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Cambridge, United Kingdom

Roos G.,Vrije Universiteit Brussel | Roos G.,Brussels Center for Redox Biology | Foloppe N.,51 Natal Road | Messens J.,Vlaams Instituut voor Biotechnologie VIB | And 2 more authors.
Antioxidants and Redox Signaling | Year: 2013

Many cellular functions involve cysteine chemistry via thiol-disulfide exchange pathways. The nucleophilic cysteines of the enzymes involved are activated as thiolate. A thiolate is much more reactive than a neutral thiol. Therefore, determining and understanding the pKas of functional cysteines are important aspects of biochemistry and molecular biology with direct implications for redox signaling. Here, we describe the experimental and theoretical methods to determine cysteine pKa values, and we examine the factors that control these pKas. Drawing largely on experience gained with the thioredoxin superfamily, we examine the roles of solvation, charge-charge, helix macrodipole, and hydrogen bonding interactions as pK a-modulating factors. The contributions of these factors in influencing cysteine pKas and the associated chemistry, including the relevance for the reaction kinetics and thermodynamics, are discussed. This analysis highlights the critical role of direct hydrogen bonding to the cysteine sulfur as a key factor modulating the equilibrium between thiol S-H and thiolate S-. This role is easily understood intuitively and provides a framework for biochemical functional insights. © 2013, Mary Ann Liebert, Inc.

Xu X.,CNRS Laboratory of Biology and Applied Pharmacology | Xu X.,East China Normal University | Ben Imeddourene A.,CNRS Laboratory of Biology and Applied Pharmacology | Ben Imeddourene A.,University Pierre and Marie Curie | And 4 more authors.
Biochemistry | Year: 2014

We investigated how the intrinsic sequence-dependent properties probed via the phosphate linkages (BI 虠 BII equilibrium) influence the preferred shape of free DNA, and how this affects the nucleosome formation. First, this exploits NMR solution studies of four B-DNA dodecamers that together cover 39 base pairs of the 5′ half of the sequence 601, of special interest for nucleosome formation. The results validate our previous prediction of a systematic, general sequence effect on the intrinsic backbone BII propensities. NMR provides new evidence that the backbone behavior is intimately coupled to the minor groove width. Second, application of the backbone behavior predictions to the full sequence 601 and other relevant sequences demonstrates that alternation of intrinsic low and high BII propensities, coupled to intrinsic narrow and wide minor grooves, largely coincides with the sinusoidal variations of the DNA minor groove width observed in crystallographic structures of the nucleosome. This correspondence is much poorer with low affinity sequences. Overall, the results indicate that nucleosome formation involves an indirect readout process implicating pre-existing DNA minor groove conformations. It also illustrates how the prediction of the intrinsic structural DNA behavior offers a powerful framework to gain explanatory insight on how proteins read DNA. © 2014 American Chemical Society.

Lounnas V.,Radboud University Nijmegen | Ritschel T.,Computational Drug Discovery | Kelder J.,Beethovengaarde 97 | McGuire R.,BioAxis Research BV | And 2 more authors.
Computational and Structural Biotechnology Journal | Year: 2013

The past decade has witnessed a paradigm shift in preclinical drug discovery with structure-based drug design (SBDD) making a comeback while high-throughput screening (HTS) methods have continued to generate disappointing results. There is a deficit of information between identified hits and the many criteria that must be fulfilled in parallel to convert them into preclinical candidates that have a real chance to become a drug. This gap can be bridged by investigating the interactions between the ligands and their receptors. Accurate calculations of the free energy of binding are still elusive; however progresses were made with respect to how one may deal with the versatile role of water. A corpus of knowledge combining X-ray structures, bioinformatics and molecular modeling techniques now allows drug designers to routinely produce receptor homology models of increasing quality. These models serve as a basis to establish and validate efficient rationales used to tailor and/or screen virtual libraries with enhanced chances of obtaining hits. Many case reports of successful SBDD show how synergy can be gained from the combined use of several techniques. The role of SBDD with respect to two different classes of widely investigated pharmaceutical targets: (a) protein kinases (PK) and (b) G-protein coupled receptors (GPCR) is discussed. Throughout these examples prototypical situations covering the current possibilities and limitations of SBDD are presented. © 2013 Lounnas et al.

Karshikoff A.,Bulgarian Academy of Science | Nilsson L.,Karolinska Institutet | Foloppe N.,51 Natal Road
Biochemistry | Year: 2013

E. coli DsbA is an intensively studied enzyme of the thioredoxin superfamily of thiol-disulfide oxidoreductases. DsbA catalyzes the disulfide bond formation and folding of proteins in the bacterial periplasm. DsbA and its mutants have highlighted the strong and puzzling influence of the -C-X1-X2-C- active site variants, found across the thioredoxin superfamily, on the ionization and redox properties of this site. However, the interpretation of these observations remains wanting, largely due to a dearth of structural information. Here, molecular dynamics simulations are used to provide extensive information on the structure and dynamics of reduced -C30-X31-X32-C33- motifs in wild type DsbA and 13 of its mutants. These simulations are combined with calculations of the pK of H32 and of the very low pK of the catalytic cysteine C30. In wild type DsbA, the titrations of C30 and H32 are shown to be coupled; the protonation states and dynamics of H32 are examined. The thiolate of C30 is stabilized by hydrogen bonds with the protein. Modulation of these hydrogen bonds by alteration of residue X32 has the greatest impact on the pK of C30, which rationalizes its higher pK in thioredoxin and tryparedoxin. Because of structural constrains, residue X31 has only an indirect and weak influence on the pK of C30. The dynamics of C30 is clearly related to its stabilizing interactions and pK value. Although relatively small differences between pKs were not reproduced in the calculations, the major trends are explained, adding new insights to our understanding of enzymes in this family. © 2013 American Chemical Society.

Foloppe N.,51 Natal Road | Vlamis-Gardikas A.,University of Patras | Nilsson L.,Karolinska Institutet
Biochemistry | Year: 2012

The -Cys-X1-X2-Cys- active site motif is central to the function of enzymes of the thioredoxin superfamily, including glutaredoxins. Their chemistry depends on the lowered pKa of the N-terminal thiolate cysteine of the -Cys-X1-X2-Cys- sequence; therefore its structure, dynamics, and electrostatics matter. Much information about the glutaredoxin structures was obtained by nuclear magnetic resonance (NMR), yet these various NMR structures produced heterogeneous and discordant views of the -Cys-X1-X2-Cys- motifs. This study addresses these inconsistencies by a computational and experimental investigation of three diverse reduced -Cys-X1-X2-Cys- motifs, from human glutaredoxin 1 (hGrx1), Escherichia coli glutaredoxin 2 (EcGrx2), and T4 virus glutaredoxin (T4Grx). The NMR models do not account for the low pKa of the N-terminal cysteine. However, extensive investigations of the NMR conformers by simulations yielded consensus structures for the -Cys-X1-X2-Cys- motif, with well-defined orientations for the cysteines. pKa calculations indicated that the consensus structure stabilizes the thiolate by local hydrogen bonds. The consensus structures of EcGrx2 and T4Grx formed the basis for predicting low pKa values for their N-terminal cysteines. Subsequent experimental titrations showed that these pKa values are <5, supporting the validity of the consensus structure. The simulations also revisited the conformational dynamics of side chains around the -Cys-X1-X2-Cys- motif, which allowed reconciliation of calculated and measured pKa values for important hGrx1 mutants. The conformational spread of these side chains, which differs between NMR and molecular dynamics models, is likely to be relevant to substrate recognition. The new structural models determined in this work should prove to be valuable in future molecular studies of the glutaredoxins. © 2012 American Chemical Society.

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