Allosteric modulation of ghrelin receptor signaling by lipids.

The membrane is an integral component of the G protein-coupled receptor signaling machinery. Here we demonstrate that lipids regulate the signaling efficacy and selectivity of the ghrelin receptor GHSR through specific interactions and bulk effects. We find that PIP2 shifts the conformational equilibrium of GHSR away from its inactive state, favoring basal and agonist-induced G protein activation. This occurs because of a preferential binding of PIP2 to specific intracellular sites in the receptor active state. Another lipid, GM3, also binds GHSR and favors G protein activation, but mostly in a ghrelin-dependent manner. Finally, we find that not only selective interactions but also the thickness of the bilayer reshapes the conformational repertoire of GHSR, with direct consequences on G protein selectivity. Taken together, this data illuminates the multifaceted role of the membrane components as allosteric modulators of how ghrelin signal could be propagated.

Conformational Equilibrium of CDK/Cyclin Complexes by Molecular Dynamics with Excited Normal Modes.

Cyclin-dependent kinases (CDKs) and their associated regulatory cyclins are central for timely regulation of cell-cycle progression. They constitute attractive pharmacological targets for development of anticancer therapeutics, since they are frequently deregulated in human cancers and contribute to sustained, uncontrolled tumor proliferation. Characterization of their structural/dynamic features is essential to gain in-depth insight into structure-activity relationships. In addition, the identification of druggable pockets or key intermediate conformations yields potential targets for the development of novel classes of inhibitors. Structural studies of CDK2/cyclin A have provided a wealth of information concerning monomeric/heterodimeric forms of this kinase. There is, however, much less structural information for other CDK/cyclin complexes, including CDK4/cyclin D1, which displays an alternative (open) position of the cyclin partner relative to CDK, contrasting with the closed CDK2/cyclin A conformation. In this study, we carried out normal-mode analysis and enhanced sampling simulations with our recently developed method, molecular dynamics with excited normal modes, to understand the conformational equilibrium on these complexes. Interestingly, the lowest-frequency normal mode computed for each complex described the transition between the open and closed conformations. Exploration of these motions with an explicit-solvent representation using molecular dynamics with excited normal modes confirmed that the closed conformation is the most stable for the CDK2/cyclin A complex, in agreement with their experimentally available structures. On the other hand, we clearly show that an open↔closed equilibrium may exist in CDK4/cyclin D1, with closed conformations resembling that captured for CDK2/cyclin A. Such conformational preferences may result from the distinct distributions of frustrated contacts in each complex. Using the same approach, the putative roles of the Thr(160) phosphoryl group and the T-loop conformation were investigated. These results provide a dynamic view of CDKs revealing intermediate conformations not yet characterized for CDK members other than CDK2, which will be useful for the design of inhibitors targeting critical conformational transitions.

An insight into the transcriptome of the digestive tract of the bloodsucking bug, Rhodnius prolixus.

The bloodsucking hemipteran Rhodnius prolixus is a vector of Chagas' disease, which affects 7-8 million people today in Latin America. In contrast to other hematophagous insects, the triatomine gut is compartmentalized into three segments that perform different functions during blood digestion. Here we report analysis of transcriptomes for each of the segments using pyrosequencing technology. Comparison of transcript frequency in digestive libraries with a whole-body library was used to evaluate expression levels. All classes of digestive enzymes were highly expressed, with a predominance of cysteine and aspartic proteinases, the latter showing a significant expansion through gene duplication. Although no protein digestion is known to occur in the anterior midgut (AM), protease transcripts were found, suggesting secretion as pro-enzymes, being possibly activated in the posterior midgut (PM). As expected, genes related to cytoskeleton, protein synthesis apparatus, protein traffic, and secretion were abundantly transcribed. Despite the absence of a chitinous peritrophic membrane in hemipterans - which have instead a lipidic perimicrovillar membrane lining over midgut epithelia - several gut-specific peritrophin transcripts were found, suggesting that these proteins perform functions other than being a structural component of the peritrophic membrane. Among immunity-related transcripts, while lysozymes and lectins were the most highly expressed, several genes belonging to the Toll pathway - found at low levels in the gut of most insects - were identified, contrasting with a low abundance of transcripts from IMD and STAT pathways. Analysis of transcripts related to lipid metabolism indicates that lipids play multiple roles, being a major energy source, a substrate for perimicrovillar membrane formation, and a source for hydrocarbons possibly to produce the wax layer of the hindgut. Transcripts related to amino acid metabolism showed an unanticipated priority for degradation of tyrosine, phenylalanine, and tryptophan. Analysis of transcripts related to signaling pathways suggested a role for MAP kinases, GTPases, and LKBP1/AMP kinases related to control of cell shape and polarity, possibly in connection with regulation of cell survival, response of pathogens and nutrients. Together, our findings present a new view of the triatomine digestive apparatus and will help us understand trypanosome interaction and allow insights into hemipteran metabolic adaptations to a blood-based diet.

A comparative proteomic analysis of Vibrio cholerae O1 wild-type cells versus a phoB mutant showed that the PhoB/PhoR system is required for full growth and rpoS expression under inorganic phosphate abundance.

PhoB/PhoR is a two-component system originally described as involved in inorganic phosphate (Pi) transport and metabolism under Pi limitation. In order to disclose other roles of this system, a proteomic analysis of Vibrio cholerae 569BSR and its phoB/phoR mutant under high Pi levels was performed. Most of the proteins downregulated by the mutant have roles in energy production and conversion and in amino acid transport and metabolism. In contrast, the phoB/phoR mutant upregulated genes mainly involved in adaptation to atypical conditions, indicating that the absence of a functional PhoB/PhoR caused increased expression of a number of genes from distinct stress response pathways. This might be a strategy to overcome the lack of RpoS, whose expression in the stationary phase cells of V. cholerae seems to be controlled by PhoB/PhoR. Moreover, compared to the wild-type strain the phoB/phoR mutant presented a reduced cell density at stationary phase of culture in Pi abundance, lower resistance to acid shock, but higher tolerance to thermal and osmotic stresses. Together our findings show, for the first time, the requirement of PhoB/PhoR for full growth under high Pi level and for the accumulation of RpoS, indicating that PhoB/PhoR is a fundamental system for the biology of V. cholerae. BIOLOGICAL SIGNIFICANCE: Certain V. cholerae strains are pathogenic to humans, causing cholera, an acute dehydrating diarrhoeal disease endemic in Southern Asia, parts of Africa and Latin America, where it has been responsible for significant mortality and economical damage. Its ability to grow within distinct niches is dependent on gene expression regulation. PhoB/PhoR is a two-component system originally described as involved in inorganic phosphate (Pi) transport and metabolism under Pi limitation. However, Pho regulon genes also play roles in virulence, motility and biofilm formation, among others. In this paper we report that the absence of a functional PhoB/PhoR caused increased expression of a number of genes from distinct stress response pathways, in Pi abundance. Moreover, we showed, for the first time, that the interrelationship between PhoB-RpoS-(p)ppGpp-poly(P) in V. cholerae, is somewhat diverse from the model of inter-regulation between those systems, described in Escherichia coli. The V. cholerae dependence on PhoB/PhoR for the RpoS mediated stress response and cellular growth under Pi abundance, suggests that this system's roles are broader than previously thought.

How does heparin prevent the pH inactivation of cathepsin B? Allosteric mechanism elucidated by docking and molecular dynamics.

BACKGROUND: Cathepsin B (catB) is a promising target for anti-cancer drug design due to its implication in several steps of tumorigenesis. catB activity and inhibition are pH-dependent, making it difficult to identify efficient inhibitor candidates for clinical trials. In addition it is known that heparin binding stabilizes the enzyme in alkaline conditions. However, the molecular mechanism of stabilization is not well understood, indicating the need for more detailed structural and dynamic studies in order to clarify the influence of pH and heparin binding on catB stability. RESULTS: Our pKa calculations of catB titratable residues revealed distinct protonation states under different pH conditions for six key residues, of which four lie in the crucial interdomain interface. This implies changes in the overall charge distribution at the catB surface, as revealed by calculation of the electrostatic potential. We identified two basic surface regions as possible heparin binding sites, which were confirmed by docking calculations. Molecular dynamics (MD) of both apo catB and catB-heparin complexes were performed using protonation states for catB residues corresponding to the relevant acidic or alkaline conditions. The MD of apo catB at pH 5.5 was very stable, and presented the highest number and occupancy of hydrogen bonds within the inter-domain interface. In contrast, under alkaline conditions the enzyme's overall flexibility was increased: interactions between active site residues were lost, helical content decreased, and domain separation was observed as well as high-amplitude motions of the occluding loop - a main target of drug design studies. Essential dynamics analysis revealed that heparin binding modulates large amplitude motions promoting rearrangement of contacts between catB domains, thus favoring the maintenance of helical content as well as active site stability. CONCLUSIONS: The results of our study contribute to unraveling the molecular events involved in catB inactivation in alkaline pH, highlighting the fact that protonation changes of few residues can alter the overall dynamics of an enzyme. Moreover, we propose an allosteric role for heparin in the regulation of catB stability in such a manner that the restriction of enzyme flexibility would allow the establishment of stronger contacts and thus the maintenance of overall structure.

Unraveling the molecular mechanisms of nitrogenase conformational protection against oxygen in diazotrophic bacteria.

BACKGROUND: G. diazotrophicus and A. vinelandii are aerobic nitrogen-fixing bacteria. Although oxygen is essential for the survival of these organisms, it irreversibly inhibits nitrogenase, the complex responsible for nitrogen fixation. Both microorganisms deal with this paradox through compensatory mechanisms. In A. vinelandii a conformational protection mechanism occurs through the interaction between the nitrogenase complex and the FeSII protein. Previous studies suggested the existence of a similar system in G. diazotrophicus, but the putative protein involved was not yet described. This study intends to identify the protein coding gene in the recently sequenced genome of G. diazotrophicus and also provide detailed structural information of nitrogenase conformational protection in both organisms. RESULTS: Genomic analysis of G. diazotrophicus sequences revealed a protein coding ORF (Gdia0615) enclosing a conserved "fer2" domain, typical of the ferredoxin family and found in A. vinelandii FeSII. Comparative models of both FeSII and Gdia0615 disclosed a conserved beta-grasp fold. Cysteine residues that coordinate the 2[Fe-S] cluster are in conserved positions towards the metallocluster. Analysis of solvent accessible residues and electrostatic surfaces unveiled an hydrophobic dimerization interface. Dimers assembled by molecular docking presented a stable behaviour and a proper accommodation of regions possibly involved in binding of FeSII to nitrogenase throughout molecular dynamics simulations in aqueous solution. Molecular modeling of the nitrogenase complex of G. diazotrophicus was performed and models were compared to the crystal structure of A. vinelandii nitrogenase. Docking experiments of FeSII and Gdia0615 with its corresponding nitrogenase complex pointed out in both systems a putative binding site presenting shape and charge complementarities at the Fe-protein/MoFe-protein complex interface. CONCLUSIONS: The identification of the putative FeSII coding gene in G. diazotrophicus genome represents a large step towards the understanding of the conformational protection mechanism of nitrogenase against oxygen. In addition, this is the first study regarding the structural complementarities of FeSII-nitrogenase interactions in diazotrophic bacteria. The combination of bioinformatic tools for genome analysis, comparative protein modeling, docking calculations and molecular dynamics provided a powerful strategy for the elucidation of molecular mechanisms and structural features of FeSII-nitrogenase interaction.

Changes in protein expression due to deleterious mutations in the FA/BRCA pathway.

Inherited deleterious mutations in one of the Fanconi anemia genes lead to a disease, characterized by bone marrow failure, myeloid leukemia, and hypersensitivity to DNA damage. We identified proteins likely associated to the molecular signaling pathways involved in DNA repair of interstrand cross-link lesions and in mechanisms of genomic stability mediated by FA/BRCA pathways. We compared protein maps resolved by bidimensional electrophoresis and analyzed differentially expressed proteins, by mass spectrometry, between FA complementation group C (FANCC)-deficient cells, and their ectopically corrected counterpart in physiological conditions or after treatment with MMC. We found six differentially expressed proteins; among them, the checkpoint mediator protein MDC1 whose expression was disrupted in FANCC-/- cells. The potential role of differentially expressed proteins in FA phenotype is discussed.

Probing the interaction between vesicular stomatitis virus and phosphatidylserine.

The entry of enveloped animal viruses into their host cells always depends on membrane fusion triggered by conformational changes in viral envelope glycoproteins. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion between the viral envelope and the endosomal membrane at the acidic environment of this compartment. In this work, we evaluated VSV interactions with membranes of different phospholipid compositions, at neutral and acidic pH, using atomic force microscopy (AFM) operating in the force spectroscopy mode, isothermal calorimetry (ITC) and molecular dynamics simulation. We found that the binding forces differed dramatically depending on the membrane phospholipid composition, revealing a high specificity of G protein binding to membranes containing phosphatidylserine (PS). In a previous work, we showed that the sequence corresponding amino acid 164 of VSV G protein was as efficient as the virus in catalyzing membrane fusion at pH 6.0. Here, we used this sequence to explore VSV-PS interaction using ITC. We found that peptide binding to membranes was exothermic, suggesting the participation of electrostatic interactions. Peptide-membrane interaction at pH 7.5 was shown to be specific to PS and dependent on the presence of His residues in the fusion peptide. The application of the simplified continuum Gouy-Chapman theory to our system predicted a pH of 5.0 at membrane surface, suggesting that the His residues should be protonated when located close to the membrane. Molecular dynamics simulations suggested that the peptide interacts with the lipid bilayer through its N-terminal residues, especially Val(145) and His(148).

Analyzing different parameters of steered molecular dynamics for small membrane interacting molecules.

The aim of this report is two-fold: First, to show the applicability of the Steered Molecular Dynamics (SMD) methodology for analyzing non-specific interactions governing the membrane affinity process of small biological molecules. Second, to point out a correlation between the system response and certain combinations of the SMD parameters (spring-elastic-constant and pulling-group). For these purposes, a simplified membrane model was used, modeled as a non-polar region limited by two polar aqueous media in a continuous dielectric representation. Polarization-induced effects at both interfaces were taken into account by the "electrostatic images" method. To perform SMD simulations a harmonic external force, representing a spring acting on a selected atom, forces the molecule to "break" its interaction with the surrounding environment by extracting it out of the membrane. With this approach, small molecules and peptides, with known affinity for the membrane environment, were studied: the zwitterionic tryptophan residue and a pentapeptide AcWLKLL. The SMD parameters, spring-elastic-constant and pulling-group, were varied and combined in order to analyze the systems responses in each case. It was observed that, the spring stiffness was crucial to reveal specific events that occur during the molecule behavior; hence, it was directly responsible for the sensitivity of this methodology. The pulling-group selected highly influenced on the reaction pathway, a fact that it was not observed with other parameters; consequently, force profiles are like the "fingerprints" of these induced pathways. The potential profile for the tryptophan was recovered from the SMD simulations being in good agreement with that estimated by an approximation method. With this rather simple model approach, SMD methodology has proven to be suitable for revealing the main interactions that govern the membrane affinity processes of small molecules and peptides.

Gene characterization and predicted protein structure of the mitochondrial chaperonin HSP10 of Trypanosoma cruzi.

Heat shock protein (HSP) 10 is a member of the highly conserved group of molecular chaperons, which are necessary for efficient folding of many proteins in normal and stress conditions and have been implicated in several human diseases. We have characterized the HSP10 genes of Trypanosoma cruzi, the causative agent of Chagas' disease. After sequence analysis of clones obtained from the T. cruzi Genome Initiative, we show that the T. cruzi HSP10 coding region is 300 bp long, encoding a polypeptide of 100 amino acids with highest sequence identity (83%) to HSP10 of Trypanosoma brucei and lowest (28%) to HSP10 of Escherichia coli. The T. cruzi HSP10 genes are arranged in 3 tandemly repeated copies, which give rise to a major mRNA of 1.0 kb that remains unaltered during heat shock; a smaller mRNA species is induced at 37 degrees C by alternate polyadenylation. Finally, the presence of a conserved 5-amino acid residue deletion in trypanosomatid HSP10s led us to generate a molecular model of the T. cruzi HSP10 structure. The oligomeric assembly of this model shows some peculiar characteristics that may have functional significance.

Electrostatic properties in the catalytic site of papain: A possible regulatory mechanism for the reactivity of the ion pair.

We present an analysis of the electrostatic properties in the catalytic site of papain (EC 3.4.22.2), an archetype enzyme of the C1 cysteine proteinase family, and we investigate their possible role in the formation, stabilization and regulation of the Cys25((-))...His159((+)) catalytic ion pair. The electrostatic properties were computed using a reassociation method based in multicentered multipolar expansions obtained from ab initio quantum calculations of overlapping protein fragments. Solvent effects were introduced by coupling the use of multicentered multipolar expansions to two continuum boundary element methods to solve the Poisson and the linearized Poisson-Boltzmann equations. The electrostatic profile found in the proton transfer region of papain showed that this enzyme has a well-defined electrostatic environment to favor the formation and stabilization of the catalytic ion pair. The papain catalytic site electrostatic profile can be considered as an electrostatic fingerprint of the papain family with the following characteristics: (i) the presence of a net electric field highly aligned in the (Cys25)-SG-->(His159)-ND1 direction; (ii) the electrostatic profile has a saddle-point character; (iii) it is basically a local environmental effect. Furthermore, our analysis describes a possible regulatory mechanism (the E(SG-->ND1) attenuation effect) controlling the ion pair reactivity and permits to infer the Asp57 acidic residue as the most probable candidate to act as the electrostatic modulator.

Stochastic molecular dynamics of peanut lectin PNA complex with T-antigen disaccharide.

The stochastic boundary molecular dynamics simulation method was applied to investigate the structure of a complex comprised of a tetrameric peanut lectin and the tumour-associated disaccharide, Galbeta1-3GalNAc (T-antigen). Only a small region encompassing the active site was explicitly included in the calculations, but the electrical contribution of most outer atoms was taken into account by adding to the effective potential a term coming from an electrostatic potential grid that was pre-calculated and used to approximate the electrostatic energy and the force at any point in the interacting site. Results of simulating the intermolecular hydrogen bond network agree fairly well with X-ray experiments. An estimation of the direct and water-mediated interaction mean lifetimes and mean water residence times around the T-antigen oxygen atoms was computed over 400 ps. Monitoring the behaviour of water molecules within the active site revealed that there is a constant exchange of water with the bulk, especially in the proximity of ASN41, ASN127 and GLU129. The temporal evolution of the glycosidic linkage was also investigated and compared with simulations of T-antigen in solution. These studies of peanut lectins-sugar complexes clearly emphasize the importance of bound water molecules in generating carbohydrate specificity.