Walter Palm

Crystallographic refinement and atomic models of the intact immunoglobulin molecule Kol and its antigen-binding fragment at 3.0 Å and 1.9 Å resolution

Abstract The crystal structures of the intact immunoglobulin G1, (λ) Kol and its Fab † fragment were crystallographically refined at 3.0 A and 1.9 A resolution, respectively. The methods used were real space refinement (RLSP) energy and residual refinement (EREF), phase combination, constrained rigid body refinement (CORELS) and difference and Fourier map inspection. The final R -values are 0.24 and 0.26. These analyses allowed the construction of atomic models of parts not seen in detail in the previous analyses at 5 A and 3 A resolution, respectively (Colman et al. , 1976; Matsushima et al. , 1978): i.e. the hinge segment, the hypervariable segments and their intimate interaction with the hinge segment of a crystallographically related molecule. The hinge segment forms a short poly- l -proline double helix from Cys527 to Cys530 (Eu numbering 226 to 230). The preceding segment forms an open turn of helix. This segment and the segment following the poly- l -proline part, which was found to be flexible in Fc fragment crystals (Deisenhofer et al. , 1976) probably allow arm and stem movement of the antibody molecule. The combining site of Kol is compared with the combining site of Fab New (Saul et al. , 1978). The narrow cleft formed by the hypervariable loops in Kol is filled with aromatic amino acid side-chains. In the crystal, the hypervariable loops contact the hinge and adjacent segments of a related molecule accompanied by a substantial loss in accessible surface area. This contact is preserved in Kol Fab crystals and presumably occurs in the Kol cryoprecipitate. A comparison of the quaternary structures of intact Kol and Fab New showed, in addition to the large change in elbow angle (Colman et al. , 1976), changes in lateral domain association. These are discussed in the context of a possible signal transmission from the combining site to the distal end. An attempt was made to model build the IgG3 hinge segment, which is quadruplicated with respect to IgG1 (Michaelsen et al. , 1977), on the basis of the Kol hinge structure. A polyproline double helix appeared to be the most plausible model. The Fc part was found to be disordered in intact Kol crystals (Colman et al. , 1976). Refinement has reduced the electron density further in the crystal space, where the Fc parts must be located. Disorder, if static, must be fourfold or more in the crystalline state. Intensity measurements on Kol F(ab′) 2 and their comparison with intact Kol crystals provide evidence that the disorder is predominantly of a static nature.

Crystal structure of the human Fab fragment Kol and its comparison with the intact Kol molecule.

The crystal structure of the Fab‡ fragment of the IgG protein Kol was analysed and an electron density map calculated at 3 A resolution based on phases obtained from multiple isomorphous replacement. An atomic model was constructed but the lack of amino acid sequence data causes some uncertainty, in particular concerning the amino acid side-chains. Several cycles of constrained crystallographic refinement (Deisenhofer & Steigemann, 1975) produced an R value of 0.36. The resulting model was compared with the intact Kol IgG crystal structure (Colman et al., 1976), which had also been subjected to constrained crystallographic refinement, and two Fab fragments (Davies et al., 1975). The elbow angle was found open and only 8 ° more bent than in intact Kol, in contrast to the other Fab fragments, which show a strongly bent elbow angle. The intramolecular longitudinal V-C contacts are very similar in Kol Fab and Kol IgG and considerably fewer in number than in the other Fab fragments. The lateral V-V and C-C association is virtually identical in Kol Fab and Kol IgG. Although Kol Fab and Kol IgG crystallize in different lattices, they exhibit a nearly identical head to tail packing of the Fab parts, with the C modules touching the hypervariable segments of the V modules. This strongly conserved particular mode of aggregation might be reflected in the property of cold precipitation of the Kol cryoglobulin.

Preliminary X-ray data from well-ordered crystals of a human immunoglobulin G molecule

Abstract Crystals of an intact immunoglobulin G molecule, which are suitable for high resolution X-ray analysis, have been grown. The space group is P 3 1 21 (or enantiomorph) and the crystallographic diad relates the two halves of one molecule.

On the isolation, characterisation, and crystallisation of a human Bence-Jones protein of kappa type.

The isolation and purification of the human Bence‐Jones protein Rei is described. Physico‐chemical data are given characterising the protein. It is shown that the protein could be crystallised. It is intended to use the crystals for single crystal X‐ray diffraction studies.

Crystallographic evidence for structurally similar domains in the human κ-type Bence-Jones protein Rei

Abstract Patterson search calculations demonstrated the presence of a local pseudo 2-fold axis in the crystalline state of the monomeric Bence-Jones protein Rei. These local diads are also observed in a preliminary Fourier synthesis at 4 A resolution. They appear to relate the variable and constant halves of neighbouring molecules. The presence of local diads is evidence that the tertiary structures of the constant and variable halves of the Bence-Jones protein are similar.

Quaternary structure of immunoglobulin m: A model based on small-angle X-ray scattering data

Abstract This paper presents some data on a human Waldenstrom immunoglobulin M.IgM GAL based on small-angle X-ray scattering data. the IgM GAL had molecular weight 970 000, volume 1760 nm 3 , radius of gyration 12 nm and maximum diameter 37 nm. The conclusions from various model calculations are discussed. A flat, star-shaped model is compatible both with X-ray scattering data and electron micrographs.

X-ray small-angle scattering on soluble antigen—antibody complexes

By ultracentrifugal and electrophoretical studies Singer and Campbell [l] have provided data on the composition and conformation of soluble antigenantibody complexes formed in excess antigen. They found that with a large antigen excess, a complex ‘A complex’ is formed composed of two antigens bound by one antibody molecule (Ag, Ab) which are arranged in a linear array. Their findings concerning the composition of the A complex were confirmed by a number of investigations [2, 31. Electron microscopic studies by Feinstein and Rowe [4] and Valentine [5] have shown DNP labelled ferritin molecules bridged by anti-DNP antibodies, the latter having a more or less flexible Yshaped structure. Unfortunately the distances between the ferritin molecules varied within too large a range for precise dimensions of the complexes to be given. All electron micrographs revealed markedly smaller dimensions for the complexes than expected from the hydrodynamic [6] and X-ray data [7] available for yG immunoglobulin. These discrepancies might be ascribed partially to the difficulties in the staining method [8] and to total dehydration in the vacuum necessary for electron microscopy. However it is now well established that the antigens within the A complex are bound on the extreme ends of the respective F(ab) parts of the antibody molecule. The present paper is a report of our attempts to obtain additional data on the conformation of the A complex with the aid of the small angle X-ray scattering method, which in the past has contributed information on the structure of immunoglobulin [7,9, lo] , The work

X-Ray Diffraction Analysis of Immunoglobulin Structure

The crystal structures of a human IgG antibody molecule Kol and a human Fc fragment have been determined at 4 A and 3.5 A resolution respectively by isomorphous replacement.