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Bactericidal Antibody Recognition of Meningococcal PorA by Induced Fit

1999; Elsevier BV; Volume: 274; Issue: 3 Linguagem: Inglês

10.1074/jbc.274.3.1495

1083-351X

Jean van den Elsen, Lucy Vandeputte-Rutten, Jan Kroon, Piet Gros,

Bacterial Genetics and Biotechnology

MN12H2 is a bactericidal antibody directed against outer membrane protein PorA epitope P1.16 of Neisseria meningitidis. Binding of MN12H2 to PorA at the meningococcal surface activates the classical complement pathway resulting in bacterial lysis. We have determined the crystal structure of the unliganded MN12H2 Fab fragment in two different crystal forms and compared it with the structure of the Fab in complex with a P1.16-derived peptide. The unliganded Fabs have elbow bend angles of 155° and 159°, whereas the liganded Fab has a more closed elbow bend of 143°. Substantial differences in quaternary and tertiary structure of the antigen binding site are observed between the unliganded and liganded MN12H2 Fab structures that can be attributed to peptide binding. The variable light and heavy chain interface of the liganded Fab is twisted by a 5° rotation along an axis approximately perpendicular to the plane of the interface. Hypervariable loops H1, H2, and framework loop FR-H3 follow this rotation. The hypervariable loop H3 undergoes conformational changes but remains closely linked to hypervariable loop L1. In contrast with the binding site expansion seen in other Fab-peptide structures, the MN12H2 binding site is narrowed upon peptide binding due to the formation of a "false floor" mediated by arginine residue 101 of the light chain. These results indicate that PorA epitope P1.16 of N. meningitidis is recognized by the complement-activating antibody MN12H2 through induced fit, allowing the formation of a highly complementary immune complex. MN12H2 is a bactericidal antibody directed against outer membrane protein PorA epitope P1.16 of Neisseria meningitidis. Binding of MN12H2 to PorA at the meningococcal surface activates the classical complement pathway resulting in bacterial lysis. We have determined the crystal structure of the unliganded MN12H2 Fab fragment in two different crystal forms and compared it with the structure of the Fab in complex with a P1.16-derived peptide. The unliganded Fabs have elbow bend angles of 155° and 159°, whereas the liganded Fab has a more closed elbow bend of 143°. Substantial differences in quaternary and tertiary structure of the antigen binding site are observed between the unliganded and liganded MN12H2 Fab structures that can be attributed to peptide binding. The variable light and heavy chain interface of the liganded Fab is twisted by a 5° rotation along an axis approximately perpendicular to the plane of the interface. Hypervariable loops H1, H2, and framework loop FR-H3 follow this rotation. The hypervariable loop H3 undergoes conformational changes but remains closely linked to hypervariable loop L1. In contrast with the binding site expansion seen in other Fab-peptide structures, the MN12H2 binding site is narrowed upon peptide binding due to the formation of a "false floor" mediated by arginine residue 101 of the light chain. These results indicate that PorA epitope P1.16 of N. meningitidis is recognized by the complement-activating antibody MN12H2 through induced fit, allowing the formation of a highly complementary immune complex. Antibody-antigen recognition is considered one of the most specific intermolecular interactions in the immune system. In addition to their antigen binding specificity, antibodies display a variety of secondary biological activities that are critical for host defense. These include virus neutralization, complement activation, opsonization, and signal transduction. Structural aspects of these antibody-associated effector functions have been described for virus-neutralizing antibodies (1Rini J.M. Schulze-Gahmen U. Wilson I.A. Science. 1992; 255: 959-965Crossref PubMed Scopus (497) Google Scholar, 2Tormo J. Blaas D. Parry N.R. Rowlands D. Stuart D. Fita I. EMBO J. 1994; 13: 2247-2256Crossref PubMed Scopus (99) Google Scholar, 3Rini J.M. Stanfield R.L. Stura E.A. Salinas P.A. Profy A.T. Wilson I.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6325-6328Crossref PubMed Scopus (213) Google Scholar, 4Ghiara J.B. Stura E.A. Stanfield R.L. Profy A.T. Wilson I.A. Science. 1994; 264: 82-85Crossref PubMed Scopus (240) Google Scholar). The atomic details of the complexes between the Fab fragments of anti-viral antibodies and peptides derived from viral epitopes show common structural features: 1) binding of the peptides induces major rearrangements of the antibody variable domains, and 2) the epitope-peptides bind in tight turn conformations. For a bacterial epitope, we have recently found that it was bound in a similar manner by complement-activating antibody MN12H2, directed against outer membrane protein PorA of Neisseria meningitidis (5van den Elsen J.M.H. Herron J.N. Hoogerhout P. Poolman J.T. Boel E. Logtenberg T. Wilting J. Crommelin D.J.A. Kroon J. Gros P. Proteins Struct. Funct. Genet. 1997; 29: 113-125Crossref PubMed Scopus (32) Google Scholar). PorA is a cation-selective transmembrane protein of 44 kDa that forms trimeric pores in the meningococcal outer membrane. According to a topology model, based on known porin structures, the protein is thought to span the membrane in a 16-strand β-barrel conformation (6van der Ley P. Heckels J.E. Virji J. Hoogerhout P. Poolman J.T. Infect. Immun. 1991; 59: 2963-2971Crossref PubMed Google Scholar). The model predicts eight extended extracellular loops. Two of these surface exposed loops (loops 1 and 4) are highly immunogenic and evoke antibodies that induce complement mediated bacterial killing. MN12H2 is a bactericidal antibody that is elicited against loop 4 of PorA. The recognition of loop 4 epitope P1.16 by MN12H2 is used as a model for studying the molecular and structural details of bactericidal antibody recognition of PorA. The first three-dimensional details of the bactericidal recognition of PorA epitope P1.16 were unveiled in the crystal structure of the MN12H2 Fab fragment in complex with a peptide derived from PorA residues 180–187 (5van den Elsen J.M.H. Herron J.N. Hoogerhout P. Poolman J.T. Boel E. Logtenberg T. Wilting J. Crommelin D.J.A. Kroon J. Gros P. Proteins Struct. Funct. Genet. 1997; 29: 113-125Crossref PubMed Scopus (32) Google Scholar). The fluorescein-labeled peptide was found in a type I β-turn conformation in the antigen binding cavity. The structure revealed several hydrophobic and electrostatic interactions between both binding partners, including a salt bridge between aspartate 182P of PorA and MN12H2 light chain residue histidine 31L. 1Amino acid residues of the MN12H2 heavy chain and light chain are indicated by H and L, respectively. The peptide residues are indicated by P. Throughout the text, a strict sequential numbering system is used. With the results from a thermodynamic study, this salt bridge was identified as the key interaction explaining the increased incidence of meningitis in United Kingdom in the early 1980s, caused by a D182N mutant strain of N. meningitidis (7van den Elsen J.M.H. van Pomeren E. Poolman J.T. Wilting J. Herron J.N. Crommelin D.J.A. Anal. Biochem. 1997; 247: 382-388Crossref PubMed Scopus (9) Google Scholar, 8van den Elsen J.M.H. van Unen L.M.A. van Bloois L. Busquets M.A. Jiskoot W. Hoogerhout P. Wilting J. Herron J.N. Crommelin D.J.A. Biochemistry. 1997; 36: 12583-12591Crossref PubMed Scopus (14) Google Scholar). The capacity of meningococcal PorA to evoke antibodies that induce complement-mediated bacterial killing has incited study to use this protein as a target in vaccine development. In clinical vaccination trials with outer membrane vesicles, it was shown that PorA was critical for the induction of bactericidal antibodies in humans (9Rouppe van der Voort E. van der Ley P. van der Biezen J. George S. Tunnella O. van Dijken H. Kuipers B. Poolman J. Infect. Immun. 1996; 64: 2745-2751Crossref PubMed Google Scholar). It was also shown that the presence of these antibodies correlate with protection against meningococcal disease (10Milagres L.G. Ramos S.R. Sacchi C.T. Melles C.E. Vieira V.S. Sat H. Brito G.S. Moraes J.C. Frash C.E. Infect. Immun. 1994; 62: 4419-4424Crossref PubMed Google Scholar). The protective activity of the immune complex between an antigen and a bactericidal antibody depends on its ability to cross-link complement factor C1q. With the binding of C1q, the classical complement pathway is activated, which leads to the formation of a multiprotein membrane-attack complex, causing bacterial membrane rupture. Here, we present the three-dimensional structure of the unliganded MN12H2 Fab fragment at 2.5 Å resolution based on two different crystal forms and compare it with the Fab-P1.16 peptide complex. With both structures available, we examine possible mechanisms by which quaternary and tertiary changes following antigen binding may activate the classical complement pathway. The murine monoclonal antibody MN12H2 was purified from hybridoma cell culture supernatant as described previously (11Jiskoot W. Van Hertrooij J.J.C.C. Hoven A.-M.V. Klein Gebbinck J.W.T.M. Van der Velden-de Groot T. Crommelin D.J.A. Beuvery E.C. J. Immunol. Methods. 1991; 138: 273-283Crossref PubMed Scopus (32) Google Scholar). The Fab fragment was obtained by papain digestion with papain-agarose beads (Sigma) at 37 °C for 4–16 h. The digestion buffer consisted of 10 mm Tris-HCl (Fluka, Buchs, Germany), pH 7.4, 1 mm EDTA, 0.02% w/v NaN3, and 1 mm dithioerythritiol. After digestion at least four prominent Fab isoforms could be identified by isoelectric focusing (IEF) 2The abbreviations used are: IEF, isoelectric focussing; CAPS, 3-(cyclohexylamino)-1-propane sulfonic acid; CH1, constant heavy domain 1; CL, constant light domain; MES, 2-morpholinoethanesulfonic acid; MPD, 2-methyl 2,4-peptanediol; VH, variable heavy domain; VL, variable light domain; V m, Matthews coefficient; Vκ, variable region κ; Jκ, joining region κ. (Pharmacia Phast system, Pharmacia LKB, Uppsala, Sweden) with approximate pI values of 8.45, 8.65, 9.1 and 9.3. The IEF pattern of MN12H2 Fab-peptide crystals showed that the crystallized Fab fragment mainly consisted of the pI 8.65 isoform. Isolation of this isoform was performed by anion exchange chromatography using a Q-Sepharose column (Amersham Pharmacia Biotech). The Fab fraction of the papain digest was loaded to the column using 20 mm3-(cyclohexylamino)-1-propane sulfonic acid (CAPS) (Fluka), pH 9.8, 0.02% w/v NaN3 as a binding buffer. The different Fab isoforms were eluted with a 0–0.15 m NaCl gradient. The recovered isoforms were analyzed by SDS-polyacrylamide gel electrophoresis, IEF, electron-spray mass spectrometry, and dynamic light scattering using a DynaPro-801 dynamic light scattering instrument (Protein Solutions Ltd., High Wycombe, Buckinghamshire, United Kingdom). Specific binding of the purified pI 8.65 Fab isoform to the P1.16 epitope was determined by means of fluorescence polarization experiments with a synthetic epitope peptide, as described earlier (8van den Elsen J.M.H. van Unen L.M.A. van Bloois L. Busquets M.A. Jiskoot W. Hoogerhout P. Wilting J. Herron J.N. Crommelin D.J.A. Biochemistry. 1997; 36: 12583-12591Crossref PubMed Scopus (14) Google Scholar). Preliminary crystallization conditions of the unliganded MN12H2 Fab were identified with a set of screening solutions using concentrations of 15–45% v/v 2-methyl 2,4 peptanediol (MPD) (Fluka) and 5–25% w/v polyethylene glycol 3000 (Fluka) as precipitating agents in combination with low molar concentrations of CaCl2, MgCl2, and CdCl2. The screening solutions were buffered using sodium acetate, 2-morpholinoethane-sulfonic acid (MES) (Fluka), HEPES (Fluka), or Tris-HCl (Fluka) with pH values of 4.5, 6.5, 7.5, and 8.5, respectively. Crystals for data collection were obtained at 4 °C in hanging drops using 20–30% v/v MPD and 20 mmCdCl2 in 50 mm MES buffer, pH 6.7. Data collection was performed at 120 K on a McScience DIP-2020 image plate detector using graphite monochromatized CuKαradiation from a Nonius FR570 rotating anode (Nonius, Delft, The Netherlands) operated at 45 mV and 95 mA. Diffraction data were auto-indexed and processed with DENZO and SCALEPACK (12Otwinowski Z. Minor W. Methods Enzymol. 1996; 276: 307-326Crossref Scopus (38771) Google Scholar). Data were collected from two different Fab crystal forms obtained under slightly different conditions (27 and 20% (v/v) MPD). Crystals grown at 27% MPD were C-centered orthorhombic C2221 with cell dimensions a = 86.0, b = 114.9,c = 153.1 Å. Diffraction to about 3.2 Å resolution was collected to 100% completeness from a single crystal of approximate size 0.2 × 0.2 × 0.15 mm3. The asymmetric unit contains one Fab molecule with a Matthews coefficient (V m) (13Matthews B.W. J. Mol. Biol. 1968; 33: 491-497Crossref PubMed Scopus (8002) Google Scholar) of 3.8 Å3/Da and a solvent content of 68%. The 20% MPD crystals belonged to the C-centered monoclinic space group C2, with unit cell dimensionsa = 114.6, b = 85.9, c= 87.1 Å and β = 122.7°. A complete data set was collected from a single crystal with dimensions of 0.2 × 0.2 × 0.15 mm3 diffracting to 2.5 Å resolution. The monoclinic crystal form also contains one Fab in the asymmetric unit,V m is 3.6 Å3/Da, with a solvent content of 66%. Crystallization and data collection details are summarized in Table I.Table ICrystallization and data collectionUnliganded Fab, orthorhombicUnliganded Fab, monoclinicFab-peptide complexCrystallization conditions27% v/v MPD20% v/v MPD15% w/v polyethylene glycol 20,00020 mm CdCl220 mm CdCl220 mm CdCl250 mm MES (pH 6.7)50 mm MES (pH 6.7)0.1 m NaAc (pH 4.6)Space groupC2221C2P21a (Å)86.0114.655.7b(Å)114.985.969.1c(Å)153.187.172.9β (°)122.7112.0Resolution (Å)3.22.52.6Solvent content (%)686656Reflections (no.)105,659176,95194,382Unique reflections (no.)12,86124,59815,404Completeness (%)aCompleteness was computed not counting the low resolution reflections up to 20 Å.10010097RsymbRsym = Σ ‖ I − 〈I〉 ‖ /Σ〈I〉.0.0710.0950.068a Completeness was computed not counting the low resolution reflections up to 20 Å.b Rsym = Σ ‖ I − 〈I〉 ‖ /Σ〈I〉. Open table in a new tab The structures of the unliganded MN12H2 Fabs were solved by molecular replacement using the MN12H2 Fab of Protein Data Bank entry 1MPA as a search model. The rotation function, the Patterson correlation function optimizing the relative orientation of the four Fab domains (VL, VH, CL, and CH1), the translation function, and structure refinement were performed with the program Crystallography and NMR System (14Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.-S. Kuszweski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D. 1998; 54: 905-921Crossref PubMed Scopus (17023) Google Scholar, 15Rice L.M. Brünger A.T. Proteins Struct. Funct. Genet. 1994; 19: 277-290Crossref PubMed Scopus (382) Google Scholar, 16Pannu N.S. Read R.J. Acta Crystallogr. Sect. A. 1996; 52: 659-668Crossref Scopus (319) Google Scholar, 17Adams P.D. Pannu N.S. Read R.J. Brünger A.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5018-5023Crossref PubMed Scopus (383) Google Scholar). Refinement used the maximum likelihood target with all experimental amplitudes except for a randomly selected test set of 10% that was used for cross-validated ςA weighting. The automated refinement procedure consisted of conjugate minimization followed by torsion angle molecular dynamics in combination with simulated annealing. Refinement was preceded by calculation of a bulk-solvent model, estimate ςA, and weight values. Rounds of refinement were followed by rebuilding of the models in O (18Jones T.A. Zou J.-Y. Cowan S.S. Kjeldgaard M.A. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13036) Google Scholar) using ςA weighted maps. Finally, the unliganded Fab models were subjected to restrained B-factor refinement. The structure of the MN12H2 Fab in complex with the P1.16-derived fluorescein-conjugated peptide was also subjected to the maximum likelihood target and torsion angle dynamics refinement method. As an indication of changes in the quaternary structure upon ligand binding for each molecule the angle between the pseudo-2-fold rotation axes of the VL-VH and CL-CH1 domains and the V and C superdomains (elbow bend angle) was determined using the program ROTMOL (kindly supplied by J. N. Herron). To further analyze quaternary and tertiary changes, the structures of the unliganded Fab and Fab-peptide complex were overlaid by least squares rotation and translation using O (18Jones T.A. Zou J.-Y. Cowan S.S. Kjeldgaard M.A. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13036) Google Scholar). The structure of the unliganded Fab was solved for two different crystal forms. The monoclinic crystal form (space group C2) diffracted to 2.5 Å resolution, whereas the orthorhombic crystal form (space group C2221) diffracted to a resolution of 3.2 Å. Crystallization and data collection statistics are given in TableI. Structures were refined to an R factor of 23% and an Rfreeof 27% for the monoclinic crystal form and to an R factor of 25% and an Rfree of 30% for the orthorhombic crystal form. Geometric parameters evaluated with PROCHECK (19Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar) and WHAT IF (20Vriend G. J. Mol. Graph. 1990; 8: 52-56Crossref PubMed Scopus (3396) Google Scholar) show acceptable values for both crystal structures. Refinement and model statistics are given in Table II. Almost 90% of the residues of the monoclinic crystal form of the unliganded Fab had main chain torsion angles that fell within the energetically most favored regions of the Ramachandran plot (21Ramachandran G.N. Sasisekharan V. Adv. Protein Chem. 1968; 23: 283-438Crossref PubMed Scopus (2799) Google Scholar), whereas none were found in the disallowed regions (Fig.1 A).Table IIRefinement/statisticsUnliganded Fab, orthorhombicUnliganded Fab, monoclinicResolution20–3.220–2.5R (%)25.323.1Rfree (%)29.827.0Cadmium ions (no.)43Water molecules (no.)5196Root mean square deviations from idealityBonds (Å)0.0140.008Angles (°)1.691.62Dihedrals (°)25.726.3Improper dihedrals (°)0.770.79Average B-factors (protein) (Å2)NDaND, not determined. B-factors were taken from the monoclinic unliganded MN12H2 Fab.23Cross-validatedςa coordinate error (Å)0.710.34a ND, not determined. B-factors were taken from the monoclinic unliganded MN12H2 Fab. Open table in a new tab The final unliganded MN12H2 Fab model in the monoclinic crystal form contains all 219 light chain amino acid residues and 221 of the 225 residues of the heavy chain as determined by electron spray mass spectrometry (molecular mass, 49,201 Da). The model also includes the classically disordered interchain-disulfide region Cys-136H–Gly-141H (Fig. 1 B), for which good density was observed. Some basic residues at the exterior of the Fab display ill-defined electron density (79L, 82L, 86L, 108L, 56H, 63H, and 69H) and the side chain conformation of Phe-106H at the top of hypervariable loop H3 must also be considered as tentative. In the orthorhombic unliganded Fab the interchain-disulfide region residues Gly-137H–Gly-141H were excluded from the model due to weak electron density. A 4° difference in elbow bend angle was found between the two unliganded Fab crystal forms (Table III). Otherwise, both structures are virtually identical.Table IIIPseudodyad and elbow bend angles for the unliganded and peptide-complexed MN12H2 FabUnliganded Fab, orthorhombicUnliganded Fab, monoclinicLiganded FabPseudodyad anglesVL-VH dimer175°176°175°CL-CH1 dimer168°168°169°Elbow bend anglesV-C domains159°155°143° Open table in a new tab As in the MN12H2 Fab-peptide complex (5van den Elsen J.M.H. Herron J.N. Hoogerhout P. Poolman J.T. Boel E. Logtenberg T. Wilting J. Crommelin D.J.A. Kroon J. Gros P. Proteins Struct. Funct. Genet. 1997; 29: 113-125Crossref PubMed Scopus (32) Google Scholar), strong electron density peaks were seen in both unliganded Fab crystal forms. Because cadmium ions were essential for crystallization and because of the vicinity of putative cadmium binding residues, the positions of these 5 ς electron density peaks are very likely to be occupied by cadmium ions. As in the 1MPA structure, a putative cadmium binding site was found near the Nδ1 of His-98L. In the monoclinic unliganded Fab structure, this cadmium is coordinated by two water molecules, as in the 1MPAstructure, and by the carboxylate oxygens of Glu-218L at the N terminus of a symmetry related molecule. A second cadmium ion interacts with the Nε2 of His-172H, the Nδ2 of Asn-143L, and a water molecule. The third cadmium is coordinated by the carboxylate oxygens of Glu-190L, the Nε2 nitrogen of His-194L, and a water molecule and via crystal packing interactions with the carboxylate moiety of a symmetry-related Asp-181H. In the orthorhombic crystal form, an additional cadmium ion was observed that occupied a special position situated on a 2-fold rotation axis parallel to b. The ion was found to be coordinated by the carboxylate oxygens of four glutamate residues, including Glu-84L, Glu-86L, and their symmetry-related residues. However, some of the densities assigned to coordinating water molecules may actually be occupied by chloride ions. Because the structures of the free and complexed MN12H2 Fab were refined using different techniques, for good comparison of both models, the Fab-peptide structure (Protein Data Bank entry 1MPA) was subjected to refinement using the maximum likelihood target. Refinement and rebuilding of heavy chain regions 42H–43H, 136H–141H and 156H–158Hresulted in a drop in free R factor by 4.5% from 30.9 to 26.4%. Rebuilding of these heavy chain regions also improved their abnormal main chain Φ and Ψ torsion angles as indicated by the Ramachandran plot (21Ramachandran G.N. Sasisekharan V. Adv. Protein Chem. 1968; 23: 283-438Crossref PubMed Scopus (2799) Google Scholar). Structure validation of the model confirm these results showing improved model geometry, as indicated by a decrease in bond and angle violations and a reduction of close contacts (see also TableIV). Because of ambiguous density, C-terminal residue Ile-225H was removed from the model. No significant conformational differences were observed for the bound fluorescein-labeled peptide and the antigen binding site.Table IVRefinement statistics of liganded FabFab-peptide complex, 1MPA (5)Fab-peptide complex, 2MPAResolution8–2.620–2.6R (%)19.420.2Rfree (%)30.926.4Cadmium ions (no.)11Water molecules (no.)1752Root mean square deviations from idealityBonds (Å)0.0110.008Angles (°)1.891.53Dihedrals (°)27.325.5Improper dihedrals (°)1.510.77Average B-factors (protein) (Å2)2844Cross-validatedςa coordinate error (Å)0.450.48The 1MPA structure of the MN12H2 Fab-P1.16 peptide complex was refined using the least squares target function. The 2MPA structure resulted from refinement of 1MPA using the maximum likelihood target and torsion angle dynamics refinement method. Open table in a new tab The 1MPA structure of the MN12H2 Fab-P1.16 peptide complex was refined using the least squares target function. The 2MPA structure resulted from refinement of 1MPA using the maximum likelihood target and torsion angle dynamics refinement method. We observed elbow bend angles of 155° and 159° for the unliganded Fab in the monoclinic crystal form and the orthorhombic crystal form, respectively. For the liganded MN12H2 Fab, a more closed elbow bend was found with an angle of 143° between the pseudo-2-fold rotation axis of the V and C superdomains (Table III). A 5° rotation was observed between the variable domains of the unliganded Fabs and the Fab-peptide complex, along an axis approximately perpendicular to the VL-VHinterface. As illustrated in Figs. 2 and3 A, the largest coordinate differences resulting from this rotation were found at the tips of the hypervariable loops (Cα-coordinate differences up to 3.6 Å for hypervariable loop H1 and up to 3.3 Å for H2). The H3 loop does not follow this domain rotation, and its backbone is relatively kept in position with only minor Cα-coordinate differences from 0.2 Å for Asp-105H and up to 1.6 Å for Ala-104H. As depicted in Fig.3 B, the H3 loop is fixed to loop L1 by a tandem of Tyr-Asp hydrogen bonds. The side chain hydroxyls of tyrosine residues 37L and 41L bind to the carboxylate oxygens of aspartate residues 102H and 109H of loop H3. These VH-VL interface interactions are further tightened by a hydrogen bond between the Nζ of hypervariable loop L2 residue Lys-55L and the carbonyl oxygen of Phe-101H (loop H3).Figure 3A, relative positions of the variable light and heavy chains in the dimers of the unliganded Fab and the Fab-peptide complex. B, conformational differences in hyper variable loop H3 between the unliganded MN12H2 Fab and the Fab-peptide complex. C, superposition of the cavity floor residues of the unliganded and liganded MN12H2 Fabs. For these figures the corresponding VL chains of the monoclinic unliganded Fab and the liganded Fab structures were overlaid as a dimer as described in Fig. 2. Light and heavy chain residues of the liganded Fab are shown in green and magenta. Unliganded Fab residues and their corresponding backbone positions are shown in yellow. These figures were produced with MOLSCRIPT and RASTER3D (30Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar, 31Merrit E.A. Murphy M.E. Acta Crystallogr. D. 1994; 50: 869-873Crossref PubMed Scopus (2859) Google Scholar).View Large Image Figure ViewerDownload (PPT) Major side chain displacements between the unliganded Fabs and the Fab-peptide complex were observed in loops L3 and H3. In both unliganded Fabs, arginine residue 101L protrudes from the floor of the antigen binding site (Fig. 3 C), thereby creating a positively charged bulge. This is also illustrated in the molecular surface of the unliganded antigen binding site in Fig.4 A, colored for electrostatic potential. In contrast, Arg-101L spans the binding pocket in the Fab-peptide complex, and its guanidinium group forms hydrogen bridges with residues Ser-97H and Tyr-41L. In the newly refined liganded Fab structure, additional interactions are seen with Arg-101L: a water-mediated hydrogen-bonding interaction with the hydroxyl group of Ser-94L (hypervariable loop L3) and a hydrogen bond with Nδ2 of His-35H (hypervariable loop H2). The largest atomic displacements between the free and the complexed Fab were observed in the H3 loop. As illustrated in Figs. 3 B and 4 A, a dramatic displacement was seen for Tyr-103H with a 9-Å difference of the hydroxyl oxygens between both structures. The hydroxyl oxygen of Tyr-100H, pointing toward the binding site in the unliganded Fab, is moved away 3.4 Å from the cavity in the Fab-peptide complex and points upward. Between both free Fab crystal forms and the Fab-peptide complex, the hypervariable loops play different roles in crystal packing interactions. In the monoclinic as well as the orthorhombic unliganded Fab L3 loop, residue His-98L is involved in head-to-tail symmetry interactions with the C terminus of the light chain, mediated by a cadmium ion (defining the variable domain as head and the constant domain as tail). In the monoclinic crystal form, 70% of all hypervariable loop crystal contacts involve additional head-to-tail van der Waals interactions between the tips of loops L1 and L2 and the interchain disulfide region. Also, several salt links with the C-terminal region of the CH1 domain of a symmetry-related molecule are observed. The interactions between loop L1 and the interchain disulfide residues Gly-137H and Thr-140H in the monoclinic unliganded Fab result in unexpectedly well defined electron density for this archetypically disordered region. In the orthorhombic Fab, no interactions are observed between the three light chain hypervariable loops and the interchain disulfide region. In both unliganded Fab structures, the H3 loop is packed head-to-head with the binding site of a symmetry-related molecule. In the complexed Fab, crystal contacts mainly involve head-to-head packing between the apices of hypervariable loop L1 and loops H1 and H3 of a symmetry-related molecule. The fluorescein label of the peptide accounts for 11 additional head-to-tail contacts with the C terminus of the CH1 domain. A single interaction is observed between loop L1 and the interchain disulfide region. In contrast with the free Fab crystal forms, the liganded Fab displays crystal contacts with elbow bend residues 112L, 113L, and 114L that are packed against the heavy chain of a symmetry-related molecule. Comparison of two crystal forms of the unliganded MN12H2 Fab with the liganded structure of the epitope-peptide Fab complex reveals significant conformational changes. The surface representations of the binding sites of both structures in Fig. 4 indicate the quaternary and tertiary changes of the Fab upon peptide binding. The antigen binding site of MN12H2 may be thought of as a left-handed baseball glove, with hypervariable loops L1 and L3 forming the thumb, and the VHdomain (with loops H3, H2, and H1) as the fingers. As in the baseball glove, the thumb (loop L1) and the forefinger (loop H3) remain connected upon peptide binding through a "two-bar" web formed by a tandem of tyrosine-aspartate interactions, whereas the other fingers (loops H2, framework region-H3, and H1) follow the shape of the peptide (see also Fig. 2). In the unliganded Fab, arginine residue 101L forms a positively charged bulge in the binding cavity. Upon peptide binding, this bulge is depressed, and its charge is neutralized due to the formation of a false floor that narrows the cavity and connects four hypervariable loops (L1, L3, H1, and H3). The side chain rearrangements in the H3 loop represented by tyrosines 100H and 103H are necessary to clear the binding site to further accommodate the peptide binding. When the P1.16 peptide is positioned in the binding site of the unliganded Fab, the side chain of these residues sterically blocks the binding of the peptide. The sterical hindrance of hypervariable loop H1 residues that is encountered by the peptide is overcome by the observed 5° twist of the VL-VH interface in the liganded Fab. This rotation brings H1 residue Tyr-33H in position to form a hydrophobic stack with peptide residue Thr-183P. The VL-VHtwist also decreases the distance between the Cα atoms of hypervariable loops L1 (Arg-101L) and H3 (Ser-97H) from 13 to 11.5 Å, thereby facilitating the formation of a Arg-101L-mediated bidentate hydrogen bond with the side chain hydroxyl oxygen of Ser-97H and the hydroxyl oxygen of Tyr-41L. The hydrogen bond between the Nη2 group of Arg-101L and Nδ2 of His-35H (hypervariable loop H2) induces an almost 90° rotation of the 35H imidazolium ring bringing the Nε2 nitrogen in position to fix the peptide backbone at the newly formed cavity floor. The formation of this false floor, directed by Arg-101L, stabilizes the topography of the complexed VL-VH dimer by locking four hypervariable loops (L1, L3, H1, and H3) into a new conformation. A thermodynamic study on the interaction with MN12H2 and the P1.16 peptide revealed that these interactions, together with the release of structured water from the binding pocket and the newly formed interactions between Fab and peptide, favor the interaction by 100 kJ mol−1 (8van den Elsen J.M.H. van Unen L.M.A. van Bloois L. Busquets M.A. Jiskoot W. Hoogerhout P. Wilting J. Herron J.N. Crommelin D.J.A. Biochemistry. 1997; 36: 12583-12591Crossref PubMed Scopus (14) Google Scholar). It largely offsets the 50 kJ mol−1 cost in free energy needed to induce the structural changes in both binding partners (as determined by the analysis of Sturtevant (22Sturtevant J.M. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 2236-2240Crossref PubMed Scopus (881) Google Scholar)). With the exposed guanidinium moiety of Arg-101L in the cavity surrounded by basic residues His-31L and Arg-59L, an overall positively charged binding site is formed in the unliganded Fab, as illustrated in Fig. 4 A. Although Arg-101L does not contribute directly to peptide binding, it could explain the medium-affinity cross-reactive binding mode of MN12H2 for negatively charged self-antigens such as single stranded DNA and cardiolipin. 3J. M. H. van den Elsen, unpublished results. The appearance of basic residues at these sites within the light chain hypervariable loops and especially at the junction between Vκ and Jκ1 genes (101L) fits the general pattern of DNA-binding antibodies described by Radic and Weigert (23Radic M.Z. Weigert M. Annu. Rev. Immunol. 1994; 12: 487-520Crossref PubMed Scopus (433) Google Scholar). Whether the binding of these autoantigens adopt different complexed structures remains to be determined. Relative disposition of VL-VH domains has been observed before in Fab structures upon binding of a peptide (1Rini J.M. Schulze-Gahmen U. Wilson I.A. Science. 1992; 255: 959-965Crossref PubMed Scopus (497) Google Scholar, 24Wilson I.A. Stanfield R.L. Curr. Opin. Struct. Biol. 1993; 3: 113-118Crossref Scopus (267) Google Scholar,25Stanfield R.L. Fieser T.M. Lerner R.A. Wilson I.A. Science. 1990; 248: 721Crossref PubMed Scopus (346) Google Scholar). It illustrates the intrinsic flexibility of antibodies in adapting to the shape of an antigen. The largest VL-VHrotations have been observed for anti-DNA Fab BV0–401 (7.5°) (26Herron J.N. He X.M. Ballard D.W. Blier P.R. Pace P.E. Bothwell A.L.M. Voss Jr., E.W. Edmundson A.B. Proteins Struct. Funct. Genet. 1991; 11: 159-175Crossref PubMed Scopus (276) Google Scholar) and for anti-HIV Fab 50.1 upon complexation with a V3 loop peptide (16°) (25Stanfield R.L. Fieser T.M. Lerner R.A. Wilson I.A. Science. 1990; 248: 721Crossref PubMed Scopus (346) Google Scholar). Unlike MN12H2, these Fab structures show a large decrease in the number of VL-VH interface contacts upon complexation with their antigen. The loss of self-contacts is mainly ascribed to rearrangements of the H3 loop, which moves out of the binding site with root mean square deviations in backbone atoms up to 5 Å. In contrast with this binding site expansion, the MN12H2 Fab shows a closer association of both domains upon peptide binding by forming a false floor. The 5° rotation of the H1 and H2 loop narrows the cavity even more and holds the peptide in a tight lock. Because the backbone of the H3 loop remains fixed, massive side-chain rearrangements of tyrosines 100H and 103H are essential for the binding site to adapt to the shape of the peptide. All of the structural changes mentioned in this section appear to be necessary for peptide binding. Because both monoclinic and orthorhombic crystal forms of the unliganded Fab display similar features in a different crystal packing environment, the conformational differences with the liganded Fab are believed to be peptide-induced. No conformational changes of fluorescein binding residue Arg-59H have been detected between the unliganded Fabs and 1MPA, suggesting no or only minor effects of the fluorescein molecule on the shaping of the binding site. Conformational changes in the elbow bend angle and the distal extremities of the constant region, such as the interchain disulfide region, are believed to be induced by the general flexibility of these regions and by their different crystal packing interactions. The elbow bend regions at the junction between the variable and constant domains are very flexible and there have been differences found in the elbow bend angle between unliganded structures as well as liganded structures of the same Fab (27Wilson I.A. Stanfield R.L. Curr. Opin. Struct. Biol. 1994; 4: 857-867Crossref PubMed Scopus (453) Google Scholar). Stanfield et al. (25Stanfield R.L. Fieser T.M. Lerner R.A. Wilson I.A. Science. 1990; 248: 721Crossref PubMed Scopus (346) Google Scholar) even reported a 13° difference in elbow bend angle between two complexed 50.1 Fab molecules found in the same asymmetric unit, also illustrating this general flexibility. There is no evidence that the peptide-induced conformational changes, other than adjusting fit and complementarity to the antigen, provide a signal that is transmitted to the constant domains triggering secondary biological activities. The accessibility and the tight-turn recognition motif of immunogenic loops might be important for evoking antibodies that are able to neutralize viral infections or activate the complement system. Several mechanisms have been postulated for virus neutralizing activities of antibodies, including interference with the viral attachment to the cell and viral internalization, induction of structural changes as a consequence of binding, and intracapsid cross-linking, preventing uncoating of the virus (28Tormo J. Centeno N.B. Fontana E. Bubendorfer T. Fita I. Blaas D. Proteins Struct. Funct. Genet. 1995; 23: 491-501Crossref PubMed Scopus (14) Google Scholar). A mechanism for the complement activating capacity of bactericidal antibody MN12H2 may be the improved complementarity between the P1.16 epitope and the binding site. This tight conformational state might decrease the off-rate of the complex, thereby augmenting the effective antibody valence on the bacterial surface for cross-linking C1q. We thank The Dutch National Institute of Public Health and the Environment in Bilthoven for providing the MN12H2 hybridoma cell-culture supernatant. We also thank Dr. J. N. Herron from the University of Utah for critically reviewing the manuscript. The technical assistance of C. Versluis from the Utrecht University with the electron-spray mass spectrometry experiments is gratefully acknowledged.