Navegando por Autor "Salinas, Roberto Kopke"
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Item CALX-CBD1 Ca2D-binding cooperativity studied by NMR spectroscopy and ITC with bayesian statistics.(2020) Cardoso, Marcus Vinícius Cangussu; Rivera, José Domingo; Vitale, Phelipe Augusto Mariano; Degenhardt, Maximilia Frazão de Souza; Abiko, Layara Akemi; Oliveira, Cristiano Luis Pinto de; Salinas, Roberto KopkeThe Naþ/Ca2þ exchanger of Drosophila melanogaster, CALX, is the main Ca2þ-extrusion mechanism in olfactory sensory neurons and photoreceptor cells. Naþ/Ca2þ exchangers have two Ca2þ sensor domains, CBD1 and CBD2. In contrast to the mammalian homologs, CALX is inhibited by Ca2þ binding to CALX-CBD1, whereas CALX-CBD2 does not bind Ca2þ at physiological concentrations. CALX-CBD1 consists of a b-sandwich and displays four Ca2þ-binding sites at the tip of the domain. In this study, we used NMR spectroscopy and isothermal titration calorimetry (ITC) to investigate the cooperativity of Ca2þ bind- ing to CALX-CBD1. We observed that this domain binds Ca2þ in the slow exchange regime at the NMR chemical shift timescale. Ca2þ binding restricts the dynamics in the Ca2þ-binding region. Experiments of 15N chemical exchange saturation transfer and 15N R2 dispersion allowed the determination of Ca2þ dissociation rates (30 s1 ). NMR titration curves of residues in the Ca2þ- binding region were sigmoidal because of the contribution of chemical exchange to transverse magnetization relaxation rates, R2. Hence, a novel, to our knowledge, approach to analyze NMR titration curves was proposed. Ca2þ-binding cooperativity was examined assuming two different stoichiometric binding models and using a Bayesian approach for data analysis. Fittings of NMR and ITC binding curves to the Hill model yielded nHill 2.9, near maximal cooperativity (nHill 1⁄4 4). By assuming a stepwise model to interpret the ITC data, we found that the probability of binding from 2 up to 4 Ca2þ is approximately three orders of magnitude higher than that of binding a single Ca2þ. Hence, four Ca2þ ions bind almost simultaneously to CALX-CBD1. Coop- erative Ca2þ binding is key to enable this exchanger to efficiently respond to changes in the intracellular Ca2þ concentration in sensory neuronal cells.Item Role of a high centrality residue in protein dynamics and thermal stability.(2021) Almeida, Vitor Medeiros; Chaudhuri, Apala; Cardoso, Marcus Vinícius Cangussu; Matsuyama, Bruno Yasui; Ferreira, Gláucio Monteiro; Trossini, Gustavo Henrique Goulart; Salinas, Roberto Kopke; Loria, J. Patrick; Marana, Sandro RobertoCentralities determined from Residue Interaction Networks (RIN) in proteins have been used to predict aspects of their structure and dynamics. Here, we correlate the Eigenvector Centrality (Ec) with the rate constant for thermal denaturation (kden) of the HisF protein from Thermotoga maritima based on 12 single alanine substitution mutants. The molecular basis for this correlation was further explored by studying a mutant containing a replacement of a high Ec residue, Y182A, which displayed increased kden at 80 ◦C. The crystallographic structure of this mutant showed few changes, mostly in two flexible loops. The 1 H-15N -HSQC showed only subtle changes of cross peak positions for residues located near the mutation site and scattered throughout the structure. However, the comparison of the RIN showed that Y182 is the vertex of a set of high centrality residues that spreads throughout the HisF structure, which is lacking in the mutant. Cross-correlation displacements of Cα calculated from a molecular dynamics simulation at different temperatures showed that the Y182A mutation reduced the correlated movements in the HisF structure above 70 ◦C. 1 H-15N NMR chemical shift covariance using temperature as perturbation were consistent with these results. In conclusion the increase in temperature drives the structure of the mutant HisF-Y182A into a less connected state, richer in non-concerted motions, located predominantly in the C-terminal half of the protein where Y182 is placed. Conversely, wild-type HisF responds to increased temperature as a single unit. Hence the replacement of a high Ec residue alters the dis- tribution of thermal energy through HisF structure.Item Structural basis for effector recognition by an antibacterial type IV secretion system.(2022) Oka, Gabriel Umaji; Souza, Diorge Paulo de; Cenens, William; Matsuyama, Bruno Yasui; Cardoso, Marcus Vinícius Cangussu; Oliveira, Luciana C.; Lima, Filipe da Silva; Cuccovia, Iolanda Midea; Carvalho, Cristiane Rodrigues Guzzo; Salinas, Roberto Kopke; Farah, Shaker ChuckMany soil-, water-, and plant-associated bacterial species from the orders Xanthomonadales, Burkholderales, and Neisseriales carry a type IV secretion system (T4SS) specialized in translocating effec- tor proteins into other gram-negative species, leading to target cell death. These effectors, known as X-Tfes, carry a carboxyl- terminal domain of ∼120 residues, termed XVIPCD, characterized by several conserved motifs and a glutamine-rich tail. Previous studies showed that the XVIPCD is required for interaction with the T4SS coupling protein VirD4 and for T4SS-dependent translo- cation. However, the structural basis of the XVIPCD–VirD4 interac- tion is unknown. Here, we show that the XVIPCD interacts with the central all-alpha domain of VirD4 (VirD4AAD). We used solution NMR spectroscopy to solve the structure of the XVIPCD of X-TfeXAC2609 from Xanthomonas citri and to map its interaction surface with VirD4AAD. Isothermal titration calorimetry and in vivo Xanthomonas citri versus Escherichia coli competition assays using wild-type and mutant X-TfeXAC2609 and X-TfeXAC3634 indicate that XVIPCDs can be divided into two regions with distinct functions: the well-folded N-terminal region contains specific conserved motifs that are responsible for interactions with VirD4AAD, while both N- and carboxyl-terminal regions are required for effective X-Tfe translocation into the target cell. The conformational stabil- ity of the N-terminal region is reduced at and below pH 7.0, a prop- erty that may facilitate X-Tfe unfolding and translocation through the more acidic environment of the periplasm.