Andrew F. Wilderspin

Identification of an inhibitory Zn2+ binding site on the human glycine receptor α1 subunit

1 Whole‐cell glycine‐activated currents were recorded from human embryonic kidney (HEK) cells expressing wild‐type and mutant recombinant homomeric glycine receptors (GlyRs) to locate the inhibitory binding site for Zn2+ ions on the human α1 subunit. 2 Glycine‐activated currents were potentiated by low concentrations of Zn2+ (<10 μm) and inhibited by higher concentrations (>100 μm) on wild‐type α1 subunit GlyRs. 3 Lowering the external pH from 7.4 to 5.4 inhibited the glycine responses in a competitive manner. The inhibition caused by Zn2+ was abolished leaving an overt potentiating effect at 10 μm Zn2+ that was exacerbated at 100 μm Zn2+. 4 The identification of residues involved in the formation of the inhibitory binding site was also assessed using diethylpyrocarbonate (DEPC), which modifies histidines. DEPC (1 mm) abolished Zn2+‐induced inhibition and also the potentiation of glycine‐activated currents by Zn2+. 5 The reduction in glycine‐induced whole‐cell currents in the presence of high (100 μm) concentrations of Zn2+ did not increase the rate of glycine receptor desensitisation. 6 Systematic mutation of extracellular histidine residues in the GlyR α1 subunit revealed that mutations H107A or H109A completely abolished inhibition of glycine‐gated currents by Zn2+. However, mutation of other external histidines, H210, H215 and H419, failed to prevent inhibition by Zn2+ of glycine‐gated currents. Thus, H107 and H109 in the extracellular domain of the human GlyR α1 subunit are major determinants of the inhibitory Zn2+ binding site. 7 An examination of Zn2+ co‐ordination in metalloenzymes revealed that the histidine‐ hydrophobic residue‐histidine motif found to be responsible for binding Zn2+ in the human GlyR α1 subunit is also shared by some of these enzymes. Further comparison of the structure and location of this motif with a generic model of the GlyR α1 subunit suggests that H107 and H109 participate in the formation of the inhibitory Zn2+ binding site at the apex of a β sheet in the N‐terminal extracellular domain.

The 3-D structure of HIV-1 proteinase and the design of antiviral agents for the treatment of AIDS

A proteinase is essential for replication of HIV. Cloning and chemical synthesis have provided a sufficient supply of HIV-1 proteinase for the determination of its three-dimensional structure. Analogies between the structures of HIV-1 proteinase and the mammalian enzyme renin, which is involved in the control of blood pressure, have given important clues concerning the design of specific inhibitors that have antiviral activity.

A pyruvate-stimulated adenylate cyclase has a sequence related to the fes/fps oncogenes and to eukaryotic cyclases

The pyruvate‐stimulated adenylate cyclase from Brevibacterium liquefaciens produces up to 450μM cyclic AMP in the culture medium when the bacterium is grown on glucose and alanine. In this paper we report the cloning, expression and sequencing of the gene for this enzyme. Residues were identified, within the C‐terminal domain, which are conserved in adenylate and guanylate cyclase sequences from eukaryotes and in the adenylate cyclase of the prokaryote Rhizobium meliloti. We have also identified a sequence of 30 residues near the N‐terminus of the protein which is homologous to part of the regulatory domain of the cellular homologues of the oncogenes fes and fps; this sequence is also present in the avian Fujinami sarcoma virus fps gene.

Three-dimensional Structure and Evolution of HIV-1 Protease

HIV-1 proteinase processes its virally encoded polyproteins into mature structural proteins and enzymes that are essential for viral propagation. As a consequence the proteinase is an attractive target for prospective antiviral agents for the treatment of AIDS, and knowledge of its tertiary structure an important step in drug design. Following the observation (Toh et al. 1985) that retroviral proteinases shared a highly conserved sequence Asp-Thr/Ser-Gly with the pepsins, it has been hypothesised (Pearl and Taylor, 1987; Blundell et al. 1988) on the basis of sequence analysis and modelling studies that these enzymes exist as dimers closely similar in three-dimensional structure to the ancestral dimeric proteinase suggested for the aspartic proteinases (Tang et al. 1978). This has now been confirmed, first by X-ray analysis of a synthetic HIV-1 proteinase in the laboratory of Wlodawer (Weber et al. 1989) and then for a recombinant enzyme in our own laboratories (Lapatto et al. 1989). These X-ray structure analyses indicated that the overall fold of the HIV-1 proteinase closely resembled that of the RSV-proteinase (Miller et al., 1989). The Asp-Thr-Gly sequences adopt a conformation closely similar to that of the pepsin-like aspartic proteinases but organised symmetrically in the dimer about the crystallographic 2-fold axis. However, the N- and C-termini together form an intermolecular four-stranded sheet, which is central to the stability of the dimer, in contrast to the inter-subunit sheet of the pepsins, which has six antiparallel strands arranged around the pseudo dyad.