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J P Mornon

Publications and source records attributed to J P Mornon.

At least 19 recordsLinked to original sources
PubMed2002

Structural features of prions explored by sequence analysis I. Sequence data.

Animal prion proteins (PrPs) form at the sequence level a very homogenous and 'closed' family. Therefore, few of their structural and functional features can be gleaned from sequence comparison as is now possible on a wide scale for other protein families. To detect putatively related proteins (at the structural and/or functional level), we used a battery of sequence analysis tools. This analysis resulted in (i) the identification of a putative 'prion-like' domain within the envelope of foamy retroviruses, (ii) the detection of putative similarities between prions and an interferon-inducible membrane protein, and (iii) the proposal that of the TATA-box-binding protein is a structural scaffold, which might allow understanding of a key event leading to the structural conversion from PrP(C) (normal cellular prion structure) towards PrP(Sc) (pathogenic structure).

Amino Acid Sequence↗
PubMed2002

Structural features of prions explored by sequence analysis. II. A PrP(Sc) model.

Prion diseases are neurodegenerative disorders associated with a conformational conversion of the prion PrP protein, in which the beta strand content increases and that of the a helix decreases. However, the structure of the pathogenous form PrP(Sc), occurring after conformational conversion of the normal cellular form PrP(C), is not yet known. From sequence analysis, we have previously proposed that helix H2 of the prion PrP(C) structure might be a key region for this structural conversion. More recently, we identified the TATA box-binding protein fold as a putative scaffold that may locally satisfy the predicted secondary-structure organisation of PrP(Sc). In the present analysis, we detail the schematic construction of PrP(Sc) monomeric and dimeric models, based on this hypothesis. These models are globally compatible with available data and therefore may provide further insights into the structurally and functionally elusive PrP protein. Some comments are also devoted to a comparison of the yeast Ure2p prion and animal prions.

Amino Acid Sequence↗
PubMed2001

uDENN, DENN, and dDENN: indissociable domains in Rab and MAP kinases signaling pathways.

DENN domains are found in a variety of signaling proteins but their exact function remains undefined. Some of the DENN-containing proteins, such as rat Rab3GEP (Rab3 GDP/GTP exchange protein) or mouse Rab6IP1 (Rab6 interacting protein 1) interact with GTPases of the Rab family. Others, such as human MADD (MAP (Mitogen-activated protein) kinase activating protein containing death domain) and human ST5 (Suppressor of tumoreginicity 5) gene products are involved in regulation of MAPKs (Mitogen-activated protein kinases) signaling pathways. Using a combination of profile-based and bidimensional analyses, we show here that DENN domains are much larger than described to date in domain databases, always encircled on both sides by more divergent domains, that we called uDENN and dDENN. These however share conserved amino acids which could play a key role in the DENN functions.

Algorithms↗
PubMed2001

Molecular and structural analysis of two novel mutations in a patient with mut(-) methylmalonyl-CoA deficiency.

Inherited defects in the gene encoding the methylmalonyl-CoA mutase (MCM) result in the mut forms of methylmalonic aciduria (MMA). Twelve mutations have been identified associated with the mut(-) phenotype. We report two novel mutations (K621N and D156N) in a compound heterozygote mut(-) patient. These two mutations and three previously published ones (H627N, A191E, Y231N) were mapped onto a three-dimensional homology model of the human MCM constructed from the crystal structure of the Propionibacterium shermanii enzyme.

Acyl Coenzyme A↗
PubMed2001

Distribution of tightened end fragments of globular proteins statistically matches that of topohydrophobic positions: towards an efficient punctuation of protein folding?

Using a set of 372 proteins representative of a variety of 56 distinct globular folds, a statistical correlation was observed between two recently revealed features of protein structures: tightened end fragments or 'closed loops', i.e. sequence fragments that are able in three-dimensional (3D) space to nearly close their ends (a current parameter of polymer physics), and 'topohydrophobic positions', i.e. positions always occupied in 3D space by strong hydrophobic amino acids for all members of a fold family. Indeed, in sequence space, the distribution of preferred lengths for tightened end fragments and that for topohydrophobic separation match. In addition to this statistically significant similarity, the extremities of these 'closed loops' may be preferentially occupied by topohydrophobic positions, as observed on a random sample of various folds. This observation may be of special interest for sequence comparison of distantly related proteins. It is also important for the ab initio prediction of protein folds, considering the remarkable topological properties of topohydrophobic positions and their paramount importance within folding nuclei. Consequently, topohydrophobic positions locking the 'closed loops' belong to the deep cores of protein domains and might have a key role in the folding process.

Models, Molecular↗
PubMed2001

Glycosidase active site mutations in human alpha-L-iduronidase.

Mucopolysaccharidosis type I (MPS I; McKusick 25280) results from a deficiency in alpha-L-iduronidase activity. Using a bioinformatics approach, we have previously predicted the putative acid/base catalyst and nucleophile residues in the active site of this human lysosomal glycosidase to be Glu182 and Glu299, respectively. To obtain experimental evidence supporting these predictions, wild-type alpha-L-iduronidase and site-directed mutants E182A and E299A were individually expressed in Chinese hamster ovary-K1 cell lines. We have compared the synthesis, processing, and catalytic properties of the two mutant proteins with wild-type human alpha-L-iduronidase. Both E182A and E299A transfected cells produced catalytically inactive human alpha-L-iduronidase protein at levels comparable to the wild-type control. The E182A protein was synthesized, processed, targeted to the lysosome, and secreted in a similar fashion to wild-type alpha-L-iduronidase. The E299A mutant protein was also synthesized and secreted similarly to the wild-type enzyme, but there were alterations in its rate of traffic and proteolytic processing. These data indicate that the enzymatic inactivity of the E182A and E299A mutants is not due to problems of synthesis/folding, but to the removal of key catalytic residues. In addition, we have identified a MPS I patient with an E182K mutant allele. The E182K mutant protein was expressed in CHO-K1 cells and also found to be enzymatically inactive. Together, these results support the predicted role of E182 and E299 in the catalytic mechanism of alpha-L-iduronidase and we propose that the mutation of either of these residues would contribute to a very severe clinical phenotype in a MPS I patient.

Amino Acid Sequence↗
PubMed2001

Molecular characterization of the major membrane skeletal protein in the ciliate Tetrahymena pyriformis suggests n-plication of an early evolutionary intermediate filament protein subdomain.

Epiplasmin C is the major protein component of the membrane skeleton in the ciliate Tetrahymena pyriformis. Cloning and analysis of the gene encoding epiplasmin C showed this protein to be a previously unrecognized protein. In particular, epiplasmin C was shown to lack the canonical features of already known epiplasmic proteins in ciliates and flagellates. By means of hydrophobic cluster analysis (HCA), it has been shown that epiplasmin C is constituted of a repeat of 25 domains of 40 residues each. These domains are related and can be grouped in two families called types I and types II. Connections between types I and types II present rules that can be evidenced in the sequence itself, thus enforcing the validity of the splitting of the domains. Using these repeated domains as queries, significant structural similarities were demonstrated with an extra six heptads shared by nuclear lamins and invertebrate cytoplasmic intermediate filament proteins and deleted in the cytoplasmic intermediate filament protein lineage at the protostome-deuterostome branching in the eukaryotic phylogenetic tree.

Amino Acid Sequence↗
PubMed2000

Voronoï tessellation reveals the condensed matter character of folded proteins.

The packing geometry of amino acids in folded proteins is analyzed via a modified Voronoï tessellation method which distinguishes bulk and surface. From a statistical analysis of the Voronoï cells over 40 representative proteins, it appears that the packings are in average similar to random packings of hard spheres encountered in condensed matter physics, with a quite strong fivefold local symmetry. Moreover, the statistics permits one to establish a classification of amino acids in terms of increasing propensity to be buried in agreement with what is known from chemical considerations.

Chemical Phenomena↗
PubMed2000

Sequence and structural features of the T-fold, an original tunnelling building unit.

A similar fold has been found in four archetype enzymes that perform different functions. This new fold has been named the T-fold because it is found in multimeric proteins crossed by a tunnel. The T-fold consists of an antiparallel beta-sheet of four sequential strands, and two antiparallel helices between the second and third strand, layered on the concave side of the beta-sheet. The presently known T-fold proteins share a high structural similarity (a mean of 1.4 A root mean square (r.m.s.) deviation on the common core) while they only exhibit a low level of sequence identity (a mean of 10.5% on the aligned regions). They bind to substrates belonging to the purine or pterin families, and share a fold-related binding site with a glutamate or glutamine residue anchoring the substrate and a lot of conserved interactions. They also share a similar oligomerization mode: several T-folds join together to form a beta(2n)alpha(n) barrel, then two barrels join together in a head-to-head fashion to made up the native enzymes. The T-fold has the characteristics of a globular domain, with a hydrophobic core and a clearly defined topohydrophobic network. It defines a new class of common folds or recurrent domains found in distantly related proteins. However, it is likely not stable in monomeric form and until now is only observed in association with other T-folds through multimerization. Proteins 2000;39:142-154.

Aldehyde-Lyases↗
PubMed2000

Structural features of normal and mutant human lysosomal glycoside hydrolases deduced from bioinformatics analysis.

Lysosomal storage diseases are due to inherited deficiencies in various enzymes involved in basic metabolic processes. As with other genetic diseases, accurate structure data for these enzymatic proteins should help in better understanding the molecular effects of mutations identified in patients with the corresponding lysosomal diseases; however, no such three-dimensional (3D) structure data are available for many lysosomal enzymes. Thus, we herein intend to illustrate for an audience of molecular geneticists how structure information can nonetheless be obtained via a bioinformatics approach in the case of five human lysosomal glycoside hydrolases. Indeed, using the two-dimensional hydrophobic cluster analysis method to decipher the sequence information available in data banks for the large group of glycoside hydrolases (clan GH-A) to which these human lysosomal enzymes belong, we could deduce structure predictions for their catalytic domains and propose explanations for the molecular effects of mutations described in patients. In addition, in the case of human beta-glucuronidase for which experimental 3D data have been reported, we also show here that bioinformatics methods relying on the available 3D structure information can be used to obtain further insights into the effects of various mutations described in patients with Sly disease. In a broader perspective, our work stresses that, in the context of a rapid increase in protein sequence information through genome sequencing, bioinformatics approaches might be highly useful for generating structure-function predictions based on sequence-structure interrelationships.

Crystallography, X-Ray↗
PubMed2000

Human glucocerebrosidase: heterologous expression of active site mutants in murine null cells.

Using bioinformatics methods, we have previously identified Glu235 and Glu340 as the putative acid/base catalyst and nucleophile, respectively, in the active site of human glucocerebrosidase. Thus, we undertook site-directed mutagenesis studies to obtain experimental evidence supporting these predictions. Recombinant retroviruses were used to express wild-type and E235A and E340A mutant proteins in glucocerebrosidase-deficient murine cells. In contrast to wild-type enzyme, the mutants were found to be catalytically inactive. We also report the results of various studies (Western blotting, glycosylation analysis, subcellular fractionation, and confocal microscopy) indicating that the wild-type and mutant enzymes are identically processed and sorted to the lysosomes. Thus, enzymatic inactivity of the mutant proteins is not the result of incorrect folding/processing. These findings indicate that Glu235 plays a key role in the catalytic machinery of human glucocerebrosidase and may indeed be the acid/base catalyst. As concerns Glu340, the results both support our computer-based predictions and confirm, at the biological level, previous identification of Glu340 as the nucleophile by use of active site labeling techniques. Finally, our findings may help to better understand the molecular basis of Gaucher disease, the human lysosomal disease resulting from deficiency in glucocerebrosidase.

Animals↗
PubMed2000

Beta-sheet modeling by helical surfaces.

We present a topological description of a beta-sheet in terms of a piece of helical surface. It requires only two easy-to-handle parameters: the twist, i.e. the turn of the helical surface per residue, and the coiling, which is a curvature along the strands or in the direction perpendicular to the strands of the sheet. This method applies fairly well to three- and four-strand sheets, forming a too limited structure to be able to build a barrel. From an analysis of beta-sheets derived from a structural database, we show that this picture can even be reduced to the use of one main value, the twist angle. The dependence of beta-sheet twisting on the number of strands in a sheet, and also on the length and direction of strands, has been demonstrated. The applications of such a description may include the rapid modeling of 3D structures.

Computational Biology↗
PubMed2000

HYR, an extracellular module involved in cellular adhesion and related to the immunoglobulin-like fold.

Domains belonging to the immunoglobulin-like fold are responsible for a wide variety of molecular recognition processes. Here we describe a new family of domains, the HYR family, which is predicted to belong to this fold, and which appears to be involved in cellular adhesion. HYR domains were identified in several eukaryotic proteins, often associated with Complement Control Protein (CCP) modules or arranged in multiple copies. Our analysis provides a sequence and structural basis for understanding the role of these domains in interaction mechanisms and leads to further characterization of heretofore undescribed repeated domains with similar folds found in several bacterial proteins involved in enzymatic activities (some chitinases) or in cell surface adhesion (streptococcal C-alpha antigen).

Amino Acid Sequence↗
PubMed2000

The uteroglobin fold.

Uteroglobin (UTG) forms a fascinating homodimeric structure that binds small- to medium-sized ligands through an internal hydrophobic cavity, located at the interface between the two monomers. Previous studies have shown that UTG fold is not limited to the UTG/CC10 family, whose sequence/structure relationships are highlighted here, but can be extended to the cap domain of Xanthobacter autotrophicus haloalkane dehalogenase. We show here that UTG fold is adopted by several other cap domains within the alpha/beta hydrolase family, making it a well-suited "geode" structure allowing it to sequester various hydrophobic molecules. Additionally, some data about a new crystal form of oxidized rabbit UTG are presented, completing previous structural studies, as well as results from molecular dynamics, suggesting an alternative way for the ligand to reach the internal cavity.

Amino Acid Sequence↗
PubMed1999

Structure modelling and site-directed mutagenesis of the rat aromatic L-amino acid pyridoxal 5'-phosphate-dependent decarboxylase: a functional study.

The pyridoxal-5'-phosphate-dependent enzymes (B6 enzymes) are grouped into three main families named alpha, beta, and gamma. Proteins in the alpha and gamma families share the same fold and might be distantly related, while those in the beta family exhibit specific structural features. The rat aromatic L-amino acid decarboxylase (AADC; EC(4.1.1.28)) catalyzes the synthesis of two important neurotransmitters: dopamine and serotonin. It binds the cofactor pyridoxal-5'-phosphate and belongs to the alpha family. Despite the low level of sequence identity (approximately 10%) shared by the rat AADC and the sequences of the enzymes belonging to the B6 enzymes family, including the known three-dimensional structures, a multiple sequence alignment was deduced. A model was built using segments belonging to seven of the eleven known structures. By homology, and based on knowledge of the biochemistry of the aspartate aminotransferase, structurally and functionally important residues were identified in the rat AADC. Site-directed mutagenesis of the conserved residues D271, T246, and C311 was carried out in order to confirm our predictions and highlight their functional role. Mutation of D271A and D271N resulted in complete loss of enzyme activity, while the D271E mutant exhibited 2% of the wild-type activity. Substitution of T246A resulted in 5% of the wild-type activity while the C311A mutant conserved 42% of the wild-type activity. A functional model of the AADC is discussed in view of the structural model and the complementary mutagenesis and labelling studies.

Amino Acid Sequence↗
PubMed1999

Functional characterization of the phosphorylating D-glyceraldehyde 3-phosphate dehydrogenase from the archaeon Methanothermus fervidus by comparative molecular modelling and site-directed mutagenesis.

Phosphorylating archaeal D-glyceraldehyde 3-phosphate dehydrogenases (GraP-DHs) share only 15-20% identity with their glycolytic bacterial and eukaryotic counterparts. Unlike the latter which are NAD-specific, archaeal GraP-DHs exhibit a dual-cofactor specificity with a marked preference for NADP. In the present study, we have constructed a three-dimensional model of the Methanothermus fervidus GraP-DH based upon the X-ray structures of the Bacillus stearothermophilus and Escherichia coli GraP-DHs. The overall structure of the archaeal enzyme is globally similar to homology modelling-derived structures, in particular for the cofactor binding domain, which might adopt a classical Rossmann fold. M. fervidus GraP-DH can be considered as a dimer of dimers which exhibits negative and positive cooperativity in binding the coenzymes NAD and NADP, respectively. As expected, the differences between the model and the templates are located mainly within the loops. Based on the predictions derived from molecular modelling, site-directed mutagenesis was performed to characterize better the cofactor binding pocket and the catalytic domain. The Lys32Ala, Lys32Glu and Lys32Asp mutants led to a drastic increase in the Km value for NADP (i.e. 165-, 500- and 1000-fold, respectively), thus demonstrating that the invariant Lys32 residue is one of the most important determinants favouring the adenosine 2'-PO42- binding of NADP. The involvement of the side chain of Asn281, which was postulated to play a role equivalent to that of the Asn313 of bacterial and eukaryotic GraP-DHs in fixing the position of the nicotinamide ring in a syn orientation [Fabry, S. & Hensel, R. (1988) Gene 64, 189-197], was ruled out. Most of the amino acids involved in catalysis and in substrate recognition in bacterial and eukaryotic GraP-DHs are not conserved in the archaeal enzyme except for the essential Cys149. Inspection of our model suggests that side chains of invariant residues Asn150, Arg176, Arg177 and His210 are located in or near the active site pocket. The Arg177Asn mutation induced strong allosteric properties with the Pi, indicating that this residue should be located near to the intersubunit interfaces. The Arg176Asn mutation led to a 10-fold decrease in the kcat, a 35-fold increase in the Km value for D-glyceraldehyde 3-phosphate and a 1000-fold decrease in the acylation rate. These results strongly suggest that Arg176 is involved in the Ps site. The His210Asn mutation increased the pKapp of the catalytic Cys149 from 6.3 to 7.6, although no Cys-/His+ ion pair was detectable [Talfournier, F., Colloc'h, N., Mornon, J.P. & Branlant, G. (1998) Eur. J. Biochem. 252, 447-457]. No other invariant amino acid which can play a role as a base catalyst to favour the hydride transfer is located in the active site. The fact that the efficiency of phosphorolysis is 1000-fold lower when compared to the B. stearothermophilus GraP-DH suggests significant differences in the nature of the Pi site. Despite these differences, it is likely that the archaeal GraP-DHs and their bacterial and eukaryotic counterparts have evolved from a common ancestor.

Archaeal Proteins↗
PubMed1999

New efficient statistical sequence-dependent structure prediction of short to medium-sized protein loops based on an exhaustive loop classification.

A bank of 13,563 loops from three to eight amino acid residues long, representing motifs between two consecutive regular secondary structures, has been derived from protein structures presenting less than 95 % sequence identity. Statistical analyses of occurrences of conformations and residues revealed length-dependent over-representations of particular amino acids (glycine, proline, asparagine, serine, and aspartate) and conformations (alphaL, epsilon, betaPregions of the Ramachandran plot). A position-dependent distribution of these occurrences was observed for N and C-terminal residues, which are correlated to the nature of the flanking regions. Loops of the same length were clustered into statistically meaningful families on the basis of their backbone structures when placed in a common reference frame, independent of the flanks. These clusters present significantly different distributions of sequence, conformations, and endpoint residue Calphadistances. On the basis of the sequence-structure correlation of this clustering, an automatic loop modeling algorithm was developed. Based on the knowledge of its sequence and of its flank backbone structures each query loop is assigned to a family and target loop supports are selected in this family. The support backbones of these target loops are then adjusted on flanking structures by partial exploration of the conformational space. Loop closure is performed by energy minimization for each support and the final model is chosen among connected supports based upon energy criteria. The quality of the prediction is evaluated by the root-mean-square deviation (rmsd) between the final model and the native loops when the whole bank is re-attributed on itself with a Jackknife test. This average rmsd ranges from 1.1 A for three-residue loops to 3.8 A for eight-residue loops. A few poorly predicted loops are inescapable, considering the high level of diversity in loops and the lack of environment data. To overcome such modeling problems, a statistical reliability score was assigned for each prediction. This score is correlated to the quality of the prediction, in terms of rmsd, and thus improves the selection accuracy of the model. The algorithm efficiency was compared to CASP3 target loop predictions. Moreover, when tested on a test loop bank, this algorithm was shown to be robust when the loops are not precisely delimited, therefore proving to be a useful tool in practice for protein modeling.

Models, Molecular↗