Crystal structure of a Fab complex formed with PfMSP1-19, the C-terminal fragment of merozoite surface protein 1 from Plasmodium falciparum: a malaria vaccine candidate.

Merozoite surface protein 1 (MSP1) is the major protein component on the surface of the merozoite, the erythrocyte-invasive form of the malaria parasite Plasmodium. Present in all species of Plasmodium, it undergoes two distinct proteolytic maturation steps during the course of merozoite development that are essential for invasion of the erythrocyte. Antibodies specific for the C-terminal maturation product, MSP1-19, can inhibit erythrocyte invasion and parasite growth. This polypeptide is therefore considered to be one of the more promising malaria vaccine candidates. We describe here the crystal structure of recombinant MSP1-19 from P.falciparum (PfMSP1-19), the most virulent species of the parasite in humans, as a complex with the Fab fragment of the monoclonal antibody G17.12. This antibody recognises a discontinuous epitope comprising 13 residues on the first epidermal growth factor (EGF)-like domain of PfMSP1-19. Although G17.12 was raised against the recombinant antigen expressed in an insect cell/baculovirus system, it binds uniformly to the surface of merozoites from the late schizont stage, showing that the cognate epitope is exposed on the naturally occurring MSP1 polypeptide complex. Although the epitope includes residues that have been mapped to regions recognised by invasion-inhibiting antibodies studied by other workers, G17.12 does not inhibit erythrocyte invasion or MSP1 processing.

Inhibition of HIV protease by monoclonal antibodies.

The protease of HIV plays a critical role in the maturation of the infectious particles of the virus. The enzyme has therefore been extensively studied with the objective of developing therapeutics that inhibit viral proliferation. We have produced monoclonal antibodies specific for the HIV-1 protease, and selected those that inhibit enzyme function for use as probes to study the enzyme's activity and as an eventual aid for the development of potential inhibitors targeted to regions other than the active site. We have characterized two such mAbs, F11.2.32 and 1696, which have inhibition constants in the low nanomolar range and which recognize epitopes from different regions of the protease. The crystal structures of the two antibodies, both in the free state as well as complexes with peptide fragments corresponding to their respective epitopes, have been solved. The structural analyses, taken together with other functional data on the antibodies, suggest mechanisms of protease inhibition by these antibodies.

Crystallization and preliminary structural analysis of an antibody complex formed with PfMSP1-19, a malaria vaccine candidate.

The 11 kDa C-terminal fragment of the proteolyticly matured surface antigen, PfMSP1, from Plasmodium falciparum is a promising malaria vaccine candidate. The soluble recombinant form of this naturally occurring fragment has been crystallized as a complex with the Fab of a specific murine monoclonal antibody. The crystals belong to the space group P2(1), with unit-cell parameters a = 51.8, b = 213.5,c = 60.0 A, beta =101.0 degrees, and with Z = 4. Diffraction data have been measured to 2.9 A resolution and a preliminary model of the complex has been determined by molecular replacement. The epitope recognised by G17.12 is located on the N-terminal EGF-like domain of the antigen.

The crystal structure of C-terminal merozoite surface protein 1 at 1.8 A resolution, a highly protective malaria vaccine candidate.

The C-terminal proteolytic processing product of merozoite surface protein 1 (MSP1) appears essential for successful erythrocyte invasion by the malarial parasite, Plasmodium. We have determined the crystal structure at 1.8 A resolution of a soluble baculovirus-recombinant form of the protein from P. cynomolgi, which confers excellent protective efficacy in primate vaccination trials. The structure comprises two EGF-like domains, and sequence comparisons strongly suggest that the same conformation is present in all species of Plasmodium, including P. falciparum and P. vivax, which are pathogenic in man. In particular, conserved interdomain contacts between the two EGF modules should preserve the compact form of the molecule in all species. Implications of the crystal structure for anti-malarial vaccine development are discussed.

Inhibition of the HIV-1 and HIV-2 proteases by a monoclonal antibody.

The monoclonal antibody 1696, directed against the HIV-1 protease, displays strong inhibitory effects toward the catalytic activity of the enzyme of both the HIV-1 and HIV-2 isolates. This antibody cross-reacts with peptides that include the N-terminus of the enzyme, a region that is well conserved in sequence among different viral strains and which, furthermore, is crucial for homodimerization to the active enzymatic form. This observation, as well as antigen-binding studies in the presence of an active site inhibitor, suggest that 1696 inhibits the HIV protease by destabilizing its active homodimeric form. To characterize further how the antibody 1696 inhibits the HIV-1 and HIV-2 proteases, we have solved the crystal structure of its Fab fragment by molecular replacement and refined it at 3.0 A resolution. The antigen binding site has a deep cavity at its center, which is lined mainly by acidic and hydrophobic residues, and is large enough to accommodate several antigen residues. The structure of the Fab 1696 could form a starting basis for the design of alternative HIV protease-inhibiting molecules of broad specificity.

Three-dimensional structure of an Fab-peptide complex: structural basis of HIV-1 protease inhibition by a monoclonal antibody.

F11.2.32, a monoclonal antibody raised against HIV-1 protease (Kd = 5 nM), which inhibits proteolytic activity of the enzyme (K(inh) = 35(+/-3)nM), has been studied by crystallographic methods. The three-dimensional structure of the complex between the Fab fragment and a synthetic peptide, spanning residues 36 to 46 of the protease, has been determined at 2.2 A resolution, and that of the Fab in the free state has been determined at 2.6 A resolution. The refined model of the complex reveals ten well-ordered residues of the peptide (P36 to P45) bound in a hydrophobic cavity at the centre of the antigen-binding site. The peptide adopts a beta hairpin-like structure in which residues P38 to P42 form a type II beta-turn conformation. An intermolecular antiparallel beta-sheet is formed between the peptide and the CDR3-H loop of the antibody; additional polar interactions occur between main-chain atoms of the peptide and hydroxyl groups from tyrosine residues protruding from CDR1-L and CDR3-H. Three water molecules, located at the antigen-antibody interface, mediate polar interactions between the peptide and the most buried hypervariable loops, CDR3-L and CDR1-H. A comparison between the free and complexed Fab fragments shows that significant conformational changes occur in the long hypervariable regions, CDR1-L and CDR3-H, upon binding the peptide. The conformation of the bound peptide, which shows no overall structural similarity to the corresponding segment in HIV-1 protease, suggests that F11.2.32 might inhibit proteolysis by distorting the native structure of the enzyme.

Preliminary crystallographic studies of an anti-HIV-1 protease antibody that inhibits enzyme activity.

F11.2.32, a monoclonal antibody directed against the HIV-1 protease, displays strong inhibitory effects toward the catalytic activity of the enzyme. The antibody cross-reacts with peptides 36-46 and 36-57 from the protease. Crystals of the Fab have been obtained both in the free state and as complexes formed with the protease peptide fragments, 36-46 and 36-57. Diffraction data have been collected for the free and complexed forms of Fab F11.2.32 and preliminary models for the crystal structures were obtained by molecular replacement.

Multiple crystal forms of endoglucanase CelD: signal peptide residues modulate lattice formation.

The crystal structure of Clostridium thermocellum endoglucanase CelD revealed an extended NH2-terminal segment (involving residues from the putative leader peptide) sticking out from the enzyme core to interact with a symmetry related molecule through an intermolecular salt bridge (Lys38-Asp201). Enzymatic digestion of CelD with various proteases emphasized the flexibility of the NH2-segment in solution. Proteolytic removal of Lys38 or the substitution of bridge-forming residues by site-directed mutagenesis promoted crystal packing arrangements that differ from that of wild type CelD. Crystals of wild-type CelD (a = 99.3 A c = 191.8 A) are trigonal, space group P3(1)21, with one molecule in the asymmetric unit (form A), whereas crystals of papain-treated CelD (a = 100.4 A, c = 248.7 A), of CelDK38M (a = 100.1 A, c = 248.4 A) and of papain-treated CelDD201A (a = 99.9 A, c = 250.0 A) are trigonal, space group P3(1)21, with two crystallographically independent molecules (form B), and crystals of chymotrypsin-treated CelD (a = 100.0 A, c = 254.3 A) and of CelDD201A (a = 99.8 A, c = 254.7 A) are hexagonal, space group P6(1)22, with one molecule in the asymmetric unit (form C). Only chymotrypsin-treated CelD (which preserves both Lys38 and Asp201) can grow in crystal form A upon macroseeding, indicating that formation of the intermolecular salt bridge is critical for stability of this crystal form. Flexible NH2- and COOH-terminal peptide extensions were found to influence crystal nucleation, but not crystal growth. The crystal structures of papain-treated CelD and chymotrypsin-treated CelD, determined at 3.5 A resolution by molecular replacement techniques, demonstrate that a small change in molecular orientation promoted by Lys38 account for the differences between crystal forms B and C.

Three-dimensional structure of a heteroclitic antigen-antibody cross-reaction complex.

Although antibodies are highly specific, cross-reactions are frequently observed. To understand the molecular basis of this phenomenon, we studied the anti-hen egg lysozyme (HEL) monoclonal antibody (mAb) D11.15, which cross-reacts with several avian lysozymes, in some cases with a higher affinity (heteroclitic binding) than for HEL. We have determined the crystal structure of the Fv fragment of D11.15 complexed with pheasant egg lysozyme (PHL). In addition, we have determined the structure of PHL, Guinea fowl egg lysozyme, and Japanese quail egg lysozyme. Differences in the affinity of D11.15 for the lysozymes appear to result from sequence substitutions in these antigens at the interface with the antibody. More generally, cross-reactivity is seen to require a stereochemically permissive environment for the variant antigen residues at the antibody-antigen interface.

Crystallization, preliminary X-ray diffraction study, and crystal packing of a complex between anti-hen lysozyme antibody F9.13.7 and guinea-fowl lysozyme.

The complex formed between the Fab fragment of a murine monoclonal antihen egg lysozyme antibody F9.13.7 and the heterologous antigen Guinea-fowl egg lysozyme has been crystallized by the hanging drop technique. The crystals, which diffract X-rays to 3 A resolution, belong to the monoclinic space group P2(1), with a = 83.7 A, b = 195.5 A, c = 50.2 A, beta = 108.5 degrees and have two molecules of the complex in the asymmetric unit. The three-dimensional structure has been determined from a preliminary data set to 4 A using molecular replacement techniques. The lysozyme-Fab complexes are arranged with their long molecular axes approximately parallel to the crystallographic unique axis. Fab F9.13.7 binds an antigenic determinant that partially overlaps the epitope recognized by antilysozyme antibody HyHEL10.

Studies of structure and specificity of some antigen-antibody complexes.

By using X-ray diffraction and immunochemical techniques, we have exploited the use of monoclonal antibodies raised against hen egg lysozyme (HEL) to study systematically those factors responsible for the high specificity of antigen-antibody interactions. HEL was chosen for our investigations because its three-dimensional structure and immunochemistry have been well characterized and because naturally occurring sequence variants from different avian species are readily available to test the fine specificity of the antibodies. The X-ray crystal structure of a complex formed between HEL and the Fab D1.3 shows a large complementary surface with close interatomic contacts between antigen and antibody. Thus single amino acid sequence changes in heterologous antigens give antigen-antibody association constants that are several orders of magnitude smaller than that of the homologous antigen. For example, a substitution of His for Glu at position 121 in the antigen is sufficient to diminish significantly the binding between D1.3 and the variant lysozyme. The conformation of HEL when complexed to D1.3 shows no significant difference from that seen in the free molecule, and immunobinding studies with other anti-HEL antibodies suggest that this observation may be generally true for the system of monoclonal antibodies that we have studied.