FYVE zinc-finger proteins in the plant model Arabidopsis thaliana: identification of PtdIns3P-binding residues by comparison of classic and variant FYVE domains.

Classic FYVE zinc-finger domains recognize the phosphoinositide signal PtdIns3P and share the basic (R/K)(1)(R/K)HHCR(6) (single-letter amino acid codes) consensus sequence. This domain is present in predicted PtdIns3P 5-kinases and lipases from Arabidopsis thaliana. Other Arabidopsis proteins, named PRAF, consist of a pleckstrin homology (PH) domain, a regulator of chromosome condensation (RCC1) guanine nucleotide exchange factor repeat domain, and a variant FYVE domain containing an Asn residue and a Tyr residue at positions corresponding to the PtdIns3P-interacting His(4) and Arg(6) of the basic motif. Dot-blot and liposome-binding assays were used in vitro to examine the phospholipid-binding ability of isolated PRAF domains. Whereas the PH domain preferentially bound PtdIns(4,5)P(2), the variant FYVE domain showed a weaker charge-dependent binding of phosphoinositides. In contrast, specificity for PtdIns3P was obtained by mutagenic conversion of the variant into a classic FYVE domain (Asn(4),Tyr(6)-->His(4),Arg(6)). Separate substitutions of the variant residues were not sufficient to impose preferential binding of PtdIns3P, suggesting a co-operative effect of these residues in binding. A biochemical function for PRAF was indicated by its ability to catalyse guanine nucleotide exchange on some of the small GTPases of the Rab family, permitting a discussion of the biological roles of plant FYVE proteins and their regulation by phosphoinositides.

Structure determination of glucose isomerase from Streptomyces murinus at 2.6 A resolution.

Glucose isomerase from Streptomyces murinus has been crystallized in space group P4(1)2(1)2, cell dimensions a = b = 137.65 and c = 132.20 A. One dimer of the tetrametric molecule is found per asymmetric unit. An initial structure solution was obtained by the molecular replacement method. The crystallographic refinement was performed using molecular dynamics techniques with X-ray restraints. The final crystallographic R value is 21.4% at 2.6 A resolution including 3023 non-H atoms, two metal ions and two water molecules per monomer.

Crystallization and preliminary investigation of xylose isomerase from Bacillus coagulans.

Xylose isomerase from Bacillus coagulans has been crystallized in two different crystal forms. One crystal form is in space group P2(1)2(1)2, cell dimensions a = 462, b = 165, c = 82 A. The other is in space group I422, cell dimensions a = b = 113, c = 153 A.

Structural determination of the functional sites of E. coli elongation factor Tu.

Recently, we have made significant progress in solving the structure of a nicked form of elongation factor (EF)-Tu complexed with GDP. The structure has been refined to an R factor of 19.2% at 2.6 A resolution, so that most of the structure is clearly visible in the electron density map. Here we describe what is known about functional sites of EF-Tu in terms of the structure, which still lacks amino acids 40-60.

Stereochemistry of peptides containing a thioacyl group.

Endothiopeptides are oligopeptides in which one or more oxoacyl moieties in peptide groups are replaced with thioacyl moieties. The stereochemical consequences of this replacement are discussed and compared with results from X-ray crystallographic determinations of small oligopeptides containing one or two thiopeptide groups. The analysis shows that the conformations normally observed around C alpha carbon atoms in proteins are also stereochemically favourable for peptides in which a thiopeptide group has been introduced. Limitations are only present in cases where the thiopeptide is part of structural elements like helices and beta-sheets. This opens the possibility of using one or more thiopeptide groups as functional modifiers of biologically active oligopeptide and protein molecules.

A model for the tertiary structure of p21, the product of the ras oncogene.

A model was developed for the structure of p21, the protein with a molecular weight of 21,000 that is produced by the ras genes. This model predicts that p21 consists of a central core of beta-sheet structure, connected by loops and alpha helices. Four of these loops comprise the guanine nucleotide binding site. The phosphoryl binding region is made up of amino acid sequences from 10 to 16 and from 57 to 63 of p21. The latter sequence may contain a site for magnesium binding. Amino acids defining guanine specificity are Asn-116 and Asp-119, and sequences around amino acid 145 may contribute to guanine binding. The model makes it possible to visualize how oncogenic mutations of p21 affect interaction with guanine nucleotides.

Structural details of the binding of guanosine diphosphate to elongation factor Tu from E. coli as studied by X-ray crystallography.

Structural details of the guanosine diphosphate binding to a modified form of elongation factor Tu from Escherichia coli, resulting from X-ray crystallographic studies, are reported. The protein elements that take part in the nucleotide binding are located in four loops connecting beta-strands with alpha-helices. These loops correspond to regions in primary sequences which show a high degree of homology when compared with other prokaryotic and eukaryotic elongation factors and initiation factor 2.

Cross-linking of tRNA at two different sites of the elongation factor Tu.

Recently, we reported on the induction by kirromycin of two tRNA binding sites on elongation factor Tu. To obtain independent information on the existence of these two sites and to characterize them further, 3' oxidized tRNA was cross-linked to elongation factor Tu by [3H]borohydride reduction. Specific cross-linking occurred exclusively in the presence of kirromycin. In the case of elongation factor Tu X GDP X kirromycin, cross-linking was found at lysine-208; in elongation factor Tu X GTP X kirromycin, cross-linking was at lysine-208 and lysine-237. In both elongation factor Tu complexes, kirromycin itself was found cross-linked to lysine-357. The tRNA cross-linking sites are in agreement with the idea of two different binding sites of tRNA on elongation factor Tu.

Specific alterations of the EF-Tu polypeptide chain considered in the light of its three-dimensional structure.

Specific alterations of the elongation factor Tu (EF-Tu) polypeptide chain have been identified in a number of mutant species of this elongation factor. In two species, Ala-375, located on domain II, was found by amino acid analysis to be replaced by Thr and Val, respectively. These replacements substantially lower the affinity of EF-Tu.GDP for the antibiotic kirromycin. Since kirromycin can be cross-linked to Lys-357, also located on domain II but structurally very far from Ala-375, these data suggest that the replacements alter the relative position of domains I and II. The Ala-375 replacements also lower the dissociation rates of the binary complexes EF-Tu.GTP and the binding constants for EF-Tu.GTP and Phe-tRNA. It is conceivable that these effects are also mediated by movements of domains I and II relative to each other. Replacement of Gly-222 by Asp has been found in another mutant by DNA sequence analysis of the cloned tufB gene, coding for this mutant EF-Tu. Gly-222 is part of a structural domain, characteristic for a variety of nucleotide binding enzymes. Its replacement by Asp does not abolish the ability of EF-Tu to sustain protein synthesis. It increases the dissociation rate of EF-Tu.GTP by approximately 30%. In the presence of kirromycin this mutant species of EF-Tu.GDP does not bind to the ribosome, in contrast to its wild-type counterpart. A possible explanation is now open for experimental verification.

The geometry of the thiopeptide unit.

The structure of the modified dipeptide Z-Glyt-Gly-OBzl is reported with special emphasis on the linear and angular dimensions of the thiopeptide unit. It is shown that, except for the differences between the carbonyl and the thioacyl bond lengths, there are no significant differences between the oxopeptide and the thiopeptide unit.

The role of arginine residues at enzyme active sites. The interaction between guanidinium ions and p-nitro-phenyl phosphate and its effect on the rate of hydrolysis of the ester.

In alcoholic solutions a relatively strong complex forms among two guanidinium ions and one p-nitrophenylphosphate dianion. The effect of this complex formation on the hydrolysis of the ester is to lower the rate by a factor of 4 in solutions containing 1 M guanidine hydrochloride when compared with solutions of the same total ionic strength containing no guanidinium ion. It is therefore suggested that, for the enzymatically catalyzed hydrolysis of phosphate compounds going via the formation of a metaphosphate intermediate, the role of any arginine residues at the active site is primarily one of binding and positioning the substrate.