Hélène Souchon

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.

Use of site-directed mutagenesis to probe the structure, function and isoniazid activation of the catalase/peroxidase, KatG, from Mycobacterium tuberculosis

A series of mutants bearing single amino acid substitutions often encountered in the catalase/peroxidase, KatG, from isoniazid-resistant isolates of Mycobacterium tuberculosis has been produced by site-directed mutagenesis. The resultant enzymes were overexpressed, purified and characterized. Replacing Cys-20 by Ser abolished disulphide-bridge formation, but did not affect either dimerization of the enzyme or catalysis. The substitution of Thr-275, which is probably involved in electron transfer from the haem, by proline resulted in a highly unstable enzyme with insignificant enzyme activities. The most commonly occurring substitution in drug-resistant clinical isolates is the replacement of Ser-315 by Thr; this lowered catalase and peroxidase activities by 50% and caused a significant decrease in the KatG-mediated inhibition of the activity of the NADH-dependent enoyl-[acyl-carrier protein] reductase, InhA, in vitro. The ability of this enzyme to produce free radicals from isoniazid was severely impaired, as judged by its loss of NitroBlue Tetrazolium reduction activity. Replacement of Leu-587 by Pro resulted in marked instability of KatG, indicating that the C-terminal domain is also important for structural and functional integrity.

The crystal structure and catalytic mechanism of cellobiohydrolase CelS, the major enzymatic component of the Clostridium thermocellum Cellulosome.

Cellobiohydrolase CelS plays an important role in the cellulosome, an active cellulase system produced by the thermophilic anaerobe Clostridium thermocellum. The structures of the catalytic domain of CelS in complex with substrate (cellohexaose) and product (cellobiose) were determined at 2.5 and 2.4 A resolution, respectively. The protein folds into an (alpha/alpha)(6) barrel with a tunnel-shaped substrate-binding region. The conformation of the loops defining the tunnel is intrinsically stable in the absence of substrate, suggesting a model to account for the processive mode of action of family 48 cellobiohydrolases. Structural comparisons with other (alpha/alpha)(6) barrel glycosidases indicate that CelS and endoglucanase CelA, a sequence-unrelated family 8 glycosidase with a groove-shaped substrate-binding region, use the same catalytic machinery to hydrolyze the glycosidic linkage, despite a low sequence similarity and a different endo/exo mode of action. A remarkable feature of the mechanism is the absence, from CelS, of a carboxylic group acting as the base catalyst. The nearly identical arrangement of substrate and functionally important residues in the two active sites strongly suggests an evolutionary relationship between the cellobiohydrolase and endoglucanase families, which can therefore be classified into a new clan of glycoside hydrolases.

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.

Three-dimensional structure and antigen binding specificity of antibodies

A number of specific Fab and Fv fragments and their complexes with antigens (avian lysozymes), haptens, and anti-idiotopic Fabs have been studied by immunochemical and crystallographic techniques. Antigen and antibody interact through closely complementary contacting surfaces, without major conformational changes. An idiotopic determinant of a monoclonal antibody is shown to include parts of most of its complementarity determining regions. The specificity of antigen recognition resides in the close complementarity of the antigenic determinant with the antibody combining site.

Crystallization of antibody fragments and their complexes with antigen

Abstract Immunoglobulins, myeloma light chains and their fragments, and Fab fragments from monoclonal antibodies of predefined specificity have been crystallized as single components or complexed with their specific antigens. The intersegmental flexibility of antibody molecules has imposed the strategy of attempting to crystallize their Fab and Fc fragments separately. Intrasegmental mobility in Fabs has not been an obstacle to their crystallization, although this has been a low frequency event, occuring in about 1 in 25 to 1 in 50 trials with different Fabs. However, the immune system provides a large functional and structural diversity of antibody molecules so that an active search may eventually reveal antibodies of the desired specificity suitable for crystallization and X-ray diffraction studies.

Crystallization and preliminary X-ray diffraction studies of antigen-antibody complexes

Monoclonal antibodies of predefined specificity have been purified and crystallized as single components or complexed with their specific antigens. The intersegmental flexibility of antibody molecules has imposed the strategy of attempting to crystallize their Fab fragments separately. Intrasegmental mobility in Fabs has rarely been an obstacle to their crystallization. The immune system, however, provides a large functional and structural diversity of antibody molecules suitable for crystallization and X-ray diffraction studies.

Nucleotide sequence of the VH,VL regions of an anti-idiotopic antibody reacting with a private idiotope of the anti-lysozyme D1.3 antibody

Antibody E225 reacts with a private idiotope of the anti-lysozyme antibody D1.3. A complex between the Fab fragments from these BALB/c monoclonal antibodies has been crystallized and the determination of the three-dimensional structure of this idiotope-anti-idiotope complex is under way. The nucleotide VH and VL sequences of E225 presented here have been determined to provide the amino acid sequence information necessary for the interpretation of the high resolution electron density maps of the complex, obtained by X-ray crystallography. The cDNAs synthesized from the Vkappa and VH mRNAs were cloned in E. coli. Both cDNA strands were sequenced by the dideoxy termination method. The translated amino acid sequence shows that Vkappa, VH correspond to groups five (V) and II(b) of mouse immunoglobulin light and heavy chains, respectively. Sequence alignments between the complementarity determining regions of E225 and the antigenic determinant of lysozyme recognized by D1.3 do not indicate whether or not the anti-idiotopic antibody structurally mimics the external antigen.