Jean-Loup Risler

Amino acid substitutions in structurally related proteins a pattern recognition approach: Determination of a new and efficient scoring matrix

Amino acid substitutions in evolutionarily related proteins have been studied from a structural point of view. We consider here that an amino acid al in a protein p1 has been replaced by the amino acid a2 in the structurally similar protein p2 if, after superposition of the p1 and p2 structures, the a1 and a2 C alpha atoms are no more than 1.2 A apart. Thirty-two proteins, grouped in 11 classes, have been analysed by this method. This produced 2860 amino acid pairs (substitutions), which were analysed by multi-dimensional statistical methods. The main results are as follows: (1) according to the observed exchangeability of amino acid side-chains, only four groups (strong clusters) could be delineated; (i) Ile and Val, (ii) Leu and Met, (iii) Lys, Arg and Gln, and (iv) Tyr and Phe. The other residues could not be classified. (2) The matrix of distances between amino acids, or scoring matrix, determined from this study, is different from any other published matrix. (3) Except for the distance matrices based on the chemical properties of amino acid side-chains, which can be grouped together, all other published matrices are different from one another. (4) The distance matrix determined in this study seems to be very efficient for aligning distantly related protein sequences.

Non mycobacterial virulence genes in the genome of the emerging pathogen Mycobacterium abscessus.

Mycobacterium abscessus is an emerging rapidly growing mycobacterium (RGM) causing a pseudotuberculous lung disease to which patients with cystic fibrosis (CF) are particularly susceptible. We report here its complete genome sequence. The genome of M. abscessus (CIP 104536T) consists of a 5,067,172-bp circular chromosome including 4920 predicted coding sequences (CDS), an 81-kb full-length prophage and 5 IS elements, and a 23-kb mercury resistance plasmid almost identical to pMM23 from Mycobacterium marinum. The chromosome encodes many virulence proteins and virulence protein families absent or present in only small numbers in the model RGM species Mycobacterium smegmatis. Many of these proteins are encoded by genes belonging to a “mycobacterial” gene pool (e.g. PE and PPE proteins, MCE and YrbE proteins, lipoprotein LpqH precursors). However, many others (e.g. phospholipase C, MgtC, MsrA, ABC Fe(3+) transporter) appear to have been horizontally acquired from distantly related environmental bacteria with a high G+C content, mostly actinobacteria (e.g. Rhodococcus sp., Streptomyces sp.) and pseudomonads. We also identified several metabolic regions acquired from actinobacteria and pseudomonads (relating to phenazine biosynthesis, homogentisate catabolism, phenylacetic acid degradation, DNA degradation) not present in the M. smegmatis genome. Many of the “non mycobacterial” factors detected in M. abscessus are also present in two of the pathogens most frequently isolated from CF patients, Pseudomonas aeruginosa and Burkholderia cepacia. This study elucidates the genetic basis of the unique pathogenicity of M. abscessus among RGM, and raises the question of similar mechanisms of pathogenicity shared by unrelated organisms in CF patients.

Crystal structure of Escherichia coli methionyl-tRNA synthetase at 2.5 Å resolution☆

Native methionyl-tRNA synthetase from Escherichia coli (a dimer of molecular weight 172,000) can be converted by mild proteolysis into a well-defined monomeric fragment of molecular weight 64,000. This fragment retains full specificity towards methionine and tRNAMet, and has unimpaired activity in both the activation and aminoacylation reactions. This paper describes the structure of the active fragment, as determined by an X-ray crystallographic study at 2.5 A resolution using five heavy-atom derivatives. The elongated molecule (90 A × 52 A × 44 A) contains several α-helices, which account for 43% of the residues. Three domains can be distinguished in the structure: (1) a central core beginning at the N-terminus, consisting of a five-stranded parallel pleated sheet with α-helices connecting the β-strands; (2) a second domain with less-ordered structure, inserted between the third and fourth strand of the central sheet; (3) a C-terminal domain, beginning after the fifth parallel strand, very rich in α-helices. These three domains are organized in a biglobular structure; one globule contains the first and the second domain (N-terminal globule), the other the third domain. The two globules, linked together by a single chain, are separated by a large cleft. The most salient feature of the structure is the presence, in the N-terminal domain, of a “nucleotide binding fold” similar to that first observed in dehydrogenases. This makes methionyl-tRNA synthetase, and possibly all aminoacyl-tRNA synthetases, a new member of this family of nucleotide binding proteins possessing the characteristic “Rossmann fold”.

Crystallisation of trypsin-modified methionyl-tRNA synthetase from Escherichia coli

The elucidation of the molecular basis for the specific interaction between aminoacyl-tRNA synthetases and their corresponding tRNAs requires prior knowledge of both the primary structure and the conformation of these molecules. Considerable progress towards this goal has been achieved in the case of tRNAs: the primary structure of close to twenty of these have been determined, and X-ray diffraction analysis of crystalline tRNAs is now beginning to provide information on their internal geometry [l] . Similar progress has not been achieved in the case of aminoacyl-tRNA synthetases: there have been no reported attempts at determining the primary structure of any of these enzymes, and progress towards the elucidation of their tertiary structure has been hampered by lack of suitable, stable crystals amenable to high resolution X-ray diffraction analysis. In this paper we report on the crystallization of a modified, fully active form of methionyl-tRNA synthetase. The best crystals obtained have properties suitable for high resolution crystallographic analysis: they provide strong reflections at 2.5 A, are stable in the X-ray beam, contain only one molecule per assymmetric unit, and retain full enzymatic activity.

Software note: Gene teams: a new formalization of gene clusters for comparative genomics

This paper describes an efficient algorithm based on a new concept called gene team for detecting conserved gene clusters among an arbitrary number of chromosomes. Within the clusters, neither the order of the genes nor their orientation need be conserved. In addition, insertion of foreign genes within the clusters are permitted to a user-defined extent. This algorithm has been implemented in a publicly available TEAM software that proves to be an efficient tool for systematic searches of conserved gene clusters. Examples of actual biological results are provided. The software is downloadable from http://www-igm.univ-mlv.fr/ approximately raffinot/geneteam.html.

Crystal structure study of Opsanus tau parvalbumin by multiwavelength anomalous diffraction

The crystal structure of a small calcium‐binding protein, the parvalbumin IIIf from Opsanus tau in which Tb was substituted for Ca, has been analysed by multiwavelength anomalous diffraction. Data at a resolution of 2.3 Å were collected at three wavelengths near the L3 absorption edge of Tb (1.645–1.650 Å), using the synchrotron radiation emitted by a storage ring and a multiwire proportional counter. The phases of the reflections were determined from this single derivative, without native data. Prior to any refinement, the resulting electron density map shows a good agreement with the model of the homologous carp parvalbumin in regions of identical amino‐acid sequence.

A structure-based multiple sequence alignment of all class I aminoacyl-tRNA synthetases.

The superimposable dinucleotide fold domains of MetRS, GlnRS and TyrRS define structurally equivalent amino acids which have been used to constrain the sequence alignments of the 10 class I aminoacyl-tRNA synthetases (aaRS). The conservation of those residues which have been shown to be critical in some aaRS enables to predict their location and function in the other synthetases, particularly: i) a conserved negatively-charged residue which binds the alpha-amino group of the amino acid substrate; ii) conserved residues within the inserted domain bridging the two halves of the dinucleotide-binding fold; and iii) conserved residues in the second half of the fold which bind the amino acid and ATP substrate. The alignments also indicate that the class I synthetases may be partitioned into two subgroups: a) MetRS, IleRS, LeuRS, ValRS, CysRS and ArgRS; b) GlnRS, GluRS, TyrRS and TrpRS.

Structure—activity relationships of methionyl-trna synthetase: graphics modelling and genetic engineering

Abstract The 3D structure of the methionyl-tRNA synthetase from E. coli has been investigated using X-ray analysis 1,2 at a resolution 1.8A. 90% of the molecule is now well defined and the zinc atom has been identified in a buried region of the molecule, close to the active site. At the same time, the refinement of the complex ATP-MetRS at 2.5 A has been carried out. The crystallographic R factor has been assigned a value of 25% at 2.5A with an overall temperature factor of 9A 2 and 22% when the individual temperature factors are refined. A Fourier difference map clearly reveals the electron density of the bound ATP, showing the phosphate groups deeply plunging into the active site. In parallel, the synthetase gene has been used to probe some of the enzyme structure-activity relationships. A series of 60 modified enzymes truncated at the C-terminus have been constructed in vitro and assayed for activity. In agreement with the graphics model, the results show that a minimum of 534 residues is necessary to sustain the aminoacylation reaction. A programme of site-directed mutagenesis is in progress: residues thought to be important for the catalytic activity, the metal coordination and tRNA interaction are being modified. Preliminary results are discussed in the light of the crystallographic model.

The Membrane-proteins Encoded By Yeast Chromosome-III Genes

Examples are given for the analysis of the 68 putative membrane proteins encoded among the 170 predicted genes identified by the systematic sequencing of yeast chromosome III [(1992) Nature 357, 38‐56].

A low-resolution model of crystalline methionyl-transfer RNA synthetase from Escherichia coli.

Abstract When submitted to a controlled proteolysis by trypsin, native methionyl-tRNA synthetase from Escherichia coli (a dimer of molecular weight 172,000) yields a well-defined fragment of molecular weight 64,000 composed of one single polypeptide chain. This fragment retains full specificity towards methionine and tRNA met , and has unimpaired activity in both the activation reaction and aminoacyl-tRNA formation. Crystals of this active fragment have been studied by X-ray crystallography and, using two isomorphous heavy-atom derivatives, a 4 A electron density map has been calculated. The molecule appears as an elongated ellipsoid of overall dimensions 90 A × 43 A × 43 A. It is clearly built of two parts separated by a large cleft. The volume of one of these “domains” is approximately twice that of the other; these results are consistent with our present knowledge of the chemistry of the protein.