David R. Davies

Phosphocholine binding immunoglobulin Fab McPC603. An X-ray diffraction study at 2.7 A.

The crystal structure of the Fab of McPC603, a phosphocholine-binding mouse myeloma protein, has been refined at 2.7 A resolution by a combination of restrained least-squares refinement and molecular modeling. The overall structure remains as previously reported, with an elbow bend angle between the variable and constant modules of 133 degrees. Some adjustments have been made in the structure of the loops as a result of the refinement. The hypervariable loops are all visible in the electron density map with the exception of three residues in the first hypervariable loop of the light chain. A sulfate ion occupies the site of binding of the phosphate moiety of phosphocholine.

The molecular structure of polyadenylic acid

The structure of fibers of polyadenylic acid at acid pH has been studied by X-ray diffraction. A model is proposed consisting of two parallel intertwined helical chains, each having a screw of 3·8 A and 45° and related to each other by a dyad axis parallel to the fiber axis. Coordinates, bond distances and angles and the calculated Fourier transform are given for this model. Reasons are given why the quite different model of Morgan & Bear is thought to be wrong.

The Molecular Basis of Substrate Channeling

Substrate channeling is the process of direct transfer of an intermediate between the active sites of two enzymes that catalyze sequential reactions in a biosynthetic pathway (for reviews see Refs. 1 and 2). The active sites can be located either on separate domains in a multifunctional enzyme or on separate subunits in a multienzyme complex. Substrate channeling has been proposed to decrease transit time of intermediates, prevent loss of intermediates by diffusion, protect labile intermediates from solvent, and forestall entrance of intermediates into competing metabolic pathways (2). Loss of an intermediate by diffusion may be especially important in the case of a neutral species, such as indole, which could escape from the cell by passive diffusion across cell membranes (2–5). Nevertheless, there has been considerable debate over whether channeling actually occurs and whether it is advantageous (5, 6). X-ray crystallographic studies on several enzyme complexes have revealed two molecular mechanisms for channeling. The discovery of an intramolecular tunnel in tryptophan synthase (7) provided the first molecular mechanism for channeling. For a long time this was a unique example, but recent structure determinations have revealed plausible evidence for tunneling of ammonia and carbamate in carbamoyl-phosphate synthase (CPS) (8) and of ammonia in phosphoribosylpyrophosphate amidotransferase (GPATase) (9). The structure of dihydrofolate reductase-thymidylate synthase, however, showed no evidence for a tunnel between the two active sites (10). Neither are the two active sites adjacent to one another. Instead, there are positively charged residues along the surface between the active sites that form an electrostatic highway sufficient to channel the negatively charged dihydrofolate with high efficiency (10, 11). The focus of this minireview will be on the structural basis of channeling in the four enzyme structures cited above and on solution studies of these systems. Scheme I summarizes the reactions catalyzed by the four enzymes. A comparison of the structural and kinetic results reveals that these enzymes frequently exhibit allosteric interactions that synchronize the reactions to prevent the build-up of excess intermediate (12–16).

X-ray fiber diffraction and model-building study of polyguanylic acid and polyinosinic acid

Abstract 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).

Structure and Function of HIV-1 Integrase

HIV-1 integrase is a multidomain enzyme which is required for the integration of viral DNA into the host genome. It is one of three enzymes of HIV, the others being the Reverse Transcriptase and the Protease. It is an attractive target for therapeutic drug design. The enzyme consists of three domains. The N-terminal domain has a His2Cys2 motif which chelates zinc, the core domain has the catalytic DDE motif which is required for its enzymatic activity, and the C-terminal domain has an SH3-like fold which binds DNA nonspecifically. We review the structures of various integrase fragments, the core domain with inhibitors bound, and propose a model for DNA binding.

Similarity of three-dimensional structure between the immunoglobulin domain and the copper, zinc superoxide dismutase subunit

A striking similarity in three-dimensional structure has been observed in two functionally unrelated, sequentially non-homologous proteins: the immunoglobulin domain and the copper, zinc superoxide dismutase subunit. The immunoglobulin molecule contains several structurally similar domains composed of antiparallel β strands forming a bilayer structure or flattened cylinder. The same topological folding pattern of the antiparallel β strands into a bilayer structure of the same overall shape is found in superoxide dismutase, and external loops occur in places equivalent to hypervariable region loops. Quantitative comparisons of the various structures have been made and are discussed in detail.

Refined crystal structure of gamma-chymotrypsin at 1.9 A resolution. Comparison with other pancreatic serine proteases.

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 A 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. The three-dimensional structures of γ- and α-chymotrypsin (Birktoft & Blow, 1972) are closely similar. The largest backbone differences occur in the “calcium binding loop” (residues 75 to 78) and in the “autolysis loop” (residues 146, 149 and 150). Ala149 and Asn150 are disordered in γ-chymotrypsin, whereas they are stabilized by intermolecular interactions in α-chymotrypsin. The conformation of Ser218 is also different, presumably the indirect result of the dimeric interactions of α-chymotrypsin. These results are discussed in terms of the slow, pH-dependent interconversion of α- and γ-chymotrypsin.

The role of disulfide bonds in the assembly and function of MD-2

MD-2 is a secreted glycoprotein that binds to the extracellular domain of Toll-like receptor 4 (TLR4) and is required for the activation of TLR4 by lipopolysaccharide (LPS). The protein contains seven Cys residues and consists of a heterogeneous collection of disulfide-linked oligomers. To investigate the role of sulfhydryls in MD-2 structure and function, we created 17 single and multiple Cys substitution mutants. All of the MD-2 mutant proteins, including one totally lacking Cys residues, were secreted and stable. SDS/PAGE analyses indicated that most Cys residues could participate in oligomer formation and that no single Cys residue was required for oligomerization. Of the single Cys substitutions, only C95S and C105S failed to confer LPS responsiveness on TLR4 when mutant and TLR4 were cotransfected into cells expressing an NF-κB reporter plasmid. Surprisingly, substitution of both C95 and C105 partially restored activity. Structural analyses revealed that C95 and C105 formed an intrachain disulfide bond, whereas C95 by itself produced an inactive dimer. In contrast to the cotransfection experiments, only WT MD-2 conferred responsiveness to LPS when secreted proteins were added directly to TLR4 reporter cells. Our data are consistent with a model in which most, possibly all sulfhydryls lie on the surface of a stable MD-2 core structure where they form both intra- and interchain disulfide bridges. These disulfide bonds produce a heterogeneous array of oligomers, including some species that can form an active complex with TLR4.

The structure of helical 5'-guanosine monophosphate

Abstract An X-ray fiber diffraction analysis of 5′-GMP has been carried out. It is concluded on the basis of very well oriented diffraction patterns that the structure consists of a continuously hydrogen-bonded helix with 15 nucleotides in four turns.

On the specificity of antibody/antigen interactions: phosphocholine binding to McPC603 and the correlation of three-dimensional structure and sequence data.

Refined three-dimensional structures of McPC603 Fab and the complex with phosphocholine permit a detailed assessment of the residues crucial to determining the antibody specificity. Correlation with sequence data suggests that the structure of the binding site is highly conserved in immunoglobulins with phosphocholine-binding specificity. There is suggestive evidence that coupling of somatic mutations occurs to preserve antigen-binding specificity. The immune response is characterized by specificity and diversity. While each antibody appears to be specific for a single antigen, the immune response can generate up to 10(9) different specificities. In order to understand, at the molecular level, the nature of the interaction between antibody and antigen, it is necessary to have a high-resolution three-dimensional picture of the complex. Today it is possible to investigate antibody/antigen interactions directly by the crystallographic analysis of hybridoma products [5, 10]; in the past, structural studies were limited to myeloma proteins which, in some cases, could be shown to complex to certain haptens. Of the four Fab structures that have been determined by X-ray diffraction, only two have been demonstrated to bind hapten in the crystal. They are Fab NEW, which was shown to bind a vitamin K1 derivative [1] and McPC603, which binds to phosphocholine [6,9]. During the last few years, the McPC603 Fab structure has been refined at 2.7 A resolution and the complex of McPC603 Fab with phosphocholine has been refined independently at 3.1 A. In this communication, we make a comparative analysis of the sequences of a number of mouse phosphocholine-binding immunoglobulins based on the refined structure of the phosphocholine-binding site in McPC603.