Crystallization and preliminary X-ray studies of the VL domain of the antibody McPC603 produced in Escherichia coli

The VL domain, obtained from a recombinant Fv fragment of the antibody McPC603 expressed in Escherichia coli, has been crystallized as a dimer from 2 M-(NH4)2SO4 (pH 4.0). The crystals are hexagonal, space group P6(1)22. The cell dimensions are a = b = 86.48 A, c = 76.64 A, with a VL monomer as the asymmetric unit. The crystals diffract to 2.0 A. The structure was solved by Patterson search using the VL domain of the Fab fragment of McPC603 and the VL dimer REI.     

The galactan-binding immunoglobulin Fab J539: an X-ray diffraction study at 2.6-A resolution

The crystal structure of the Fab of the galactan-binding immunoglobulin J539 (a mouse IgA,kappa) has been determined at a resolution of approximately 2.6 A by X-ray diffraction. The starting model was that obtained from the real space search described previously (Navia, M.A., Segal, D.M., Padlan, E.A., Davies, D.R., Rao, D.N., Rudikoff, S. and Potter, M. "Crystal structure of galactan-binding mouse immunoglobulin J539 Fab at 4.5 A resolution." Proc. Nat. Acad. Sci. USA, 76:4071-4074, 1979). This Fab structure has now been refined by restrained least-squares procedures to an R-value of 19% for the 11,690 unique reflections between 8.0 A and 2.6 A. The rms deviation from ideal bond lengths is 0.025 A. The overall structure differs from McPC603 Fab, another mouse IgA,kappa antibody, in that the elbow bend, relating the variable and constant parts of the molecule, is 145 degrees vs. 133 degrees for McPC603. The region of the molecule expected to be the antigen binding site contains a large cavity with two clefts leading away from it. This has been fitted with a model of an oligo-galactan.     

Crystal structure of an anti-Lewis alpha Fab determined by molecular replacement methods

The anti-Lewis alpha mouse immunoglobulin CF4C4 (IgGl, k) Fab has been crystallized from 58% saturated ammonium sulfate in space group Pl; unit cell dimensions a = 43.4 A b = 41.7 A, c = 62.0 A, a = 72.7 degrees, beta = 96.6 degrees, gamma = 100.1 degrees. X-ray diffraction data have been measured beyond 3.0 A Bragg spacing. The crystal structure has been determined by molecular replacement methods, using as search models the constant and variable domains of the mouse immunoglobulin McPC603 (IgA, kappa) Fab. The crystallographic residual for the data 5.0 to 4.0 A, is 0.47. The approximate 2-fold axis relating the VL and the VH domains forms an angle of 164 degrees with the 2-fold axis relating the constant domains. The crystal packing is reasonable.     

Crystal structure to 2.45 A resolution of a monoclonal Fab specific for the Brucella A cell wall polysaccharide antigen.

The atomic structure of an antibody antigen-binding fragment (Fab) at 2.45 A resolution shows that polysaccharide antigen conformation and Fab structure dictated by combinatorial diversity and domain association are responsible for the fine specificity of the Brucella-specific antibody, YsT9.1. It discriminates the Brucella abortus A antigen from the nearly identical Brucella melitensis M antigen by forming a groove-type binding site, lined with tyrosine residues, that accommodates the rodlike A antigen but excludes the kinked structure of the M antigen, as envisioned by a model of the antigen built into the combining site. The variable-heavy (VH) and variable-light (VL) domains are derived from genes closely related to two used in previously solved structures, M603 and R19.9, respectively. These genes combine in YsT9.1 to form an antibody of totally different specificity. Comparison of this X-ray structure with a previously built model of the YsT9.1 combining site based on these homologies highlights the importance of VL:VH association as a determinant of specificity and suggests that small changes at the VL:VH interface, unanticipated in modeling, may cause significant modulation of binding-site properties.     

Complex between Peptostreptococcus magnus protein L and a human antibody reveals structural convergence in the interaction modes of Fab binding proteins

BACKGROUND: Peptostreptococcus magnus protein L (PpL) is a multidomain, bacterial surface protein whose presence correlates with virulence. It consists of up to five homologous immunoglobulin binding domains that interact with the variable (VL) regions of kappa light chains found on two thirds of mammalian antibodies. RESULTS: We refined the crystal structure of the complex between a human antibody Fab fragment (2A2) and a single PpL domain (61 residues) to 2.7 A. The asymmetric unit contains two Fab molecules sandwiching a single PpL domain, which contacts similar VL framework regions of two light chains via independent interfaces. The residues contacted on VL are remote from the hypervariable loops. One PpL-Vkappa interface agrees with previous biochemical data, while the second is novel. Site-directed mutagenesis and analytical-centrifugation studies suggest that the two PpL binding sites have markedly different affinities for VL. The PpL residues in both interactions are well conserved among different Peptostreptococcus magnus strains. The Fab contact positions identified in the complex explain the high specificity of PpL for antibodies with kappa rather than lambda chains. CONCLUSIONS: The PpL-Fab complex shows the first interaction of a bacterial virulence factor with a Fab light chain outside the conventional combining site. Structural comparison with two other bacterial proteins interacting with the Fab heavy chain shows that PpL, structurally homologous to streptococcal SpG domains, shares with the latter a similar binding mode. These two bacterial surface proteins interact with their respective immunoglobulin regions through a similar beta zipper interaction.     

A fully synthetic human Fab antibody library based on fixed VH/VL framework pairings with favorable biophysical properties

This report describes the design, generation and testing of Ylanthia, a fully synthetic human Fab antibody library with 1.3E+11 clones. Ylanthia comprises 36 fixed immunoglobulin (Ig) variable heavy (VH)/variable light (VL) chain pairs, which cover a broad range of canonical complementarity-determining region (CDR) structures. The variable Ig heavy and Ig light (VH/VL) chain pairs were selected for biophysical characteristics favorable to manufacturing and development. The selection process included multiple parameters, e.g., assessment of protein expression yield, thermal stability and aggregation propensity in fragment antigen binding (Fab) and IgG1 formats, and relative Fab display rate on phage. The framework regions are fixed and the diversified CDRs were designed based on a systematic analysis of a large set of rearranged human antibody sequences. Care was taken to minimize the occurrence of potential posttranslational modification sites within the CDRs. Phage selection was performed against various antigens and unique antibodies with excellent biophysical properties were isolated. Our results confirm that quality can be built into an antibody library by prudent selection of unmodified, fully human VH/VL pairs as scaffolds.     

Conformational dynamics of complementarity-determining region H3 of an anti-dansyl Fv fragment in the presence of its hapten

Antigen-induced structural changes in the Fv fragment of an anti-dansyl immunoglobulin G were studied by X-ray crystallography and stopped-flow fluorescence measurement. The crystal structure of the Fv fragment complexed with dansyl-lysine was determined at a resolution of 1.85 A. The dansyl-lysine molecule bound to a narrow cavity formed by the complementarity-determining regions H3 and H1, the N-terminal region of the VH domain and L2 of the VL domain. The structure of the binding site in the crystal structure explained well the results of the previous nuclear magnetic resonance measurements. The hapten binding caused remarkable conformational changes in H3 and its environmental structures, including the hydration structure from those observed in the unliganded state. The tip of H3 moved about 12 A from its position in the unliganded state. In addition, because of the contacts of H3 with the VL domain at the domain interface, the conformational changes of H3 resulted in the relative rotation of the variable domains by 5 degrees from their association observed in the unliganded state. The hydrophobic interactions at the domain interface seemed to be particularly important for the mutual rotation of the domains. The stopped-flow fluorescence measurement monitoring the interaction of the dansyl group and the binding pocket revealed that H3 was in a conformational equilibrium of three consecutive conformational states in the presence of dansyl-lysine in solution; an unliganded state preventing the access of the hapten, another unliganded state able to bind the hapten and the complex. The conformational dynamics of H3 in recognizing and binding the hapten molecule are discussed on the basis of the structural information from the present and previous studies.     

The disulfide bonds in antibody variable domains: effects on stability, folding in vitro, and functional expression in Escherichia coli.

The formation of the disulfide bonds in the variable domains VH and VL of the antibody McPC603 was found to be essential for the stability of all antigen binding fragments investigated. Exposure of the Fv fragment to reducing conditions in vitro resulted in irreversible denaturation of both VH and VL. In vitro refolding of the reduced Fv fragment was only possible when the disulfide bonds were allowed to form under oxidizing conditions. The analysis of a series of mutants of the Fv fragment, the Fab fragment and the single-chain Fv fragment, all secreted into the periplasm of Escherichia coli, in which each of the cysteine residues of the variable domains was replaced by a series of other amino acids, showed that functional antigen binding fragments required the presence of both the disulfide bond in VH and the one in VL. These results were also used to devise an alternative expression system based on the production of insoluble fusion proteins consisting of truncated beta-galactosidase and antibody domains, enzymatic cleavage, and refolding and assembly in vitro. This strategy should be useful for providing access to unstable antibody domains and fragments.     

Possible three-dimensional backbone folding around antibody combining site of immunoglobulin MOPC167

Using a recently developed method of predicting possible three dimensional foldings of immunoglobulin backbones around antibody combining sites, we have attempted to construct the structure of immunoglobulin MOPC167 which could bind phosphorylcholine. A small pocket was present in the middle of the predicted structure similar to that of another phosphorylcholine binding immunoglobulin, McPC603, the tertiary structure of which has been determined by X-ray diffraction studies. Although detailed atomic co-ordinates of McPC603 were not available, a rough comparison between these structures strongly suggested that the foldings of complementarity determining regions were alike only for those with identical lengths and similar amino acid sequences. The predicted structure of MOPC167 as compared with the previously predicted structure of MOPC315, a 2,4-dinitrophenol binding immunoglobulin, further suggested that the light chain of one immunoglobulin could indeed combine with the heavy chain of another immunoglobulin, but might not result in a reasonable site for binding antigens.     

The three-dimensional structure of a T-cell antigen receptor V alpha V beta heterodimer reveals a novel arrangement of the V beta domain.

The crystal structure of a mouse T-cell antigen receptor (TCR) Fv fragment complexed to the Fab fragment of a specific anti-clonotypic antibody has been determined to 2.6 A resolution. The polypeptide backbone of the TCR V alpha domain is very similar to those of other crystallographically determined V alphas, whereas the V beta structure is so far unique among TCR V beta domains in that it displays a switch of the c" strand from the inner to the outer beta-sheet. The beta chain variable region of this TCR antigen-binding site is characterized by a rather elongated third complementarity-determining region (CDR3beta) that packs tightly against the CDR3 loop of the alpha chain, without leaving any intervening hydrophobic pocket. Thus, the conformation of the CDR loops with the highest potential diversity distinguishes the structure of this TCR antigen-binding site from those for which crystallographic data are available. On the basis of all these results, we infer that a significant conformational change of the CDR3beta loop found in our TCR is required for binding to its cognate peptide-MHC ligand.     

Domain association in immunoglobulin molecules. The packing of variable domains

We have analyzed the structure of the interface between VL and VH domains in three immunoglobulin fragments: Fab KOL, Fab NEW and Fab MCPC 603. About 1800 A2 of protein surface is buried between the domains. Approximately three quarters of this interface is formed by the packing of the VL and VH beta-sheets in the conserved "framework" and one quarter from contacts between the hypervariable regions. The beta-sheets that form the interface have edge strands that are strongly twisted (coiled) by beta-bulges. As a result, the edge strands fold back over their own beta-sheet at two diagonally opposite corners. When the VL and VH domains pack together, residues from these edge strands form the central part of the interface and give what we call a three-layer packing; i.e. there is a third layer composed of side-chains inserted between the two backbone side-chain layers that are usually in contact. This three-layer packing is different from previously described beta-sheet packings. The 12 residues that form the central part of the three observed VL-VH packings are absolutely or very strongly conserved in all immunoglobulin sequences. This strongly suggests that the structure described here is a general model for the association of VL and VH domains and that the three-layer packing plays a central role in forming the antibody combining site.     

Domain interactions in the Fab fragment: a comparative evaluation of the single-chain Fv and Fab format engineered with variable domains of different stability

Recombinant antibody fragments, most notably Fab and scFv, have become important tools in research, diagnostics and therapy. Since different recombinant antibody formats exist, it is crucial to understand the difference in their respective biophysical properties. We assessed the potential stability benefits of changing the scFv into the Fab format, the influence of the variable domains on the stability of the Fab fragment, and the influence of the interchain disulfide bond in the Fab fragment. To analyze domain interactions, the Fab fragment was broken down into its individual domains, several two-domain assemblies and one three-domain assembly. The equilibrium denaturation properties of these constructs were then compared to those of the Fab fragment. It was found that mutual stabilization occurred across the VH/VL and the CH1/CL interface, whereas the direct interaction between the V) and the CL domain had no influence on the stability of either domain. This observation can be explained by the different interfaces used for interaction. In contrast, the whole CH1CL and VHVL unit showed significant mutual stabilization, indicating a high degree of cooperation between the VH/VL and CH1/CL interface. The interchain disulfide bond in the Fab fragment plays an essential role in this stabilization. In addition to the effects of domain association on the thermodynamic (equilibrium) stability, Fab fragments differ from scFv fragments of similar equilibrium stability by having a very slow unfolding rate. This kinetic stabilization may increase significantly the resistance of Fab fragments against short time exposure to adverse conditions.     

Structural diversity in a human antibody germline library

To support antibody therapeutic development, the crystal structures of a set of 16 germline variants composed of 4 different kappa light chains paired with 4 different heavy chains have been determined. All four heavy chains of the antigen-binding fragments (Fabs) have the same complementarity-determining region (CDR) H3 that was reported in an earlier Fab structure. The structure analyses include comparisons of the overall structures, canonical structures of the CDRs and the VH:VL packing interactions. The CDR conformations for the most part are tightly clustered, especially for the ones with shorter lengths. The longer CDRs with tandem glycines or serines have more conformational diversity than the others. CDR H3, despite having the same amino acid sequence, exhibits the largest conformational diversity. About half of the structures have CDR H3 conformations similar to that of the parent; the others diverge significantly. One conclusion is that the CDR H3 conformations are influenced by both their amino acid sequence and their structural environment determined by the heavy and light chain pairing. The stem regions of 14 of the variant pairs are in the ‘kinked’ conformation, and only 2 are in the extended conformation. The packing of the VH and VL domains is consistent with our knowledge of antibody structure, and the tilt angles between these domains cover a range of 11 degrees. Two of 16 structures showed particularly large variations in the tilt angles when compared with the other pairings. The structures and their analyses provide a rich foundation for future antibody modeling and engineering efforts.     

Crystallization and structure determination of an autoimmune anti-poly(dT) immunoglobulin Fab fragment at 3.0 A resolution

HED10 is an autoimmune antibody (IgG) which shows considerable specificity for the single-stranded DNA poly(dT). Production of Fab fragments by papain digestion resulted in heterogeneity as judged by isoelectric focusing gels, which had a marked negative effect on crystallization. However, a single species of Fab with a pI of 7.6 could be isolated in good yield by DEAE-cellulose chromatography, and good crystals were produced by the hanging drop vapor diffusion method. The space group was P21 with cell dimensions, a = 64.2, b = 90.0, c = 42.3 A, and beta = 96.7 degrees. These crystals diffract to about 2.2 A resolution. The structure of Fab HED10 was solved by the molecular replacement method using the known structure of McPC603 and is refined to R = 27.2% at 3.0 A resolution. Fab HED10 is more extended than McPC603 and has an elbow angle (between the variable and constant domains) of 162 degrees, very similar to that observed in Fab KOL. The majority of the hypervariable regions are visible in the model.     

Novel arrangement of immunoglobulin variable domains: X-ray crystallographic analysis of the lambda-chain dimer Bence-Jones protein Loc

We have characterized and crystallized a human lambda I light-chain dimer, Bence-Jones protein Loc, which has variable (V) region antigenic determinants characteristic for the lambda I subgroup and constant (C) region determinants of the C lambda I gene Mcg. The crystal structure was determined to 3-A resolution; the R factor is 0.27. The angle formed by the twofold axes of the V and C domains, the "elbow bend", is 97 degrees, the smallest found so far for an antibody fragment. The antigen-binding site formed by the two V domains of the Loc light chain differs significantly from those of other immunoglobulin molecules (light-chain dimers and Fab fragments) for which X-ray crystallographic data are available. Whereas, in other antibody fragments, the V domains are related by a local twofold axis, a local twofold screw axis with a translational component of 3.5 A relates the V domains in protein Loc. In contrast to the classic antigen binding "pocket" formed by V domain interactions in the previously characterized antibody structures, the V region associations in protein Loc result in a central protrusion in the binding site, with grooves on two sides of the protrusion. The structure of protein Loc indicates that immunoglobulins are physically capable of forming a more diverse spectrum of antigen-binding sites than has been heretofore apparent. Moreover, the unusual protruding nature of the binding site may be analogous to structures required for some anti-idiotypic antibodies. Further, the complementarity-determining residues form parts of two independent grooves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular modeling of antibody-antigen complexes between the Brucella abortus O-chain polysaccharide and a specific monoclonal antibody.

A molecular model of the binding site of an anti-carbohydrate antibody (YsT9.1) has been developed using computer-assisted modeling techniques and molecular dynamics calculations. Sequence homologies among YsT9.1 and the Fv regions of McPC603, J539 and human Bence--Jones protein REI, all of which have solved crystal structures, provided the basis for the modeling. The groove-type combining site model had a topography which was complementary to low energy conformers of the polysaccharide, a Brucella O-antigen, and the site could be almost completely filled by a pentasaccharide epitope in either of two docking modes. Putative interactions between this epitope and the antibody are consistent with the known structural requirements for binding and lead to the design of oligosaccharide inhibitors that probe the veracity of the modeled docked complex. Ultimately both the Fv model and the docked complex will be compared with independent crystal structures of YsT9.1 Fab with and without pentasaccharide inhibitor, currently at the stage of refinement.     

Secretion and in vivo folding of the Fab fragment of the antibody McPC603 in Escherichia coli: influence of disulphides and cis-prolines.

Using the well-characterized antibody McPC603 as a model, we had found that the Fv fragment can be isolated from Escherichia coli as a functional protein in good yields, whereas the amount of the correctly folded Fab fragment of the same antibody produced under identical conditions is significantly lower. In this paper, we analyse the reasons for this difference. We found that a variety of signal sequences function in the secretion of the isolated chains of the Fab fragment or in the co-secretion of both chains in E.coli. The low yield of functional Fab fragment is not caused by inefficient expression or secretion in E.coli, but by inefficient folding and/or assembly in the periplasm. We compared the folding yields for the Fv and the Fab fragment in the periplasm under various conditions. Several diagnostic framework variants were constructed and their folding yields measured. The results show that substitutions affecting cis-proline residues and those affecting various disulphide bonds in the protein are by themselves insufficient to dramatically change the partitioning of the folding pathway to the native structure, and the cause must lie in a facile aggregation of folding intermediates common to all structural variants. However, all structural variants could be obtained in native form, demonstrating the general utility of the secretory expression strategy.     

Crystal structure of an in vitro affinity- and specificity-matured anti-testosterone Fab in complex with testosterone. Improved affinity results from small structural changes within the variable domains

A highly selective, high affinity recombinant anti-testosterone Fab fragment has been generated by stepwise optimization of the complementarity-determining regions (CDRs) by random mutagenesis and phage display selection of a monoclonal antibody (3-C(4)F(5)). The best mutant (77 Fab) was obtained by evaluating the additivity effects of different independently selected CDR mutations. The 77 Fab contains 20 mutations and has about 40-fold increased affinity (K(d) = 3 x 10(-10) m) when compared with the wild-type (3-C(4)F(5)) Fab. To obtain structural insight into factors, which are needed to improve binding properties, we have determined the crystal structures of the mutant 77 Fab fragment with (2.15 A) and without testosterone (2.10 A) and compared these with previously determined wild-type structures. The overall testosterone binding of the 77 Fab is similar to that of the wild-type. The improved affinity and specificity of the 77 Fab fragment are due to more comprehensive packing of the testosterone with the protein, which is the result of small structural changes within the variable domains. Only one important binding site residue Glu-95 of the heavy chain CDR3 is mutated to alanine in the 77 Fab fragment. This mutation, originally selected from the phage library based on improved specificity, provides more free space for the testosterone D-ring. The light chain CDR1 of 77 Fab containing eight mutations has the most significant effect on the improved affinity, although it has no direct contact with the testosterone. The mutations of CDR-L1 cause a rearrangement in its conformation, leading to an overall fine reshaping of the binding site.     

The scFv fragment of the antibody hu4D5-8: evidence for early premature domain interaction in refolding

Fluorescence spectroscopy and 1H/2H-exchange techniques have been applied to characterize the folding of an scFv fragment, derived from the humanized anti-HER2 antibody hu4D5-8. A stable intermediate, consisting of a native VL domain and an unfolded VH domain, is populated under equilibrium unfolding conditions. A partially structured intermediate, with 1H/2H-exchange protection significantly less than that of the two isolated domains together, is detectable upon refolding the equilibrium-denatured scFv fragment. This means that the domains in the heterodimer do not fold independently. Rather, they associate prematurely before full 1H/2H-exchange protection can be gained. The formation of the native heterodimer from the non-native intermediate is a slow, cooperative process, which is rate-limited by proline cis/trans-isomerization. Unproductive domain association is also detectable after short-term denaturation, i.e. with the proline residues in native conformation. Only a fraction of the short-term denatured protein folds into the native protein in a fast, proline-independent reaction, because of spontaneous proline cis/trans-reisomerization in the early non-native intermediate. The comparison with the previously studied antibody McPC603 has now allowed us to delineate similarities in the refolding pathway of scFv fragments.     

Structural consequences of target epitope-directed functional alteration of an antibody. The case of anti-hen lysozyme antibody,HyHEL-10

Decreased affinity of an antibody for a mutated epitope in an antigen can be enhanced and reversed by mutations in certain antibody residues. Here we describe the crystal structures of (a) the complex between a naturally mutated proteinaceous antigen and an antibody that was mutated and selected in vitro, and (b) the complex between the normal antigen and the mutated antibody. The mutated and selected antibody recognizes essentially the same epitope as in the wild-type antibody, indicating successful target site-directed functional alteration of the antibody. In comparing the structure of the mutated antigen-mutant antibody complex with the previously established structure of the wild-type antigen-wild-type antibody complex, we found that the enhanced affinity of the mutated antibody for the mutant antigen originated not from improvements in local complementarity around the mutated sites but from subtle and critical structural changes in nonmutated sites, including an increase in variable domain interactions. Our findings indicate that only a few mutations in the antigen-binding region of an antibody can lead to some structural changes in its paratopes, emphasizing the critical roles of the plasticity of loops in the complementarity-determining region and also the importance of the plasticity of the interaction between the variable regions of immunoglobulin heavy and light chains in determining the specificity of an antibody.