The structure of the complex between hen egg-white lysozyme and the Fab HyHEL-5 at 2.7 A resolution has previously been reported [Cohen et al. (1996), Acta Cryst. D52, 315-326]. With the availability of recombinant Fab, the X-ray structure of the complex has been re-evaluated at 1.7 A resolution. The refined structure has yielded a detailed picture of the Fab-lysozyme interface, showing the high complementarity of the protein surfaces as well as several water molecules within the interface that complete the good fit. The model of the full complex has improved significantly, yielding an R(work) of 19.5%. With this model, the structural results can be compared with the results of isothermal titration calorimetry. An attempt has been made to estimate the changes in bound waters that accompany complex formation and the difficulties inherent in using the crystal structures to provide the information necessary to make this calculation are discussed.

We have determined the three-dimensional structure oftwo crystal forms ofan antilysozyme Fab-lysozyme complex by x-ray crystallography. The epitope on lysozyme consists of three sequentially separated subsites, including one long, nearly continuous, site from Gln-41 through Tyr-53 and one from Gly-67 through Pro-70. Antibody residues interacting with lysozyme occur in each of the six complementaritydetermining regions and also include one framework residue. Arg-45 and Arg-68 form a ridge on the surface of lysozyme, which binds in a groove on the antibody surface. Otherwise the surface of interaction between the two proteins is relatively flat, although it curls at the edges. The surface of interaction is approximately 26 x 19 A. No water molecules are found in the interface. The positive charge on the two arginines is complemented by the negative charge of Glu-35 and Glu-50 from the heavy chain of the antibody. The backbone structure of the antigen, lysozyme, is mostly unperturbed, although there are some changes in the epitope region, most notably Pro-70. One side chain not in the epitope, Trp-63, undergoes a rotation of -180° about the CO-CY bond. The Fab elbow bends in the two crystal forms differ by 7°. Until recently knowledge of the structural aspects of antibody-antigen interactions has been based on the x-ray analysis of four Fab structures and on some complexes with hapten (1-5). Haptens were observed to bind in grooves or pockets in the combining sites of the New and McPC603 Fabs, and these occupied a small fraction of the total available area of these sites. When haptens bind to these Fabs, no large conformational change occurs. However, one cannot rule out the possibility that the behavior of antibodies would be different when they are bound to larger antigens, such as proteins. For example, the interaction with a much greater fraction of the combining site might in itself be sufficient to induce conformational changes in the antibody. Also, the interacting surfaces might not possess the grooves and pockets observed for haptens, but might resemble more closely the kind of surface observed in other protein-protein interfaces, where exclusion ofbound water is believed to play a key role. For this reason we undertook several years ago to investigate the crystal structures of complexes of the Fabs of several monoclonal antibodies to hen egg white lysozyme complexed with the lysozyme (6). In this paper we report the analysis of two different crystal forms of one of these

The search for a malaria vaccine has been directed mainly towards combating Plasmodium falciparum, the species responsible for most of the morbidity and mortality arising from the disease in man.Proteins exposed on the surface of the Plasmodium merozoite, the erythrocyte-invasive form of the parasite, are prime candidates for vaccine development since many of these proteins are targets of the humoral immune response from persons with naturally acquired immunity.The most abundant surface protein of P. Falciparum is Merozoite Surface Protein 1 (PfMSP1), a 195 kDa molecule attached to the membrane by a glycosyl-phosphatidylinisotol (GPI) anchor.The protein undergoes proteolytic maturation that ultimately leaves only the C-terminal 11 kDa segment, PfMSP1-19, attached by the GPI moiety to the surface of the invading parasite.Vaccination trials in animal model systems have shown that immunisation with PfMSP1-19 can lead to protective immunity.These observations, and the limited polymorphism of PfMSP1-19 (less than 5% between different strains), have made this naturally occurring polypeptide fragment a leading vaccine candidate.In order to gain further understanding of the nature of immune protection given by this antigen, we are engaged in structural studies of PfMSP1-19 and its complexes with specific monoclonal antibodies (mAb).Here, we report the crystal structure of the complex formed between PfMSP1-19 and the Fab fragment of the murine monoclonal antibody G17.12, determined by molecular replacement and refined at 2.9 Å resolution.The mAb recognised a discontinuous epitope on the first EGF domain of PfMSP1-19.

MFE-23 is a single-chain Fv (scFv) antibody ti•agment that was selected from a phage-display library for high affinity binding to carcinoembryonic antigen (CEA), and has important clinical uses in the detection, monitming and targetting of colon cancer.CEA is a seven-domain cell adhesion molecule which is expressed at high levels on colon carcinoma cells.150: 256 residues) consists of anN-terminal VH domain, a IS-residue linker, a VL domain and a 12-residue tag.MFE-23 was expressed in E. coli and purified on CEA coupled to Sepharose.Cr-ystals were produced by the hanging-drop method.MFE-23 at 2 mg/ml was mixed 1:1 with precipitant (100 mM Tris-HCI at pH 6.5 containing 45% saturated ammonium sulfate), and a 10 ~ll drop of this mixture was equilibrated against 0.5 ml of precipitant at l8°C.Well-fom1ed crystals fom1ed within several days, and diffracted to 0.2 nm resolution.Diffraction data were collected using an R-A.'CIS-ITC mounted on an RU200 rotating anode X -ray source.The crystal belongs to the trigonal space group P3221 and has unit cell dimensions of a= b = 6.170 nm and c = J 2.794 nm.A single crystal was used for structure detennination.Data to a resolution of 0.28 nm were processed using DENZO and the CCP4 program package.The structure was solved by molecular replacement using the Fv coordinates from a mmine IgA Fab fragment (Brookhaven code 2FBJ).The antibody complementarity determining regions, the linker and the tag were omitted for the calculation of initial 2Fo-Fc electron density maps.Refinement of the MFE-23 structure is cunently in progress, utilizing the rigid-body refinement and the positional refinement algorithms ofX-PLOR ar1d manual rebuilding of the model in 0. PS04.15.12 ARE "HOT SPOTS" [SUPER- FLEXIBLE REGIONS]

The structure of the complex between the Fab HyHEL-5 and chicken lysozyme revealed a large interface region containing 23 lysozyme and 28 Fab residues. Arg68 of the lysozyme is centrally placed in this interface and theoretical studies together with binding assays of this Fab to different avian lysozymes have previously shown that this arginine residue is an important contributor to the binding. The Arg68-->Lys mutant binds 10(3) times less well to the HyHEL-5 Fab. We have examined the refined crystal structure of the complex of this mutant lysozyme with the Fab. No global changes occur, but there is an introduction of a new water molecule into the interface that mediates the hydrogen bonding interactions between the lysine and residues on the Fab. These data are compared with the effects of similar changes on the inhibition of serine proteases such as trypsin where the energetic effects of this substitution are small.

We have analyzed the nature of the CH1 : CL interaction to see how it can be used to permit unrestricted association of the different heavy and light chain classes. A cavity has been observed in the interface between the two domains. When Cλ is taken from association with Cγ and combined with Cα a conformational adjustment is required that moves a bulky side chain into this cavity.

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.

A single-stage computer procedure to calculate an electron density map suitable to detect errors in a tentative macromolecular model has been developed. In this procedure, an atom of the tentative model does not contribute to the phases used to calculate electron density values at or near its current position, that is within the region containing it and a neighborhood surrounding that region. In this way, the phases used to calculate electron density values within a region are not biased by the model atoms contained within that region or its neighborhood. The number of atoms which are omitted for a given region is maintained at a small fraction of the total structure so that the phases used to calculate electron density values may still be a good approximation to the phases of the complete structure. The procedure was used to improve the model of the Fab portion of the mouse galactan-binding immunoglobulin J539 (IgA2, κ), which contains 431 residues.

The crystal structure of γ-chymotrypsin, the monomeric form of chymotrypsin, has been determined and refined to a crystallographic R-factor of 0.18 at 1.9 Å resolution. The details of the catalytic triad involving Asp102, His57 and Ser195 agree well with the results found for trypsin (Chambers & Stroud, 1979) and Streptomyces griseus protease A (Sielecki et al., 1979). As in many of the other serine proteases, the Oγ of Ser195 does not appear to be hydrogen-bonded to His57.

X-ray diffraction patterns of fibers of polyriboguanylic acid and polyriboinosinic acid are shown to be virtually identical. These diffraction patterns are consistent only with three or four-stranded models. Model-building studies on a computer-assisted interactive display system favor the four-stranded model. In addition, the greater thermal stability of poly(rG) relative to poly(rI) can be accounted for by a four-stranded model in which there are two hydrogen bonds per base for poly(rG) versus one for poly(rI).

The structure of the Fab of McPC 603, a mouse myeloma protein with phosphorylcholine binding activity, has been determined to 3.1-A resoltuion. The four domains are found to be structurally similar with a well-defined double-layer structure. A large cavity exists at one end of the fragment, the walls of which are formed exclusively of hypervariable residues. Phosphorylcholine binds in this cavity and forms specific interactions with several well-defined amino-acid side chains of the protein. The hapten is bound asymmetrically and interacts more with the heavy chain than with the light chain.

The structure of the Fab fragment of McPC 603 protein has been determined by crystallographic analysis to 3.1 å resolution. The site of phosphorylcholine binding was located in a cleft formed by the three heavy chain hypervariable loops and the first and third hypervariable regions of the light chain. There are strong interactions between the phosphate group of the hapten and the side groups of Tyr 33 and Arg 52 both of the heavy chain. Acidic side groups from both light and heavy chains are in the immediate vicinity of the positively charged choline group. The hapten is in van der Waals contact with various portions of the hypervariable loops. Phosphorylcholine occupies only a small portion of the cleft between the variable domains.

The criteria for elution of proteins from hydroxyapatite columns were examined as a function of (1) protein isoelectric point (22 proteins with isoelectric points between 3.5 and 11.0); (2) ionic nature of eluant (Na salts of PO4, F−, Cl−, SCN−, ClO4−, and CaCl2); and (3) structural differences between related proteins. It was found that proteins can be classified into three groups: (1) basic proteins, which elute at similar, moderate molarities of PO4, F−, Cl−, SCN−, and ClO4−, and low (<0.003 m) Ca2+; (2) acidic proteins which elute at about equal moderate molarities of PO4 and F−, but do not elute with Ca2+ and usually not with Cl−; (3) neutral proteins, which elute with PO4, F−, and Cl−, but show a strong anion specificity, and do not elute with Ca2+ or SCN−. Furthermore, individual specific polar groups are not in general crucial to binding or desorption, and variations in structure, other than major loosening, do not influence strongly the pattern of protein-hydroxyapatite interaction.