A genomic view of yeast membrane transporters.

The yeast membrane transporters play crucial roles in functions as diverse as nutrient uptake, drug resistance, salt tolerance, control of cell volume, efflux of undesirable metabolites and sensing of extracellular nutrients. A significant fraction of the many transporters inventoried after sequencing of the yeast genome has been characterised by classical experimental approaches. Post-genomic analysis has allowed a more extensive characterisation of transporter categories less tractable by genetics, for instance of transporters of intracellular membranes or transporters encoded by multigene families and displaying overlapping substrate specificities. A complete view of the role of membrane transporters in the metabolism of yeast may not be far off.

Role of salt bridges in homeodomains investigated by structural analyses and molecular dynamics simulations.

Homeodomains are a class of helix-turn-helix DNA-binding protein motifs that play an important role in the control of cellular development in eukaryotes. They fold in a three alpha-helix structural module, where the third helix is the recognition helix that fits into the major groove of DNA. Structural analysis of the members of the homeodomain family led to the identification of interactions likely to stabilize the protein domains. Linking the helices pairwise, three salt bridges were found to be well preserved within the family. Also well conserved were two cation-pi interactions between aromatic and positively charged side chains. To analyze the structural role of the salt bridges, molecular dynamics simulations (MD) were carried out on the wild-type homeodomain from the Drosophila paired protein (1fjl) and on three mutants, which lack one or two salt bridges and mimic natural mutations in other homeodomains. Analysis of the trajectories revealed only small structural rearrangements of the three helices in all MD simulations, thereby suggesting that the salt bridges have no essential stabilizing role at room temperature, but rather might be important for improving thermostability. The latter hypothesis is supported by a good correlation between the melting midpoint temperatures of several homeodomains and the number of salt bridges and cation-pi interactions that connect secondary structures.

Independent adaptation to riverine habitats allowed survival of ancient cetacean lineages.

The four species of "river dolphins" are associated with six separate great river systems on three subcontinents and have been grouped for more than a century into a single taxon based on their similar appearance. However, several morphologists recently questioned the monophyly of that group. By using phylogenetic analyses of nucleotide sequences from three mitochondrial and two nuclear genes, we demonstrate with statistical significance that extant river dolphins are not monophyletic and suggest that they are relict species whose adaptation to riverine habitats incidentally insured their survival against major environmental changes in the marine ecosystem or the emergence of Delphinidae.

Modeling of Enterococcus faecalis D-alanine:D-alanine ligase: structure-based study of the active site in the wild-type enzyme and in glycopeptide-dependent mutants.

A model for the 3-D structure of Enterococcus faecalis D-Ala:D-Ala ligase was produced using the X-ray structure of the Escherichia coli enzyme complexed with ADP and the methylphosphinophosphate inhibitor as a template. The model passed critical validation criteria with an accuracy similar to that of the template crystallographic structure and showed that ADP and methylphosphinophosphate were positioned in a large empty pocket at the interface between the central and the C-terminal domains, as in E. coli. It evidenced the residues important for substrate binding and catalytic activity in the active site and demonstrated a large body of conserved interactions between the active sites of the E. faecalis and the E. coli D-Ala:D-Ala ligase, the major differences residing in the balance between the hydrophobic and aromatic environment of the adenine. The model also successfully explained the inactivity of four spontaneous mutants (D295 --> V, which impairs interactions with Mg2+ and R293, which are both essential for binding and catalytic activity; S319 --> I, which perturbs recognition of D-Ala2; DAK251-253 --> E, in which the backbone conformation in the vicinity of the deletion remains unaltered but phosphate transfer from ATP is perturbed because of lack of K253; T316 --> I, which causes the loss of a hydrogen bond affecting the positioning of S319 and therefore the binding of D-Ala2). Since D-Ala:D-Ala ligase is an essential enzyme for bacteria, this approach, combining molecular modeling and molecular biology, may help in the design of specific ligands which could inhibit the enzyme and serve as novel antibiotics.

Pathways of ligand clearance in acetylcholinesterase by multiple copy sampling.

The clearance of seven different ligands from the deeply buried active-site of Torpedo californica acetylcholinesterase is investigated by combining multiple copy sampling molecular dynamics simulations, with the analysis of protein-ligand interactions, protein motion and the electrostatic potential sampled by the ligand copies along their journey outwards. The considered ligands are the cations ammonium, methylammonium, and tetramethylammonium, the hydrophobic methane and neopentane, and the anionic product acetate and its neutral form, acetic acid. We find that the pathways explored by the different ligands vary with ligand size and chemical properties. Very small ligands, such as ammonium and methane, exit through several routes. One involves the main exit through the mouth of the enzyme gorge, another is through the so-called back door near Trp84, and a third uses a side door at a direction of approximately 45 degrees to the main exit. The larger polar ligands, methylammonium and acetic acid, leave through the main exit, but the bulkiest, tetramethylammonium and neopentane, as well as the smaller acetate ion, remain trapped in the enzyme gorge during the time of the simulations. The pattern of protein-ligand contacts during the diffusion process is highly non-random and differs for different ligands. A majority is made with aromatic side-chains, but classical H-bonds are also formed. In the case of acetate, but not acetic acid, the anionic and neutral form, respectively, of one of the reaction products, specific electrostatic interactions with protein groups, seem to slow ligand motion and interfere with protein flexibility; protonation of the acetate ion is therefore suggested to facilitate clearance. The Poisson-Boltzmann formalism is used to compute the electrostatic potential of the thermally fluctuating acetylcholinesterase protein at positions actually visited by the diffusing ligand copies. Ligands of different charge and size are shown to sample somewhat different electrostatic potentials during their migration, because they explore different microscopic routes. The potential along the clearance route of a cation such as methylammonium displays two clear minima at the active and peripheral anionic site. We find moreover that the electrostatic energy barrier that the cation needs to overcome when moving between these two sites is small in both directions, being of the order of the ligand kinetic energy. The peripheral site thus appears to play a role in trapping inbound cationic ligands as well as in cation clearance, and hence in product release.

Effect of mutations within the peripheral anionic site on the stability of acetylcholinesterase.

Torpedo acetylcholinesterase is irreversibly inactivated by modifying a buried free cysteine, Cys231, with sulfhydryl reagents. The stability of the enzyme, as monitored by measuring the rate of inactivation, was reduced by mutating a leucine, Leu282, to a smaller amino acid residue. Leu282 is located within the "peripheral" anionic site, at the entrance to the active-site gorge. Thus, loss of activity was due to the increased reactivity of Cys231. This was paralleled by an increased susceptibility to thermal denaturation, which was shown to be due to a large decrease in the activation enthalpy. Similar results were obtained when either of two other residues in contact with Leu282 in Torpedo acetylcholinesterase, Trp279 and Ser291, was replaced by an amino acid with a smaller side chain. We studied the effects of various ligands specific for either the active or peripheral sites on both thermal inactivation and on inactivation by 4,4'-dithiodipyridine. The wild-type and mutated enzymes could be either protected or sensitized. In some cases, opposite effects of the same ligand were observed for chemical modification and thermal denaturation. The mutated residues are within a conserved loop, W279-S291, at the top of the active-site gorge, that contributes to the peripheral anionic site. Theoretical analysis showed that Torpedo acetylcholinesterase consists of two structural domains, each comprising one contiguous polypeptide segment. The W279-S291 loop, located in the first domain, makes multiple contacts with the second domain across the active-site gorge. We postulate that the mutations to residues with smaller side chains destabilize the conserved loop, thus disrupting cross-gorge interactions and, ultimately, the entire structure.

Conformational analysis of GpA and GpAp in aqueous solution by molecular dynamics and statistical methods.

Barnase, an extracellular endoribonuclease from Bacillus amyloliquefaciens, hydrolyses single-stranded RNA. Its very low catalytic activity toward GpN dinucleotides, where N stands for any nucleoside, is markedly increased when a phosphate is added to the 3'-end, as in GpNp. Here we investigate the conformational properties of GpA and GpAp in solution, in order to determine whether differences in these properties may be related to the changes in enzymatic activity. Two independent 1.3 ns molecular dynamics trajectories are generated for each dinucleotide in the presence of explicit water molecules and counter ions. These trajectories are analysed by monitoring molecular properties, such as the solvent accessible surface area, the distance and orientation between the bases, the behaviour of torsion angles and formation of intramolecular H-bonds. To identify relevant correlations between these parameters, statistical techniques, comprising multiple regression, clustering and discriminant analysis are used. Results show that GpA has a significant propensity to form folded conformations (approximately 50%), fostered by a small number of intramolecular H-bonds, whereas GpAp remains essentially extended. The latter behaviour seems to be due to an H-bond between the terminal phosphate and adenosine ribose group, which restricts rotation about the adenine Agamma angle. We also find that GpA folding is induced by a concerted motion of specific torsion angles, which is closely coupled to the formation of a network of flexible hydrogen bonds. Finally, on the basis of an expression for barnase KM, which incorporates the folded/extended conformational equilibria of the dinucleotide substrates, it is argued that our findings on the differences between these equilibria, can qualitatively rationalize the experimentally measured differences in enzymatic properties.

Effect of nucleotide substrate binding on the pKa of catalytic residues in barnase.

A thermodynamic cycle is used to describe barnase catalysis, which considers explicitly the presence of different ionic states of the catalytic residues Glu-73 and His-102 in barnase during the enzyme-substrate recognition process. Reinterpretation of published experimental data using rate equations derived from this cycle provides estimates of the ionization constants of these catalytic side chains, in the free enzyme and in the barnase-GpA complex. In addition, the electrostatic properties of the barnase-d(CGAC) crystal complex and of a barnase-5'3'(AAGAAp)-O-methyl ester modeled complex are investigated by means of a continuum approach to account for solvent polarization effects. Taking GpA as a reference substrate, it is shown that increasing the length of the bound nucleotide induces pKa shifts in the catalytic side chains, which modulate the fraction of enzyme in the correct ionic form for achieving the transesterification reaction. The computed results are in good agreement with the experimental variation of the optimum pH of barnase activity. The present analysis underscores the influence of pH effects on the kcat and KM kinetic constants of barnase and provides the basic formalism for linking the effective kinetic parameters, which usually depend on the pH, to the theoretical estimates of the true kinetic constants.

Transfer nuclear Overhauser effect study of the conformation of oxytocin bound to bovine neurophysin I.

This study reports the structure of the peptide hormone oxytocin bound to its carrier protein, neurophysin I, obtained by nuclear magnetic resonance techniques. At the pH value of 2.1 in our experiments, the ligand is in fast exchange with its carrier protein, allowing the use of transfer-NOE methods. The number of distance constraints for the peptide being limited, considerable attention has been paid to an accurate distance determination. The resulting accurate distance limits were used as input for a distance geometry calculation followed by a restrained molecular dynamics run. Convergence to a well-defined family of structures for oxytocin in its bound state was reached. Both the backbone and the side-chain conformations differ between the bound form and the crystal structure of free oxytocin [Wood, S. P., et al. (1986) Science 232, 633]. These differences, as well as other structural features of the bound form, are discussed in terms of interactions made with the carrier protein. Transfer-NOE experiments at low peptide protein ratios provide direct experimental evidence for contacts between the oxytocin Tyr2 residue and an aromatic residue of neurophysin. The resonance assignments of the aromatic groups [Whittaker, B. A., et al. (1985) Biochemistry 24, 2782] together with the recently published X-ray structure of the neurophysin II protein complexed with a dipeptide [Chen et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 4240] allow us to assign the aromatic signal on the protein to the neurophysin Phe22 residue.

Protein engineering of xylose (glucose) isomerase from Actinoplanes missouriensis. 1. Crystallography and site-directed mutagenesis of metal binding sites.

The structure and function of the xylose (glucose) isomerase from Actinoplanes missouriensis have been analyzed by X-ray crystallography and site-directed mutagenesis after cloning and overexpression in Escherichia coli. The crystal structure of wild-type enzyme has been refined to an R factor of 15.2% against diffraction data to 2.2-A resolution. The structures of a number of binary and ternary complexes involving wild-type and mutant enzymes, the divalent cations Mg2+, Co2+, or Mn2+, and either the substrate xylose or substrate analogs have also been determined and refined to comparable R factors. Two metal sites are identified. Metal site 1 is four-coordinated and tetrahedral in the absence of substrate and is six-coordinated and octahedral in its presence; the O2 and O4 atoms of linear inhibitors and substrate bind to metal 1. Metal site 2 is octahedral in all cases; its position changes by 0.7 A when it binds O1 of the substrate and by more than 1 A when it also binds O2; these bonds replace bonds to carboxylate ligands from the protein. Side chains involved in metal binding have been substituted by site-directed mutagenesis. The biochemical properties of the mutant enzymes are presented. Together with structural data, they demonstrate that the two metal ions play an essential part in binding substrates, in stabilizing their open form, and in catalyzing hydride transfer between the C1 and C2 positions.

Calculations of electrostatic properties in proteins. Analysis of contributions from induced protein dipoles.

The calculation of induced dipole moments and of their contribution to electrostatic effects in proteins is implemented following the approach of Warshel. Isotropic polarizabilities are assigned to individual atoms, and the resulting deviation from pairwise interactions is treated by a self-consistent iterative procedure. We give a detailed description of how the formalism is implemented in molecular mechanics and molecular dynamics simulation procedures, and report results based on calculations performed on crystal structures of crambin, liver alcohol dehydrogenase and ribonuclease T1. We focus our analysis on evaluating the contribution of polarizability of the protein matrix to electrostatic energies, local fields, to dipole moments of peptide groups and of secondary structure elements in the polypeptide chain. Our calculations confirm that induced dipole moments in proteins provide important stabilizing contributions to electrostatic energies, and that these contributions cannot be mimicked by the usual approximations where either a continuum dielectric constant, or a distance-dependent dielectric function is used. We find that induced protein dipoles appreciably affect the magnitude and direction of local electrostatic fields in a manner that is strongly influenced by the microscopic environment in the protein. Most strongly affected are fields in charged groups that are involved in close interactions with other charged groups, while the influence on local fields of aliphatic groups is marginal. We find, moreover, that induction effects from surrounding protein atoms tend on average to increase peptide dipoles and helix macro-dipoles by about 16%, again reflecting electrostatic stabilization by the protein matrix, and show that (at least in the alpha/beta domain of alcohol dehydrogenase) the contribution of side-chains to this stabilization is significant.