Similarity between class A and class B G-protein-coupled receptors exemplified through calcitonin gene-related peptide receptor modelling and mutagenesis studies

Modelling class B G-protein-coupled receptors (GPCRs) using class A GPCR structural templates is difficult due to lack of homology. The plant GPCR, GCR1, has homology to both class A and class B GPCRs. We have used this to generate a class A-class B alignment, and by incorporating maximum lagged correlation of entropy and hydrophobicity into a consensus score, we have been able to align receptor transmembrane regions. We have applied this analysis to generate active and inactive homology models of the class B calcitonin gene-related peptide (CGRP) receptor, and have supported it with site-directed mutagenesis data using 122 CGRP receptor residues and 144 published mutagenesis results on other class B GPCRs. The variation of sequence variability with structure, the analysis of polarity violations, the alignment of group-conserved residues and the mutagenesis results at 27 key positions were particularly informative in distinguishing between the proposed and plausible alternative alignments. Furthermore, we have been able to associate the key molecular features of the class B GPCR signalling machinery with their class A counterparts for the first time. These include the [K/R]KLH motif in intracellular loop 1, [I/L]xxxL and KxxK at the intracellular end of TM5 and TM6, the NPXXY/VAVLY motif on TM7 and small group-conserved residues in TM1, TM2, TM3 and TM7. The equivalent of the class A DRY motif is proposed to involve Arg(2.39), His(2.43) and Glu(3.46), which makes a polar lock with T(6.37). These alignments and models provide useful tools for understanding class B GPCR function.

Biochemical and Structural Studies on PrfA, the Transcriptional Regulator of Virulence in Listeria monocytogenes

Abstract

Listeria monocytogenes is a gram-positive soil saprophytic bacterium that is capable of causing fatal infection in humans. The main virulence regulator PrfA, a member of the Crp/FNR family of transcriptional regulators, activates the expression of essential proteins required for host cell invasion and cell-to-cell spread. The mechanism of PrfA activation and the identity of its small molecule coactivator have remained a mystery for more than 20 years, but it is hypothesized that PrfA shares mechanistic similarity to the E. coli cAMP binding protein, Crp. Crp activates gene expression by binding cAMP, increasing the DNA binding affinity of the protein and causing a significant DNA bend that facilitates RNA polymerase binding and downstream gene activation. Our data suggests PrfA activates virulence protein expression through a mechanism distinct from the canonical Crp activation mechanism that involves a combination of cysteine residue reduction and glutathione (GSH) binding.

Listeria lacking glutathione synthase (ΔgshF) is avirulent in mice; however virulence is rescued when the bacterium expresses the constitutively active PrfA mutant G145S. Interestingly, Listeria expressing a PrfA mutant in which its four cysteines are mutated to alanine (Quad PrfA), demonstrate a 30-fold decrease in virulence. The Quad and ΔgshF double mutant strains are avirulent. DNA-binding affinity, measured through fluorescence polarization assays, indicate reduction of the cysteine side chains is sufficient to allow PrfA to binds its physiological promoters Phly and PactA with low nanomolar affinity. Oxidized PrfA binds the promoters poorly.

Unexpectedly, Quad also binds promoter DNA with nanomolar affinity, suggesting that the cysteines play a role in transcription efficiency in addition to DNA binding. Both PrfA and Quad bind GSH at physiologically relevant and comparable affinities, however GSH did not affect DNA binding in either case. Thermal denaturation assays suggest that Quad and wild-type PrfA differ structurally upon binding GSH, which supports the in vivo difference in infection between the regulator and its mutant.

Structures of PrfA in complex with cognate DNA, determined through X-ray crystallography, further support the disparity between PrfA and Crp activation mechanisms as two structures of reduced PrfA bound to Phly (PrfA-Phly30 and PrfA-Phly24) suggest the DNA adopts a less bent DNA conformation when compared to Crp-cAMP- DNA. The structure of Quad-Phly30 confirms the DNA-binding data as the protein-DNA complex adopts the same overall conformation as PrfA-Phly.

From these results, we hypothesize a two-step activation mechanism wherein PrfA, oxidized upon cell entry and unable to bind DNA, is reduced upon its intracellular release and binds DNA, causing a slight bend in the promoter and small increase in transcription of PrfA-regulated genes. The structures of PrfA-Phly30 and PrfA-Phly24 likely visualize this intermediate complex. Increasing concentrations of GSH shift the protein to a (PrfA-GSH)-DNA complex which is fully active transcriptionally and is hypothesized to resemble closely the transcriptionally active structure of the cAMP-(Crp)-DNA complex. Thermal denaturation results suggest Quad PrfA is deficient in this second step, which explains the decrease in virulence and implicates the cysteine residues as critical for transcription efficiency. Further structural and biochemical studies are on-going to clarify this mechanism of activation.

Structural and Functional Characterization of the Human Tribbles Homologue 2 Pseudokinase

The Tribbles Homologues are a family of three eukaryotic pseudokinases (Trb1, Trb2, Trb3) that act as allosteric inhibitors and regulatory scaffold sites in pathways governing adipogenesis, cell proliferation and insulin signaling. The Tribbles Homologues have the same overall tertiary structure of the eukaryotic protein kinase domain, but lack multiple residues necessary to catalysis in the nucleotide-binding P-loop and the Mg2+-coordinating DFG motif. Trb1 has been shown conclusively to be incapable of binding ATP, whereas a recent study presents evidence that Trb2 autophosphorylates independently of Mg2+ in vitro. This finding is surprising given the high degree of sequence similarity between the two proteins (71%), and suggests unique nucleotide binding and phosphotransfer mechanisms. The goal of this project was to investigate whether Trb2 possesses kinase activity or not and determine its structural basis. A method for the high-yield recombinant expression and purification of stable Trb2 was developed. Trb2 nucleotide binding and autophosphorylation could not be detected across multiple experimental approaches, including thermal shift assays, MANT-ATP fluorescence, radiolabeled phosphate incorporation, and nonspecific ATPase activity assays. Further characterization also revealed that Trb2 forms homomultimers with possible functional consequences, and extensive crystallization screening has yielded multiple promising conditions that could produce diffraction-quality crystals with further optimization. This project explores the difficulties in functionally characterizing putatively active pseudokinases, and proposes a structural basis for the conserved pseudokinase features of the Tribbles homologues.

Structural Aspects of the Distinct Biochemical Properties of Glutaredoxin 1 and Glutaredoxin 2 from Saccharomyces cerevisiae

Glutaredoxins (Grxs) are small (9-12 kDa) heat-stable proteins that are ubiquitously distributed. In Saccharomyces cerevisiae, seven Grx enzymes have been identified. Two of them (yGrx1 and yGrx2) are dithiolic, possessing a conserved Cys-Pro-Tyr-Cys motif. Here, we show that yGrx2 has a specific activity 15 times higher than that of yGrx1, although these two oxidoreductases share 64% identity and 85% similarity with respect to their amino acid sequences. Further characterization of the enzymatic activities through two-substrate kinetics analysis revealed that yGrx2 possesses a lower Km for glutathione and a higher turnover than yGrx1. To better comprehend these biochemical differences, the pK(a) of the N-terminal active-site cysteines (Cys27) of these two proteins and of the yGrx2-C30S mutant were determined. Since the pK(a) values of the yGrx1 and yGix2 Cys27 residues are very similar, these parameters cannot account for the difference observed between their specific activities. Therefore, crystal structures of yGrx2 in the oxidized form and with a glutathionyl mixed disulfide were determined at resolutions of 2.05 and 1.91 angstrom, respectively. Comparisons of yGrx2 structures with the recently determined structures of yGrx1 provided insights into their remarkable functional divergence. We hypothesize that the substitutions of Ser23 and Gln52 in yGrx1 by Ala23 and Glu52 in yGrx2 modify the capability of the active-site C-terminal cysteine to attack the mixed disulfide between the N-terminal active-site cysteine and the glutathione molecule. Mutagenesis studies supported this hypothesis. The observed structural and functional differences between yGrx1 and yGrx2 may reflect variations in substrate specificity. (C) 2008 Elsevier Ltd. All rights reserved.

Lung Age Is Related To Carotid Structural Alterations In Hypertensive Subjects.

Hypertensive patients exhibit higher cardiovascular risk and reduced lung function compared with the general population. Whether this association stems from the coexistence of two highly prevalent diseases or from direct or indirect links of pathophysiological mechanisms is presently unclear. This study investigated the association between lung function and carotid features in non-smoking hypertensive subjects with supposed normal lung function. Hypertensive patients (n = 67) were cross-sectionally evaluated by clinical, hemodynamic, laboratory, and carotid ultrasound analysis. Forced vital capacity, forced expired volume in 1 second and in 6 seconds, and lung age were estimated by spirometry. Subjects with ventilatory abnormalities according to current guidelines were excluded. Regression analysis adjusted for age and prior smoking history showed that lung age and the percentage of predicted spirometric parameters associated with common carotid intima-media thickness, diameter, and stiffness. Further analyses, adjusted for additional potential confounders, revealed that lung age was the spirometric parameter exhibiting the most significant regression coefficients with carotid features. Conversely, plasma C-reactive protein and matrix-metalloproteinases-2/9 levels did not influence this relationship. The present findings point toward lung age as a potential marker of vascular remodeling and indicate that lung and vascular remodeling might share common pathophysiological mechanisms in hypertensive subjects.

Structural Connectivity Of The Default Mode Network And Cognition In Alzheimer׳s Disease.

Disconnectivity between the Default Mode Network (DMN) nodes can cause clinical symptoms and cognitive deficits in Alzheimer׳s disease (AD). We aimed to examine the structural connectivity between DMN nodes, to verify the extent in which white matter disconnection affects cognitive performance. MRI data of 76 subjects (25 mild AD, 21 amnestic Mild Cognitive Impairment subjects and 30 controls) were acquired on a 3.0T scanner. ExploreDTI software (fractional Anisotropy threshold=0.25 and the angular threshold=60°) calculated axial, radial, and mean diffusivities, fractional anisotropy and streamline count. AD patients showed lower fractional anisotropy (P=0.01) and streamline count (P=0.029), and higher radial diffusivity (P=0.014) than controls in the cingulum. After correction for white matter atrophy, only fractional anisotropy and radial diffusivity remained significantly lower in AD compared to controls (P=0.003 and P=0.05). In the parahippocampal bundle, AD patients had lower mean and radial diffusivities (P=0.048 and P=0.013) compared to controls, from which only radial diffusivity survived for white matter adjustment (P=0.05). Regression models revealed that cognitive performance is also accounted for by white matter microstructural values. Structural connectivity within the DMN is important to the execution of high-complexity tasks, probably due to its relevant role in the integration of the network.

Electronic And Structural Study Of Pt-modified Au Vicinal Surfaces: A Model System For Pt-au Catalysts.

Two single crystalline surfaces of Au vicinal to the (111) plane were modified with Pt and studied using scanning tunneling microscopy (STM) and X-ray photoemission spectroscopy (XPS) in ultra-high vacuum environment. The vicinal surfaces studied are Au(332) and Au(887) and different Pt coverage (θPt) were deposited on each surface. From STM images we determine that Pt deposits on both surfaces as nanoislands with heights ranging from 1 ML to 3 ML depending on θPt. On both surfaces the early growth of Pt ad-islands occurs at the lower part of the step edge, with Pt ad-atoms being incorporated into the steps in some cases. XPS results indicate that partial alloying of Pt occurs at the interface at room temperature and at all coverage, as suggested by the negative chemical shift of Pt 4f core line, indicating an upward shift of the d-band center of the alloyed Pt. Also, the existence of a segregated Pt phase especially at higher coverage is detected by XPS. Sample annealing indicates that the temperature rise promotes a further incorporation of Pt atoms into the Au substrate as supported by STM and XPS results. Additionally, the catalytic activity of different PtAu systems reported in the literature for some electrochemical reactions is discussed considering our findings.

A New Platinum Complex With Tryptophan: Synthesis, Structural Characterization, Dft Studies And Biological Assays In Vitro Over Human Tumorigenic Cells.

A new platinum(II) complex with the amino acid L-tryptophan (trp), named Pt-trp, was synthesized and characterized. Elemental, thermogravimetric and ESI-QTOF mass spectrometric analyses led to the composition [Pt(C11H11N2O2)2]⋅6H2O. Infrared spectroscopic data indicate the coordination of trp to Pt(II) through the oxygen of the carboxylate group and also through the nitrogen atom of the amino group. The (13)C CP/MAS NMR spectroscopic data confirm coordination through the oxygen atom of the carboxylate group, while the (15)N CP/MAS NMR data confirm coordination of the nitrogen of the NH2 group to the metal. Density functional theory (DFT) studies were applied to evaluate the cis and trans coordination modes of trp to platinum(II). The trans isomer was shown to be energetically more stable than the cis one. The Pt-trp complex was evaluated as a cytotoxic agent against SK-Mel 103 (human melanoma) and Panc-1 (human pancreatic carcinoma) cell lines. The complex was shown to be cytotoxic over the considered cells.

Structural And Biomechanical Changes In The Achilles Tendon After Chronic Treatment With Statins.

Cases of tendinopathy and tendon ruptures have been reported as side effects associated with statin therapy. This work assessed possible changes in the structural and biomechanical properties of the tendons after chronic treatment with statins. Wistar rats were divided into the following groups: treated with atorvastatin (A-20 and A-80), simvastatin (S-20 and S-80) and the group that received no treatment (C). The doses of statins were calculated using allometric scaling, based on the doses of 80 mg/day and 20 mg/day recommended for humans. The morphological aspect of the tendons in A-20, S-20 and S-80 presented signals consistent with degeneration. Both the groups A-80 and S-80 showed a less pronounced metachromasia in the compression region of the tendons. Measurements of birefringence showed that A-20, A-80 and S-80 groups had a lower degree of organization of the collagen fibers. In all of the groups treated with statins, the thickness of the epitenon was thinner when compared to the C group. In the biomechanical tests the tendons of the groups A-20, A-80 and S-20 were less resistant to rupture. Therefore, statins affected the organization of the collagen fibers and decreased the biomechanical strength of the tendons, making them more predisposed to ruptures.

Kinase Inhibitor Profile For Human Nek1, Nek6, And Nek7 And Analysis Of The Structural Basis For Inhibitor Specificity.

Human Neks are a conserved protein kinase family related to cell cycle progression and cell division and are considered potential drug targets for the treatment of cancer and other pathologies. We screened the activation loop mutant kinases hNek1 and hNek2, wild-type hNek7, and five hNek6 variants in different activation/phosphorylation statesand compared them against 85 compounds using thermal shift denaturation. We identified three compounds with significant Tm shifts: JNK Inhibitor II for hNek1(Δ262-1258)-(T162A), Isogranulatimide for hNek6(S206A), andGSK-3 Inhibitor XIII for hNek7wt. Each one of these compounds was also validated by reducing the kinases activity by at least 25%. The binding sites for these compounds were identified by in silico docking at the ATP-binding site of the respective hNeks. Potential inhibitors were first screened by thermal shift assays, had their efficiency tested by a kinase assay, and were finally analyzed by molecular docking. Our findings corroborate the idea of ATP-competitive inhibition for hNek1 and hNek6 and suggest a novel non-competitive inhibition for hNek7 in regard to GSK-3 Inhibitor XIII. Our results demonstrate that our approach is useful for finding promising general and specific hNekscandidate inhibitors, which may also function as scaffolds to design more potent and selective inhibitors.

Chemical And Structural Analyses Of Titanium Plates Retrieved From Patients.

The aim of this study was to evaluate the microscopic structure and chemical composition of titanium bone plates and screws retrieved from patients with a clinical indication and to relate the results to the clinical conditions associated with the removal of these devices. Osteosynthesis plates and screws retrieved from 30 patients between January 2010 and September 2013 were studied by metallographic, gas, and energy dispersive X-ray (EDX) analyses and the medical records of these patients were reviewed. Forty-eight plates and 238 screws were retrieved. The time elapsed between plate and screw insertion and removal ranged between 11 days and 10 years. Metallographic analysis revealed that all the plates were manufactured from commercially pure titanium (CP-Ti). The screw samples analyzed consisted of Ti-6Al-4V alloy, except four samples, which consisted of CP-Ti. Titanium plates studied by EDX analysis presented greater than 99.7% titanium by mass. On gas analysis of Ti-6Al-4V screws, three samples were outside the standard values. One CP-Ti screw sample and one plate sample also presented an oxygen analysis value above the standard. The results indicated that the physical properties and chemical compositions of the plates and screws did not correspond with the need to remove these devices or the time of retention.

Towards A Deeper Understanding Of Structural Biomass Recalcitrance Using Phase-contrast Tomography.

The development of technological routes to convert lignocellulosic biomass to liquid fuels requires an in-depth understanding of the cell wall architecture of substrates. Essential pretreatment processes are conducted to reduce biomass recalcitrance and usually increase the reactive surface area. Quantitative three-dimensional information about both bulk and surface structural features of substrates needs to be obtained to expand our knowledge of substrates. In this work, phase-contrast tomography (PCT) was used to gather information about the structure of a model lignocellulosic biomass (piassava fibers). The three-dimensional cellular organization of piassava fibers was characterized by PCT using synchrotron radiation. This technique enabled important physical features that describe the substrate piassava fibers to be visualized and quantified. The external surface area of a fiber and internal surface area of the pores in a fiber could be determined separately. More than 96% of the overall surface area available to enzymes was in the bulk substrate. The pore surface area and length exhibited a positive linear relationship, where the slope of this relationship depended on the plant tissue. We demonstrated that PCT is a powerful tool for the three-dimensional characterization of the cell wall features related to biomass recalcitrance. Original and relevant quantitative information about the structural features of the analyzed material were obtained. The data obtained by PCT can be used to improve processing routes to efficiently convert biomass feedstock into sugars.

Key Participants Of The Tumor Microenvironment Of The Prostate: An Approach Of The Structural Dynamic Of Cellular Elements And Extracellular Matrix Components During Epithelial-stromal Transition.

Cancer is a multistep process that begins with the transformation of normal epithelial cells and continues with tumor growth, stromal invasion and metastasis. The remodeling of the peritumoral environment is decisive for the onset of tumor invasiveness. This event is dependent on epithelial-stromal interactions, degradation of extracellular matrix components and reorganization of fibrillar components. Our research group has studied in a new proposed rodent model the participation of cellular and molecular components in the prostate microenvironment that contributes to cancer progression. Our group adopted the gerbil Meriones unguiculatus as an alternative experimental model for prostate cancer study. This model has presented significant responses to hormonal treatments and to development of spontaneous and induced neoplasias. The data obtained indicate reorganization of type I collagen fibers and reticular fibers, synthesis of new components such as tenascin and proteoglycans, degradation of basement membrane components and elastic fibers and increased expression of metalloproteinases. Fibroblasts that border the region, apparently participate in the stromal reaction. The roles of each of these events, as well as some signaling molecules, participants of neoplastic progression and factors that promote genetic reprogramming during epithelial-stromal transition are also discussed.

Structural aspects of the p.P222Q homozygous mutation of HSD3B2 gene in a patient with congenital adrenal hyperplasia

Type II 3β-hydroxysteroid dehydrogenase/Δ5-Δ4-isomerase (3β-HSD2), encoded by the HSD3B2 gene, is a key enzyme involved in the biosynthesis of all the classes of steroid hormones. Deleterious mutations in the HSD3B2 gene cause the classical deficiency of 3β-HSD2, which is a rare autosomal recessive disease that leads to congenital adrenal hyperplasia (CAH). CAH is the most frequent cause of ambiguous genitalia and adrenal insufficiency in newborn infants with variable degrees of salt losing. Here we report the molecular and structural analysis of the HSD3B2 gene in a 46,XY child, who was born from consanguineous parents, and presented with ambiguous genitalia and salt losing. The patient carries a homozygous nucleotide c.665C>A change in exon 4 that putatively substitutes the proline at codon 222 for glutamine. Molecular homology modeling of normal and mutant 3β-HSD2 enzymes emphasizes codon 222 as an important residue for the folding pattern of the enzyme and validates a suitable model for analysis of new mutations.

Enzyme kinetics, structural analysis and molecular modeling studies on a series of Schistosoma mansoni PNP inhibitors

The enzyme purine nucleoside phosphorylase from Schistosoma mansoni (SmPNP) is an attractive molecular target for the development of novel drugs against schistosomiasis, a neglected tropical disease that affects about 200 million people worldwide. In the present work, enzyme kinetic studies were carried out in order to determine the potency and mechanism of inhibition of a series of SmPNP inhibitors. In addition to the biochemical investigations, crystallographic and molecular modeling studies revealed important molecular features for binding affinity towards the target enzyme, leading to the development of structure-activity relationships (SAR).