Roberto J. Poljak

The three-dimensional structure of the aspartyl protease from the HIV-1 isolate BRU

The crystal structure of the aspartyl protease encoded by the gene pol of the human immunodeficiency virus (HIV-1, isolate BRU) has been determined to 2.7 A resolution. The enzyme, expressed as an insoluble denatured polypeptide in inclusion bodies of Escherichia coli has been renatured and crystallized. It differs by several amino acid replacements from the homologous enzymes of other HIV-1 isolates. A superposition of the C alpha-backbone of the BRU protease with that of the SF2 protease gives a roots mean square positional difference of 0.45 A. Thus, neither the denaturation/renaturation process nor the amino acid replacements have a noticeable effect on the three-dimensional structure of the BRU protease or on the detailed conformation of the catalytic site, which is very similar to that of other aspartyl proteases.

Three-dimensional structures of the free and the antigen-complexed Fab from monoclonal anti-lysozyme antibody D44.1.

The three-dimensional structures of the free and antigen-complexed Fabs from the mouse monoclonal anti-hen egg white lysozyme antibody D44.1 have been solved and refined by X-ray crystallographic techniques. The crystals of the free and lysozyme-bound Fabs were grown under identical conditions and their X-ray diffraction data were collected to 2.1 and 2.5 A, respectively. Two molecules of the Fab-lysozyme complex in the asymmetric unit of the crystals show nearly identical conformations and thus confirm the essential structural features of the antigen-antibody interface. Three buried water molecules enhance the surface complementarity at the interface and provide hydrogen bonds to stabilize the complex. Two hydrophobic buried holes are present at the interface which, although large enough to accommodate solvent molecules, are void. The combining site residues of the complexed FabD44.1 exhibit reduced temperature factors compared with those of the free Fab. Furthermore, small perturbations in atomic positions and rearrangements of side-chains at the combining site, and a relative rearrangement of the variable domains of the light (VL) and the heavy (VH) chains, detail a Fab accommodation of the bound lysozyme. The amino acid sequence of the VH domain, as well as the epitope of lysozyme recognized by D44.1 are very close to those previously reported for the monoclonal antibody HyHEL-5. A feature central to the FabD44.1 and FabHyHEL-5 complexes with lysozyme are three salt bridges between VH glutamate residues 35 and 50 and lysozyme arginine residues 45 and 68. The presence of the three salt bridges in the D44.1-lysozyme interface indicates that these bonds are not responsible for the 1000-fold increase in affinity for lysozyme that HyHEL-5 exhibits relative to D44.1.

Antigen specificity and cross-reactivity of monoclonal anti-lysozyme antibodies

Twenty-seven murine monoclonal anti-hen egg-white lysozyme (HEL) antibodies were tested with a panel of nine antigens; eight avian lysozymes and human lysozyme. The antibodies were arranged into 10 groups based on their antigen specificity and cross-reactivity. Antigenic determinants recognized by each group of antibodies were tentatively identified. They are located at different points of the HEL accessible surface in agreement with the notion that its entire surface has an antigenic potential. The affinity constants of antibodies representative of seven of the 10 groups range from 0.79 X 10(7) to 5.3 X 10(7) M-1. Heteroclitic antibodies occurring in some of the groups bind heterologous lysozymes with somewhat higher association constants than those for the homologous antigen (HEL). Their broader specificities do not correlate with overall lower association constants, but rather with the occurrence of public epitopes in the panel of avian lysozymes. Although the reaction of two antibodies with the solid-phase coupled antigen is not always additive, detailed interpretation of these results and the observation of actual ternary complexes Fab-HEL-Fab rules out the occurrence of conformational changes in the complexed antigen.

Production and structure of diabodies.

The first crystal structure of a diabody, a bivalent antibody fragment, confirms previous predicted structures and techniques for generating bispecific bivalent antibody fragments of considerable therapeutic potential.

Anatomy of an antibody molecule: structure, kinetics, thermodynamics and mutational studies of the antilysozyme antibody D1.3

Summary: Using site‐directed mutagenesis, x‐ray crystallograpby, micro‐calorimetric, equilibrium sedimentation and surface plasmon resonance detection techniques, we have examined the structure of an antibody‐antigen complex and (he structural and thermodynamic consequences of removing specific hydrogen bonds and van der Waals interactions in the antibody‐antigen interface. These observations show that the complex is considerably tolerant, both structurally and thermodynamically, to the truncation of antibody and antigen side chains that form contacts. Alterations ill interface solvent structure for two of the mutant complexes appear to compensate for the unfavorable enthalpy changes when antibody‐antigen interactions are removed. These changes in solvent structure, along with the increased mobility of side chains near the mutation site, probably contribute to the observed entropy compensation. In concert, data from structural studies, reaction rates, calorimetric measurements and site‐directed mutations are beginning to detail the nature of antibody‐protein antigen interactions.

Crystallization and preliminary X-ray diffraction study of the bacterially expressed Fv from the monoclonal anti-lysozyme antibody D1.3 and of its complex with the antigen, lysozyme.

The associated heavy (VH) and light (VL) chain variable domains (Fv) of the monoclonal anti-lysozyme antibody D1.3, secreted from Escherichia coli, have been crystallized in their antigen-bound and free forms. FvD1.3 gives tetragonal crystals, space group P4(1)2(1)2 (or P4(3)2(1)2), with a = 90.6 A, c = 56.4 A. The FvD1.3-lysozyme complex crystallizes in space group C2, with a = 129.2 A, b = 60.8 A, c = 56.9 A and beta = 119.3 degrees. The crystals contain one molecule of Fv or of the Fv-lysozyme complex in their asymmetric units and diffract X-rays to high resolution, making them suitable for X-ray crystallographic studies.

Structure of antibodies and their complexes with antigens

This is a narrative account and a personal recollection of some of the studies on the three-dimensional structure of antibodies conducted in my laboratory. It is not intended to be a critical or detailed account of developments in this field. In the 1950s and subsequently, research on the molecular basis of immune responses and their diversity attracted the attention of many molecular biologists, immunologists and protein chemists. The observations that stimulated my first attempts in this fascinating field were: (1) the classical demonstration by Rodney Porter (1959) that polyclonal rabbit antibodies could be cleaved into Fab and Fc fragments of which the Fc could be readily crystallized; (2) the demonstration (Hill et al., 1966) of internal homologies in immunoglobulin sequences indicating gene duplication events and suggesting the presence of discrete three-dimensional domains in antibody molecules. It is perhaps unnecessary to state here that in the 1960s protein crystallography had not reached the high level of automation that we know today. In addition, monoclonal myeloma proteins were not universally accepted as good models of antibodies. The first analysis of immunoglobulins and their fragments by X-ray diffraction in my laboratory were done on the crystalline Fc fragments from rabbit antibodies and from human myeloma proteins. When the Fab and Fab’ from human immunoglobulins were first crystallized by Rossi and Nisonoff (1968) and subsequently analysed by us by X-ray diffraction (Avey et al., 1968; Humphrey et al., 1969) it became clear that they were highly ordered and better suited for X-ray analysis than the Fc crystals. Since then, our efforts have been almost exclusively directed to the study of Fabs, since these fragments retain the antigen-binding properties of the antibody molecules from which they are derived.

Crystallization and preliminary X-ray diffraction studies of two new antigen-antibody (lysozyme-Fab) complexes☆

The complexes between the Fab fragments of two monoclonal anti-lysozyme antibodies, Fab10.6.6 (high affinity) and D44.2 (lower affinity), and their specific antigen, hen egg-white lysozyme, have been crystallized. The antibodies recognize an antigenic determinant including Arg68, but differ significantly in their association constants for the antigen. Two crystalline forms were obtained for the complex with FabF10.6.6, the higher affinity antibody. One of them is monoclinic, space group P21, with unit cell dimensions a = 145.6 A, b = 78.1 A, c = 63.1 A, beta = 89.05 degrees, consistent with the presence of two molecules of the complex in the asymmetric unit. These crystals diffract X-rays beyond 3 A making this form suitable for high-resolution X-ray diffraction studies. The second form crystallizes in the triclinic space group P1, with unit cell dimensions a = 134.0 A, b = 144.7 A, c = 98.6 A, alpha = 90.30 degrees, beta = 97.1 degrees, gamma = 90.20 degrees, consistent with the presence of 10 to 12 molecules of the complex in the unit cell. These crystals do not diffract X-rays beyond 5 A resolution. The antigen-antibody complex between FabD44.2, the lower affinity antibody, and hen egg-white lysozyme crystallizes in space group P2(1)2(1)2(1), with unit cell dimensions a = 99.7 A, b = 167.3 A, c = 84.7 A, consistent with the presence of two molecules of the complex in the asymmetric unit. These crystals diffract X-rays beyond 2.5 A resolution.

Crystallization and preliminary X-ray diffraction study of an endoglucanase from Clostridium thermocellum

Endoglucanase D, a cellulose degradation enzyme from Clostridium thermocellum has been cloned in Escherichia coli, purified and crystallized. The crystals are trigonal, space group P3(1)12 (or P3(2)12) with a = 57.7 (+/- 0.1) A, c = 192.1 (+/- 0.2) A, and diffract X-rays to a resolution of 2.8 A. They are suitable for a high-resolution X-ray diffraction analysis.

Preliminary crystallographic study of a complex between the Fab fragment of a monoclonal anti-lysozyme antibody (D1.3) and the Fab fragment from an anti-idiotopic antibody against D1.3.

An anti-lysozyme antibody, D1.3, was used as immunogen to obtain syngeneic (Balb/c) monoclonal anti-idiotopic antibodies. The complex between Fab D1.3 and the Fab fragment from the anti-idiotopic antibody E225 has been crystallized. The crystals are monoclinic, space group P2(1), with a = 75.7 A, b = 77.4 A, c = 97.2 A, beta = 111.90 degrees and one molecule of the complex in the asymmetric unit. X-ray photographs show reflections extending to a resolution of about 3 A. Although twinning occurs frequently in the large crystals obtained, this material is suitable for high-resolution X-ray analysis.