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) and that of RR6 llama VHH (8Spinelli S. Frenken L. Hermans P.W.J.J. Verrips T. Brown K. Tegoni M. Cambillau C. Biochem. 2000; 39: 1217-1222Crossref PubMed Scopus (141) Google Scholar).Table IICDR3 residues contacting the remaining VHH or antigenAntibody residues contacting the CDR3 residues2-aResidues of the antibody having atoms within 4.0 Å to CDR3; the residues in bold belong to CDR.Interacting CDR3-residues 2-bCDR3 residues contacting the remaining VHH, the antigen, or both are aligned in the column on the left, right, or middle, respectively.Antigen residues contacting the CDR3 residues2-cResidues of the antigen having atoms within 4.0 Å to the antibody.Tyr-27, Tyr-32,Cys-33Ser-95Tyr-32Thr-96Leu-47, Ser-48, Val-49Cys-33Val-97Val-49, Leu-189Thr-31Ala-98Val-49, Ser-50, Tyr-51, Asp-52, Arg-182Leu-52Ser-99Asp-180, Arg-182Leu-52, Tyr-58Thr-100Asp-180, Arg-182Gly-100aAsp-180Trp-100bArg-182, Pro-186, Glu-187Cys-332-dCys residues connected by a disulfide bond., Thr-50,Ile-51Cys-100c2-dCys residues connected by a disulfide bond.Tyr-58Ser-100dArg-100eArg-182, Gly-183, Leu-185, Glu-187Gly-47, Val-48, Tyr-59, Leu-60Gly-100fArg-100gGlu-187Gly-35, Trp-36, Phe-37, Thr-50Pro-100hPhe-37, Arg-45, Gly-47Tyr-100iAsp-100jGlu-187Cys-33, Met-34, Thr-50Tyr-100kHis-101Pro-46Val-2, Leu-4Tyr-102Leu-4, Phe-37Arg-1032-a Residues of the antibody having atoms within 4.0 Å to CDR3; the residues in bold belong to CDR.2-b CDR3 residues contacting the remaining VHH, the antigen, or both are aligned in the column on the left, right, or middle, respectively.2-c Residues of the antigen having atoms within 4.0 Å to the antibody.2-d Cys residues connected by a disulfide bond. Open table in a new tab The N-terminal part of the CDR3 of cAb-CA05 and cAb-Lys3 are in a different environment. In cAb-Lys3, it forms a protruding loop (Fig.4 D) inserting in the catalytic site of the lysozyme (21Transue T.R. De Genst E. Ghahroudi M.A. Wyns L. Muyldermans S. Proteins: Structure, Function, and Genetics. 1998; 32: 515-522Crossref PubMed Scopus (95) Google Scholar). In cAb-CA05, this part of the loop does not extend into the solvent but associates with the residues of the remaining hypervariable loops (Table II). Thus, the N-terminal and C-terminal half of the CDR3 of cAb-CA05 contact different parts of the domain. Furthermore, the entire (long) CDR3 of cAb-CA05 appears to be well fixed by these abundant contacts and by the covalent Cys33-Cys100c disulfide bond. In addition to the antigen-free cAb-CA05 crystal, crystals of the complex of cAb-CA05 with its antigen, carbonic anhydrase, were obtained and diffracted to 3.5 Å using synchrotron radiation. The structure was solved by molecular replacement (see "Experimental Procedures") and the data and refinement statistics for the antibody-antigen complex structure are shown in Table I. Binding of the antibody has little influence on the overall structure of the carbonic anhydrase. The rmsd is 0.7 Å for the Cα ato

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