MEPSAnd: minimum energy path surface analysis over n-dimensional surfaces.

SUMMARY: n-dimensional energy surfaces are becoming computationally accessible, yet interpreting their information is not straightforward. We present minimum energy path surface analysis over n-dimensional surfaces (MEPSAnd), an open source GUI-based program that natively calculates minimum energy paths across energy surfaces of any number of dimensions. Among other features, MEPSAnd can compute the path through lowest barriers and automatically provide a set of alternative paths. MEPSAnd offers distinct plotting solutions as well as direct python scripting. AVAILABILITY AND IMPLEMENTATION: MEPSAnd is freely available (under GPLv3 license) at: http://bioweb.cbm.uam.es/software/MEPSAnd/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The only exoribonuclease present in Haloferax volcanii has an unique response to temperature changes.

BACKGROUND: Little is known regarding mRNA degradation mechanisms in archaea. In some of these single-cell organisms the existence of a complex of exoribonucleases called the exosome has been demonstrated. However, in halophilic archaea the RNase R homologue is essential since it is the only enzyme described with exoribonucleolytic activity. METHODS: In this work we have characterized the mechanism of action of Haloferax volcanii RNase R and its implications for the RNA degradation process. We have determined the salt, pH and divalent ion preference, and set the best conditions for the activity assays. Furthermore, we have determined the activity of the protein at different temperatures using different substrates. The dissociation constants were also calculated by Surface Plasmon Resonance. Finally, we have built a model and compared it with the Escherichia coli counterparts. RESULTS: The results obtained showed that at 37°C, in spite of being named RNase R, this protein behaves like an RNase II protein, halting when it reaches secondary structures, and releasing a 4 nt end-product. However, at 42°C, the optimum temperature of growth, this protein is able to degrade secondary structures, acting like RNase R. GENERAL SIGNIFICANCE: This discovery has a great impact for RNA degradation, since this is the first case reported where a single enzyme has two different exoribonucleolytic activities according to the temperature. Furthermore, the results obtained are very important to help to decipher the RNA degradation mechanisms in H. volcanii, since RNase R is the only exoribonuclease involved in this process.

Mutations that hamper dimerization of foot-and-mouth disease virus 3A protein are detrimental for infectivity.

Foot-and-mouth disease virus (FMDV) nonstructural protein 3A plays important roles in virus replication, virulence, and host range. In other picornaviruses, homodimerization of 3A has been shown to be relevant for its biological activity. In this work, FMDV 3A homodimerization was evidenced by an in situ protein fluorescent ligation assay. A molecular model of the FMDV 3A protein, derived from the nuclear magnetic resonance (NMR) structure of the poliovirus 3A protein, predicted a hydrophobic interface spanning residues 25 to 44 as the main determinant for 3A dimerization. Replacements L38E and L41E, involving charge acquisition at residues predicted to contribute to the hydrophobic interface, reduced the dimerization signal in the protein ligation assay and prevented the detection of dimer/multimer species in both transiently expressed 3A proteins and in synthetic peptides reproducing the N terminus of 3A. These replacements also led to production of infective viruses that replaced the acidic residues introduced (E) by nonpolar amino acids, indicating that preservation of the hydrophobic interface is essential for virus replication. Replacements that favored (Q44R) or impaired (Q44D) the polar interactions predicted between residues Q44 and D32 did not abolish dimer formation of transiently expressed 3A, indicating that these interactions are not critical for 3A dimerization. Nevertheless, while Q44R led to recovery of viruses that maintained the mutation, Q44D resulted in selection of infective viruses with substitution D44E with acidic charge but with structural features similar to those of the parental virus, suggesting that Q44 is involved in functions other than 3A dimerization.

The Role of Gln61 in HRas GTP hydrolysis: a quantum mechanics/molecular mechanics study.

Activation of the water molecule involved in GTP hydrolysis within the HRas·RasGAP system is analyzed using a tailored approach based on hybrid quantum mechanics/molecular mechanics (QM/MM) simulation. A new path emerges: transfer of a proton from the attacking water molecule to a second water molecule, then a different proton is transferred from this second water molecule to the GTP. Gln(61) will stabilize the transient OH(-) and H(3)O(+) molecules thus generated. This newly proposed mechanism was generated by using, for the first time to our knowledge, the entire HRas-RasGAP protein complex in a QM/MM simulation context. It also offers a rational explanation for previous experimental results regarding the decrease of GTPase rate found in the HRas Q61A mutant and the increase exhibited by the HRas Q61E mutant.

Characterization of a novel HMG-CoA lyase enzyme with a dual location in endoplasmic reticulum and cytosol.

A novel lyase activity enzyme is characterized for the first time: HMG-CoA lyase-like1 (er-cHL), which is a close homolog of mitochondrial HMG-CoA lyase (mHL). Initial data show that there are nine mature transcripts for the novel gene HMGCLL1, although none of them has all its exons. The most abundant transcript is called "variant b," and it lacks exons 2 and 3. Moreover, a three-dimensional model of the novel enzyme is proposed. Colocalization studies show a dual location of the er-cHL in the endoplasmic reticulum (ER) and cytosol, but not in mitochondria or peroxisomes. Furthermore, the dissociation experiment suggests that it is a nonendoplasmic reticulum integral membrane protein. The kinetic parameters of er-cHL indicate that it has a lower V(max) and a higher substrate affinity than mHL. Protein expression and lyase activity were found in several tissues, and were particularly strong in lung and kidney. The occurrence of er-cHL in brain is surprising, as mHL has not been found there. Although mHL activity is clearly associated with energy metabolism, the results suggest that er-cHL is more closely related to another metabolic function, mostly at the pulmonary and brain level.

The African swine fever virus lectin EP153R modulates the surface membrane expression of MHC class I antigens.

We have modeled a 3D structure for the C-type lectin domain of the African swine fever virus protein EP153R, based on the structure of CD69, CD94 and Ly49A cell receptors, and this model predicts that a dimer of EP153R may establish an asymmetric interaction with one MHC-I molecule. A functional consequence of this interaction could be the modulation of MHC-I expression. By using both transfection and virus infection experiments, we demonstrate here that EP153R inhibits MHC-I membrane expression, most probably by impairing the exocytosis process, without affecting the synthesis or glycosylation of MHC antigens. Interestingly, the EP153-mediated control of MHC requires the intact configuration of the lectin domain of the viral protein, and specifically the R133 residue. Interference of EP153R gene expression during virus infection and studies using virus recombinants with the EP153R gene deleted further support the inhibitory role of the viral lectin on the expression of MHC-I antigens.

Determination of key residues for catalysis and RNA cleavage specificity: one mutation turns RNase II into a "SUPER-ENZYME".

RNase II is the prototype of a ubiquitous family of enzymes that are crucial for RNA metabolism. In Escherichia coli this protein is a single-stranded-specific 3'-exoribonuclease with a modular organization of four functional domains. In eukaryotes, the RNase II homologue Rrp44 (also known as Dis3) is the catalytic subunit of the exosome, an exoribonuclease complex essential for RNA processing and decay. In this work we have performed a functional characterization of several highly conserved residues located in the RNase II catalytic domain to address their precise role in the RNase II activity. We have constructed a number of RNase II mutants and compared their activity and RNA binding to the wild type using different single- or double-stranded substrates. The results presented in this study substantially improve the RNase II model for RNA degradation. We have identified the residues that are responsible for the discrimination of cleavage of RNA versus DNA. We also show that the Arg-500 residue present in the RNase II active site is crucial for activity but not for RNA binding. The most prominent finding presented is the extraordinary catalysis observed in the E542A mutant that turns RNase II into a "super-enzyme."

Mutations and variants in the cohesion factor genes NIPBL, SMC1A, and SMC3 in a cohort of 30 unrelated patients with Cornelia de Lange syndrome.

Cornelia de Lange syndrome (CdLS) manifests facial dysmorphic features, growth and cognitive impairment, and limb malformations. Mutations in three genes (NIPBL, SMC1A, and SMC3) of the cohesin complex and its regulators have been found in affected patients. Here, we present clinical and molecular characterization of 30 unrelated patients with CdLS. Eleven patients had mutations in NIPBL (37%) and three patients had mutations in SMC1A (10%), giving an overall rate of mutations of 47%. Several patients shared the same mutation in NIPBL (p.R827GfsX2) but had variable phenotypes, indicating the influence of modifiers in CdLS. Patients with NIPBL mutations had a more severe phenotype than those with mutations in SMC1A or those without identified mutations. However, a high incidence of palate defects was noted in patients with SMC1A mutations. In addition, we observed a similar phenotype in both male and female patients with SMC1A mutations. Finally, we report the first patient with an SMC1A mutation and the Sandifer complex.

Ten novel HMGCL mutations in 24 patients of different origin with 3-hydroxy-3-methyl-glutaric aciduria.

3-Hydroxy-3-methylglutaric aciduria is a rare autosomal recessive genetic disorder that affects ketogenesis and L-leucine catabolism. The clinical acute symptoms include vomiting, convulsions, metabolic acidosis, hypoketotic hypoglycaemia and lethargy. To date, 33 mutations in 100 patients have been reported in the HMGCL gene. In this study 10 new mutations in 24 patients are described. They include: 5 missense mutations: c.109G>A, c.425C>T, c.521G>A, c.575T>C and c.598A>T, 2 nonsense mutations: c.242G>A and c.559G>T, one small deletion: c.853delC, and 2 mutations in intron regions: c.497+4A>G and c.750+1G>A. Two prevalent mutations were detected, 109G>T (E37X) in 38% of disease alleles analyzed and c.504_505delCT in 10% of them. Although patients are mainly of European origin (71%) and mostly Spanish (54%), the group is ethnically diverse and includes, for the first time, patients from Pakistan, Palestine and Ecuador. We also present a simple, efficient method to express the enzyme and we analyze the possible functional effects of missense mutations. The finding that all identified missense mutations cause a >95% decrease in the enzyme activity, indicates that the disease appears only in very severe genotypes."

An evolutionary and structure-based docking model for glucocerebrosidase-saposin C and glucocerebrosidase-substrate interactions - relevance for Gaucher disease.

Gaucher disease, the most prevalent lysosomal storage disorder, is principally caused by malfunction of the lysosomal enzyme glucocerebrosidase (GBA), a 497-amino acid membrane glycoprotein that catalyzes the hydrolysis of glucosylceramide to ceramide and glucose in the presence of an essential 84-residue activator peptide named saposin C (SapC). Knowledge of the GBA structure, a typical (beta/alpha)(8) TIM barrel, explains the effect of few mutations, directly affecting or located near the catalytic site. To identify new regions crucial for proper GBA functionality, we analyzed the interactions of the enzyme with a second (substrate) and a third (cofactor) partner. We build 3D docking models of the GBA-SapC and the GBA-ceramide interactions, by means of methodologies that integrate both evolutive and structural information. The GBA-SapC docking model confirm the implication of three spatially closed regions of the GBA surface (TIM barrel-helix 6 and helix 7, and the Ig-like domain) in binding the SapC molecule. This model provides new basis to understand the pathogenicity of several mutations, such as the prevalent Leu444Pro, and the additive effect of Glu326Lys in the double mutant Glu326Lys-Leu444Pro. Overall, 39 positions in which amino acid changes are known to cause Gaucher disease were localized in the GBA regions identified in this work. Our model is discussed in relation to the phenotype (pathogenic effect) of these mutations, as well as to the enzymatic activity of the recombinant proteins when available. Both data fully correlates with the proposed model, which will provide a new tool to better understand Gaucher disease and to design new therapy strategies.

Structural properties of the human respiratory syncytial virus P protein: evidence for an elongated homotetrameric molecule that is the smallest orthologue within the family of paramyxovirus polymerase cofactors.

The oligomeric state and the hydrodynamic properties of human respiratory syncytial virus (HRSV) phosphoprotein (P), a known cofactor of the viral RNA-dependent RNA polymerase (L), and a trypsin-resistant fragment (X) that includes its oligomerization domain were analyzed by sedimentation equilibrium and velocity using analytical ultracentrifugation. The results obtained demonstrate that both P and fragment X are homotetrameric with elongated shapes, consistent with electron micrographs of the purified P protein in which thin rod-like molecules of approximately 12.5 +/- 1.0 nm in length were observed. A new chymotrypsin resistant fragment (Y*) included in fragment X has been identified and purified by gel filtration chromatography. Fragment Y* may represent a minimal version of the P oligomerization domain. Thermal denaturation curves based on circular dichroism data of P protein showed a complex behavior. In contrast, melting data generated for fragments X and particularly fragment Y* showed more homogeneous transitions indicative of simpler structures. A three-dimensional model of X and Y* fragments was built based on the atomic structure of the P oligomerization domain of the related Sendai virus, which is in good agreement with the experimental data. This model will be an useful tool to make rational mutations and test the role of specific amino acids in the oligomerization and functional properties of the HRSV P protein.

Computational approaches to model ligand selectivity in drug design.

To be effective, a designed drug must discriminate successfully the macromolecular target from alternative structures present in the organism. The last few years have witnessed the emergence of different computational tools aimed to the understanding and modeling of this process at molecular level. Although still rudimentary, these methods are shaping a coherent approach to help in the design of molecules with high affinity and specificity, both in lead discovery and in lead optimization. It is the purpose of this review to illustrate the array of computational tools available to consider selectivity in the design process, to summarize the most relevant applications, and to sketch the challenges ahead.

Skipping of exon 2 and exons 2 plus 3 of HMG-CoA lyase (HL) gene produces the loss of beta sheets 1 and 2 in the recently proposed (beta-alpha)8 TIM barrel model of HL.

HMG-CoA lyase (HL) deficiency is a rare autosomal recessive genetic disorder that affects ketogenesis and leucine catabolism. We report a new Spanish patient who bears the frequent nonsense mutation G109T (Mediterranean mutation). This mutation can produce aberrant splicing with three mRNA variants: one of the expected size, the second with deletion of exon 2, and the third with deletion of exons 2 and 3. Recently our group proposed a 3D model for human HL containing a (beta-alpha)(8) (TIM) barrel structure. We have studied the effect of the deletions of exon 2 and exons 2 plus 3 on the proposed HL model. Exon 2 skipping led to the loss of beta-sheet 1, and the skipping of exons 2 and 3 caused the disappearance of alpha helix 1 and beta-sheets 1 and 2.-

Structural model of carnitine palmitoyltransferase I based on the carnitine acetyltransferase crystal.

CPT I (carnitine palmitoyltransferase I) catalyses the conversion of palmitoyl-CoA into palmitoylcarnitine in the presence of L-carnitine, facilitating the entry of fatty acids into mitochondria. We propose a 3-D (three-dimensional) structural model for L-CPT I (liver CPT I), based on the similarity of this enzyme to the recently crystallized mouse carnitine acetyltransferase. The model includes 607 of the 773 amino acids of L-CPT I, and the positions of carnitine, CoA and the palmitoyl group were assigned by superposition and docking analysis. Functional analysis of this 3-D model included the mutagenesis of several amino acids in order to identify putative catalytic residues. Mutants D477A, D567A and E590D showed reduced L-CPT I activity. In addition, individual mutation of amino acids forming the conserved Ser685-Thr686-Ser687 motif abolished enzyme activity in mutants T686A and S687A and altered K(m) and the catalytic efficiency for carnitine in mutant S685A. We conclude that the catalytic residues are His473 and Asp477, while Ser687 probably stabilizes the transition state. Several conserved lysines, i.e. Lys455, Lys505, Lys560 and Lys561, were also mutated. Only mutants K455A and K560A showed decreases in activity of 50%. The model rationalizes the finding of nine natural mutations in patients with hereditary L-CPT I deficiencies.

Modulating the RNA processing and decay by the exosome: altering Rrp44/Dis3 activity and end-product.

In eukaryotes, the exosome plays a central role in RNA maturation, turnover, and quality control. In Saccharomyces cerevisiae, the core exosome is composed of nine catalytically inactive subunits constituting a ring structure and the active nuclease Rrp44, also known as Dis3. Rrp44 is a member of the ribonuclease II superfamily of exoribonucleases which include RNase R, Dis3L1 and Dis3L2. In this work we have functionally characterized three residues located in the highly conserved RNB catalytic domain of Rrp44: Y595, Q892 and G895. To address their precise role in Rrp44 activity, we have constructed Rrp44 mutants and compared their activity to the wild-type Rrp44. When we mutated residue Q892 and tested its activity in vitro, the enzyme became slightly more active. We also showed that when we mutated Y595, the final degradation product of Rrp44 changed from 4 to 5 nucleotides. This result confirms that this residue is responsible for the stacking of the RNA substrate in the catalytic cavity, as was predicted from the structure of Rrp44. Furthermore, we also show that a strain with a mutation in this residue has a growth defect and affects RNA processing and degradation. These results lead us to hypothesize that this residue has an important biological role. Molecular dynamics modeling of these Rrp44 mutants and the wild-type enzyme showed changes that extended beyond the mutated residues and helped to explain these results.

Structural and functional model for ionic (K(+)/Na(+)) and pH dependence of GTPase activity and polymerization of FtsZ, the prokaryotic ortholog of tubulin.

Bacterial cell division occurs through the formation of a protein ring (division ring) at the site of division, with FtsZ being its main component in most bacteria. FtsZ is the prokaryotic ortholog of eukaryotic tubulin; it shares GTPase activity properties and the ability to polymerize in vitro. To study the mechanism of action of FtsZ, we used molecular dynamics simulations of the behavior of the FtsZ dimer in the presence of GTP-Mg(2+) and monovalent cations. The presence of a K(+) ion at the GTP binding site allows the positioning of one water molecule that interacts with catalytic residues Asp235 and Asp238, which are also involved in the coordination sphere of K(+). This arrangement might favor dimer stability and GTP hydrolysis. Contrary to this, Na(+) destabilizes the dimer and does not allow the positioning of the catalytic water molecule. Protonation of the GTP gamma-phosphate, simulating low pH, excludes both monovalent cations and the catalytic water molecule from the GTP binding site and stabilizes the dimer. These molecular dynamics predictions were contrasted experimentally by analyzing the GTPase and polymerization activities of purified Methanococcus jannaschii and Escherichia coli FtsZ proteins in vitro.

C75 is converted to C75-CoA in the hypothalamus, where it inhibits carnitine palmitoyltransferase 1 and decreases food intake and body weight.

Central nervous system administration of C75 produces hypophagia and weight loss in rodents identifying C75 as a potential drug against obesity and type 2 diabetes. However, the mechanism underlying this effect is unknown. Here we show that C75-CoA is generated chemically, in vitro and in vivo from C75 and that it is a potent inhibitor of carnitine palmitoyltranferase 1 (CPT1), the rate-limiting step of fatty-acid oxidation. Three-D docking and kinetic analysis support the inhibitory effect of C75-CoA on CPT1. Central nervous system administration of C75 in rats led to C75-CoA production, inhibition of CPT1 and lower body weight and food intake. Our results suggest that inhibition of CPT1, and thus increased availability of fatty acids in the hypothalamus, contribute to the pharmacological mechanism of C75 to decrease food intake.

New insights into the mechanism of RNA degradation by ribonuclease II: identification of the residue responsible for setting the RNase II end product.

RNase II is a key exoribonuclease involved in the maturation, turnover, and quality control of RNA. RNase II homologues are components of the exosome, a complex of exoribonucleases. The structure of RNase II unraveled crucial aspects of the mechanism of RNA degradation. Here we show that mutations in highly conserved residues at the active site affect the activity of the enzyme. Moreover, we have identified the residue that is responsible for setting the end product of RNase II. In addition, we present for the first time the models of two members of the RNase II family, RNase R from Escherichia coli and human Rrp44, also called Dis3. Our findings improve the present model for RNA degradation by the RNase II family of enzymes.

The structure of CCT-Hsc70 NBD suggests a mechanism for Hsp70 delivery of substrates to the chaperonin.

Chaperones, a group of proteins that assist the folding of other proteins, seem to work in a coordinated manner. Two major chaperone families are heat-shock protein families Hsp60 and Hsp70. Here we show for the first time the formation of a stable complex between chaperonin-containing TCP-1 (CCT) and Hsc70, two eukaryotic representatives of these chaperone families. This interaction takes place between the apical domain of the CCT beta subunit and the nucleotide binding domain of Hsc70, and may serve to deliver the unfolded substrate from Hsc70 to the substrate binding region of CCT. We also show that a similar interaction does not occur between their prokaryotic counterparts GroEL and DnaK, suggesting that in eukarya the two types of chaperones have evolved to a concerted action that makes the folding task more efficient.