Insights into the structural basis for zinc inhibition of the glycine receptor

Histidines 107 and 109 in the glycine receptor ( GlyR) alpha(1) subunit have previously been identified as determinants of the inhibitory zinc-binding site. Based on modeling of the GlyR alpha(1) subunit extracellular domain by homology to the acetylcholine-binding protein crystal structure, we hypothesized that inhibitory zinc is bound within the vestibule lumen at subunit interfaces, where it is ligated by His(107) from one subunit and His(109) from an adjacent subunit. This was tested by co-expressing alpha(1) subunits containing the H107A mutation with alpha(1) subunits containing the H109A mutation. Although sensitivity to zinc inhibition is markedly reduced when either mutation is individually incorporated into all five subunits, the GlyRs formed by the co-expression of H107A mutant subunits with H109A mutant subunits exhibited an inhibitory zinc sensitivity similar to that of the wild type alpha(1) homomeric GlyR. This constitutes strong evidence that inhibitory zinc is coordinated at the interface between adjacent alpha(1) subunits. No evidence was found for beta subunit involvement in the coordination of inhibitory zinc, indicating that a maximum of two zinc-binding sites per alpha(1)beta receptor is sufficient for maximal zinc inhibition. Our data also show that two zinc-binding sites are sufficient for significant inhibition of alpha(1) homomers. The binding of zinc at the interface between adjacent alpha(1) subunits could restrict intersubunit movements, providing a feasible mechanism for the inhibition of channel activation by zinc.

Phylogeny of the Sporobolus indicus complex, based on internal transcribed spacer (ITS) sequences

The entire internal transcribed spacer ( ITS) region, including the 5.8S subunit of the nuclear ribosomal DNA ( rDNA), was sequenced by direct double-stranded sequencing of polymerase chain reaction (PCR) amplified fragments. The study included 40 Sporobolus ( Family Poaceae, subfamily Chloridoideae) seed collections from 14 putative species ( all 11 species from the S. indicus complex and three Australian native species). These sequences, along with those from two out-group species [ Pennisetum alopecuroides ( L.) Spreng. and Heteropogon contortus ( L.) P. Beauv. ex Roemer & Schultes, Poaceae, subfamily Panicoideae], were analysed by the parsimony method (PAUP; version 4.0b4a) to infer phylogenetic relationships among these species. The length of the ITS1, 5.8S subunit and ITS2 region were 222, 164 and 218 base pairs ( bp), respectively, in all species of the S. indicus complex, except for the ITS2 region of S. diandrus P. Beauv. individuals, which was 217 bp long. Of the 624 characters included in the analysis, 245 ( 39.3%) of the 330 variable sites contained potential phylogenetic information. Differences in sequences among the members of the S. pyramidalis P. Beauv., S. natalensis (Steud.) Dur & Schinz and S. jacquemontii Kunth. collections were 0%, while differences ranged from 0 to 2% between these and other species of the complex. Similarly, differences in sequences among collections of S. laxus B. K. Simon, S. sessilis B. K. Simon, S. elongatus R. Br. and S. creber De Nardi were 0%, compared with differences of 1-2% between these four species and the rest of the complex. When comparing S. fertilis ( Steud.) Clayton and S. africanus (Poir.) Robyns & Tourney, differences between collections ranged from 0 to 1%. Parsimony analysis grouped all 11 species of the S. indicus complex together, indicating a monophyletic origin. For the entire data set, pair-wise distances among members of the S. indicus complex varied from 0.00 to 1.58%, compared with a range of 20.08-21.44% among species in the complex and the Australian native species studied. A strict consensus phylogenetic tree separated 11 species of the S. indicus complex into five major clades. The phylogeny, based on ITS sequences, was found to be congruent with an earlier study on the taxonomic relationship of the weedy Sporobolus grasses revealed from random amplified polymorphic DNA ( RAPD). However, this cladistic analysis of the complex was not in agreement with that created on past morphological analyses and therefore gives a new insight into the phylogeny of the S. indicus complex.

Structural characterization of respiratory syncytial virus fusion inhibitor escape mutants: homology model of the F protein and a syncytium formation assay

Respiratory syncytial virus (RSV) is a ubiquitous human pathogen and the leading cause of lower respiratory tract infections in infants. Infection of cells and subsequent formation of syncytia occur through membrane fusion mediated by the RSV fusion protein (RSV-F). A novel in vitro assay of recombinant RSV-F function has been devised and used to characterize a number of escape mutants for three known inhibitors of RSV-F that have been isolated. Homology modeling of the RSV-F structure has been carried out on the basis of a chimera derived from the crystal structures of the RSV-F core and a fragment from the orthologous fusion protein from Newcastle disease virus (NDV). The structure correlates well with the appearance of RSV-F in electron micrographs, and the residues identified as contributing to specific binding sites for several monoclonal antibodies are arranged in appropriate solvent-accessible clusters. The positions of the characterized resistance mutants in the model structure identify two promising regions for the design of fusion inhibitors. (C) 2003 Elsevier Science (USA). All rights reserved.