Publications

2012

• Nucleotide recognition by the initiation factor aIF5B: Free energy simulations of a neoclassical GTPase.
  
  • Simonson Thomas
  • Satpati Priyadarshi
  Proteins - Structure, Function and Bioinformatics, Wiley, 2012, 80 (12), pp.2742-57. The GTPase aIF5B is a universally conserved initiation factor that assists ribosome assembly. Crystal structures of its nucleotide complexes, X-ray(GTP) and X-ray(GDP), are similar in the nucleotide vicinity, but differ in the orientation of a distant domain IV. This has led to two, contradictory, mechanistic models. One postulates that X-ray(GTP) and X-ray(GDP) are, respectively, the active, "ON" and the inactive, "OFF" states; the other postulates that both structures are OFF, whereas the ON state is still uncharacterized. We study GTP/GDP binding using molecular dynamics and a continuum electrostatic free energy method. We predict that X-ray(GTP) has a ≈ 3 kcal/mol preference to bind GDP, apparently contradicting its assignment as ON. However, the preference arises mainly from a single, nearby residue from the switch 2 motif: Glu81, which becomes protonated upon GTP binding, with a free energy cost of about 4 kcal/mol. We then propose a different model, where Glu81 protonation/deprotonation defines the ON/OFF states. With this model, the X-ray(GTP):GTP complex, with its protonated Glu81, is ON, whereas X-ray(GTP):GDP is OFF. The model postulates that distant conformational changes such as domain IV rotation are "uncoupled" from GTP/GDP exchange and do not affect the relative GTP/GDP binding affinities. We analyze the model using a general thermodynamic framework for GTPases. It yields rather precise predictions for the nucleotide specificities of each state, and the state specificities of each nucleotide, which are roughly comparable to the homologues IF2 and aIF2, despite the lack of any conformational switching in the model. © 2012 Wiley Periodicals, Inc. (10.1002/prot.24158)
  DOI : 10.1002/prot.24158
• Structure and cellular dynamics of Deinococcus radiodurans single-stranded DNA (ssDNA)-binding protein (SSB)-DNA complexes.
  
  • Ngo Khanh V.
  • Chitteni-Pattu Sindhu
  • Norais Cedric A.
  • Cox Michael M.
  • George Nicholas P.
  • Keck James L.
  • Battista John R.
  Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2012, 287 (26), pp.22123-32. The single-stranded DNA (ssDNA)-binding protein from the radiation-resistant bacterium Deinococcus radiodurans (DrSSB) functions as a homodimer in which each monomer contains two oligonucleotide-binding (OB) domains. This arrangement is exceedingly rare among bacterial SSBs, which typically form homotetramers of single-OB domain subunits. To better understand how this unusual structure influences the DNA binding and biological functions of DrSSB in D. radiodurans radiation resistance, we have examined the structure of DrSSB in complex with ssDNA and the DNA damage-dependent cellular dynamics of DrSSB. The x-ray crystal structure of the DrSSB-ssDNA complex shows that ssDNA binds to surfaces of DrSSB that are analogous to those mapped in homotetrameric SSBs, although there are distinct contacts in DrSSB that mediate species-specific ssDNA binding. Observations by electron microscopy reveal two salt-dependent ssDNA-binding modes for DrSSB that strongly resemble those of the homotetrameric Escherichia coli SSB, further supporting a shared overall DNA binding mechanism between the two classes of bacterial SSBs. In vivo, DrSSB levels are heavily induced following exposure to ionizing radiation. This accumulation is accompanied by dramatic time-dependent DrSSB cellular dynamics in which a single nucleoid-centric focus of DrSSB is observed within 1 h of irradiation but is dispersed by 3 h after irradiation. These kinetics parallel those of D. radiodurans postirradiation genome reconstitution, suggesting that DrSSB dynamics could play important organizational roles in DNA repair. (10.1074/jbc.M112.367573)
  DOI : 10.1074/jbc.M112.367573
• Sampling the conformational energy landscape of a hyperthermophilic protein by engineering key substitutions.
  
  • Colletier Jacques-Philippe
  • Aleksandrov Alexey
  • Coquelle Nicolas
  • Mraihi S.
  • Mendoza-Barber E.
  • Field Martin J
  • Madern D.
  Molecular Biology and Evolution, Oxford University Press (OUP), 2012, 29 (6), pp.1683-94. Proteins exist as a dynamic ensemble of interconverting substates, which defines their conformational energy landscapes. Recent work has indicated that mutations that shift the balance between conformational substates (CSs) are one of the main mechanisms by which proteins evolve new functions. In the present study, we probe this assertion by examining phenotypic protein adaptation to extreme conditions, using the allosteric tetrameric lactate dehydrogenase (LDH) from the hyperthermophilic bacterium Thermus thermophilus (Tt) as a model enzyme. In the presence of fructose 1, 6 bis-phosphate (FBP), allosteric LDHs catalyze the conversion of pyruvate to lactate with concomitant oxidation of nicotinamide adenine dinucleotide, reduced form (NADH). The catalysis involves a structural transition between a low-affinity inactive "T-state" and a high-affinity active "R-state" with bound FBP. During this structural transition, two important residues undergo changes in their side chain conformations. These are R171 and H188, which are involved in substrate and FBP binding, respectively. We designed two mutants of Tt-LDH with one ("1-Mut") and five ("5-Mut") mutations distant from the active site and characterized their catalytic, dynamical, and structural properties. In 1-Mut Tt-LDH, without FBP, the K(m)(Pyr) is reduced compared with that of the wild type, which is consistent with a complete shifting of the CS equilibrium of H188 to that observed in the R-state. By contrast, the CS populations of R171, k(cat) and protein stability are little changed. In 5-Mut Tt-LDH, without FBP, K(m)(Pyr) approaches the values it has with FBP and becomes almost temperature independent, k(cat) increases substantially, and the CS populations of R171 shift toward those of the R-state. These changes are accompanied by a decrease in protein stability at higher temperature, which is consistent with an increased flexibility at lower temperature. Together, these results show that the thermal properties of an enzyme can be strongly modified by only a few or even a single mutation, which serve to alter the equilibrium and, hence, the relative populations of functionally important native-state CSs, without changing the nature of the CSs themselves. They also provide insights into the types of mutational pathways by which protein adaptation to temperature is achieved. (10.1093/molbev/mss015)
  DOI : 10.1093/molbev/mss015
• Conformational selection through electrostatics: Free energy simulations of GTP and GDP binding to archaeal initiation factor 2.
  
  • Satpati Priyadarshi
  • Simonson Thomas
  Proteins - Structure, Function and Bioinformatics, Wiley, 2012, 80 (5), pp.1264-82. Archaeal Initiation Factor 2 is a GTPase involved in protein biosynthesis. In its GTP-bound, "ON" conformation, it binds an initiator tRNA and carries it to the ribosome. In its GDP-bound, "OFF" conformation, it dissociates from tRNA. To understand the specific binding of GTP and GDP and their dependence on the conformational state, molecular dynamics free energy simulations were performed. The ON state specificity was predicted to be weak, with a GTP/GDP binding free energy difference of -1 kcal/mol, favoring GTP. The OFF state specificity is larger, 4 kcal/mol, favoring GDP. The overall effects result from a competition among many interactions in several complexes. To interpret them, we use a simpler, dielectric continuum model. Several effects are robust with respect to the model details. Both nucleotides have a net negative charge, so that removing them from solvent into the binding pocket carries a desolvation penalty, which is large for the ON state, and strongly disfavors GTP binding compared to GDP. Short-range interactions between the additional GTP phosphate group and ionized sidechains in the binding pocket offset most, but not all of the desolvation penalty; more distant groups also contribute significantly, and the switch 1 loop only slightly. The desolvation penalty is lower for the more open, wetter OFF state, and the GTP/GDP difference much smaller. Short-range interactions in the binding pocket and with more distant groups again make a significant contribution. Overall, the simulations help explain how conformational selection is achieved with a single phosphate group. (10.1002/prot.24023)
  DOI : 10.1002/prot.24023
• Structure of the ternary initiation complex aIF2-GDPNP-methionylated initiator tRNA.
  
  • Schmitt Emmanuelle
  • Panvert Michel
  • Lazennec-Schurdevin Christine
  • Coureux Pierre-Damien
  • Perez J.
  • Thompson A.
  • Mechulam Yves
  Nature Structural and Molecular Biology, Nature Publishing Group, 2012, 19 (4), pp.450-4. Eukaryotic and archaeal translation initiation factor 2 (e/aIF2) is a heterotrimeric GTPase that has a crucial role in the selection of the correct start codon on messenger RNA. We report the 5-Å resolution crystal structure of the ternary complex formed by archaeal aIF2 from Sulfolobus solfataricus, the GTP analog GDPNP and methionylated initiator tRNA. The 3D model is further supported by solution studies using small-angle X-ray scattering. The tRNA is bound by the α and γ subunits of aIF2. Contacts involve the elbow of the tRNA and the minor groove of the acceptor stem, but not the T-stem minor groove. We conclude that despite considerable structural homology between the core γ subunit of aIF2 and the elongation factor EF1A, these two G proteins of the translation apparatus use very different tRNA-binding strategies. (10.1038/nsmb.2259)
  DOI : 10.1038/nsmb.2259
• Conformational selection by the aIF2 GTPase: a molecular dynamics study of functional pathways.
  
  • Satpati Priyadarshi
  • Simonson Thomas
  Biochemistry, American Chemical Society, 2012, 51 (1), pp.353-61. Archaeal initiation factor 2 (aIF2) is a GTPase involved in protein biosynthesis. In its GTP-bound, "ON" conformation, it binds an initiator tRNA and carries it to the ribosome. In its GDP-bound, "OFF" conformation, it dissociates from tRNA. To improve our understanding of the role of each conformational state in the aIF2 "life cycle", we start from the state immediately after GTP hydrolysis, ON:GDP:P(i) (where P(i) is inorganic phosphate), and consider the possible next steps on the pathway to the OFF:GDP product. The first possibility is P(i) dissociation, leading to ON:GDP, which could then relax into OFF:GDP. We use molecular dynamics simulations to compute the P(i) dissociation free energy and show that dissociation is highly favorable. The second possibility is conformational relaxation into the OFF state before P(i) dissociation, to form OFF:GDP:P(i). We estimate the corresponding free energy approximately, 2 ± 3.5 kcal/mol, so that this is an uphill or weakly downhill process. A third possibility is relaxation into another conformation, neither ON nor OFF. Indeed, a third, "MIXED" conformation was seen recently in a crystal structure of the aIF2:GDP:P(i) complex. For this conformational state, P(i) dissociation is weakly unfavorable, in contrast to the ON and OFF states. From this, we will deduce that if the MIXED:GDP complex is not too unstable, the ON:GDP:P(i) → MIXED:GDP:P(i) transformation is a downhill process, which can occur spontaneously. This suggests that the MIXED state could be a functional intermediate. (10.1021/bi201675n)
  DOI : 10.1021/bi201675n
• Sodium selenide toxicity is mediated by O2-dependent DNA breaks.
  
  • Peyroche Gerald
  • Saveanu Cosmin
  • Dauplais Marc
  • Lazard Myriam
  • Beuneu Francois
  • Decourty Laurence
  • Malabat Christophe
  • Jacquier Alain
  • Blanquet Sylvain
  • Plateau Pierre
  PLoS ONE, Public Library of Science, 2012, 7 (5), pp.e36343. Hydrogen selenide is a recurrent metabolite of selenium compounds. However, few experiments studied the direct link between this toxic agent and cell death. To address this question, we first screened a systematic collection of Saccharomyces cerevisiae haploid knockout strains for sensitivity to sodium selenide, a donor for hydrogen selenide (H(2)Se/HSe(-/)Se(2-)). Among the genes whose deletion caused hypersensitivity, homologous recombination and DNA damage checkpoint genes were over-represented, suggesting that DNA double-strand breaks are a dominant cause of hydrogen selenide toxicity. Consistent with this hypothesis, treatment of S. cerevisiae cells with sodium selenide triggered G2/M checkpoint activation and induced in vivo chromosome fragmentation. In vitro, sodium selenide directly induced DNA phosphodiester-bond breaks via an O(2)-dependent reaction. The reaction was inhibited by mannitol, a hydroxyl radical quencher, but not by superoxide dismutase or catalase, strongly suggesting the involvement of hydroxyl radicals and ruling out participations of superoxide anions or hydrogen peroxide. The (*)OH signature could indeed be detected by electron spin resonance upon exposure of a solution of sodium selenide to O(2). Finally we showed that, in vivo, toxicity strictly depended on the presence of O(2). Therefore, by combining genome-wide and biochemical approaches, we demonstrated that, in yeast cells, hydrogen selenide induces toxic DNA breaks through an O(2)-dependent radical-based mechanism. (10.1371/journal.pone.0036343)
  DOI : 10.1371/journal.pone.0036343
• Surveillance pathways rescuing eukaryotic ribosomes lost in translation
  
  • Graille Marc
  • Séraphin Bertrand
  Nature Reviews Molecular Cell Biology, Nature Publishing Group, 2012, 13 (11), pp.727-735. Living cells require the continuous production of proteins by the ribosomes. Any problem enforcing these protein factories to stall during mRNA translation may then have deleterious cellular effects. To minimize these defects, eukaryotic cells have evolved dedicated surveillance pathways: non-stop decay (NSD), no-go decay (NGD) and non-functional 18S-rRNA decay (18S-NRD). Recent studies support a general molecular framework for these surveillance pathways, the mechanisms of which are intimately related to translation termination. Cop. 2012 Macmillan Publishers Limited. All rights reserved. (10.1038/nrm3457)
  DOI : 10.1038/nrm3457
• A hybrid elastic band string algorithm for studies of enzymatic reactions
  
  • Aleksandrov Alexey
  • Field Martin J
  Physical Chemistry Chemical Physics, Royal Society of Chemistry, 2012, 14 (36), pp.12544. A common challenge in theoretical biophysics is the identification of a minimum energy path (MEP) for the rearrangement of a group of atoms from one stable configuration to another. The structure with maximum energy along the MEP approximates the transition state for the process and the energy profile itself permits estimation of the transition rates. In this work we describe a computationally efficient algorithm for the identification of minimum energy paths in complicated biosystems. The algorithm is a hybrid of the nudged elastic band (NEB) and string methods. It has been implemented in the pDynamo simulation program and tested by examining elementary steps in the reaction mechanisms of three enzymes: citrate synthase, RasGAP, and lactate dehydrogenase. Good agreement is found for the energies and geometries of the species along the reaction profiles calculated using the new algorithm and previous versions of the NEB and string techniques, and also those obtained by the common method of adiabatic exploration of the potential energy surface as a function of predefined reaction coordinates. Precisely refined structures of the saddle points along the paths may be subsequently obtained with the climbing image variant of the NEB algorithm. Directions in which the utility of the methods that we have implemented can be further improved are discussed. (10.1039/c2cp40918f)
  DOI : 10.1039/c2cp40918f
• The inverse protein folding problem: protein design and structure prediction in the genomic era.
  
  • Schmidt Am Busch Marcel
  • Lopes Anne
  • Mignon David
  • Gaillard Thomas
  • Simonson Thomas
  , 2012, pp.121-140. Millions of proteins are being identified every year by high throughput genome sequencing projects. Many others can potentially be created by protein engineering and design methods. Here, we review a method for computational protein design (CPD), which starts from a known protein and its 3D structure, and seeks to modify it by mutating some or all of the amino acid sidechains. The mutations are selected to provide stability, and possibly other properties, such as ligand binding. For each set of candidate mutations, the 3D structure is modeled, with an assumption of small, localized perturbations; in particular, we assume the backbone conformation does not change significantly. As in other CPD implementations, the structure is modeled using a classical, molecular mechanics approach along with a simple, implicit description of solvent. Some of the calculations have been distributed to volunteers on the Internet, through our Proteins@Home volunteer computing project. The method and selected results are described, which show that the designed sequences share important properties of natural proteins. (10.1007/978-94-007-4948-1)
  DOI : 10.1007/978-94-007-4948-1
• Lys53 of Ribosomal Protein L36AL and the CCA End of atRNA at the P/E Hybrid Site Are in Close Proximity on the Human Ribosome
  
  • Hountondji Codjo
  • Bulygin Konstantin
  • Woisard Anne
  • Tufféry Pierre
  • Créchet Jean-Bernard
  • Pech Markus
  • Nierhaus Knud H.
  • Karpova Galina
  • Baouz Soria
  ChemBioChem, Wiley-VCH Verlag, 2012, 13 (12), pp.1791-1797. Previously we have shown that the CCA end of aP-tRNA can be crosslinked with the RPL36ALprotein of the large subunit of mammalian ribosomes;itbelongs to the L44e protein family presentina ll eukaryotic andarchaeal ribosomes. Here we confirm and extend this finding and demonstrate that: 1) this crosslink is specific for atRNA at the P/E hybrid site, as atRNA in all other tRNA positions of pre-translocational ribosomes could not be crosslinked with aribosomal protein, 2) the crosslink was formed most efficiently with C74 and C75 of P/E-tRNA, but could also connectt he ultimateAof this tRNA with Lys53 of protein RPL36AL, 3) this protein contains seven monomethylated residues (three lysyl and three arginyl residues,asw ell as glutaminyl residue 51), 4) Q51 is part of aconserved GGQ motif in the L44e proteins in eukaryotic 80S ribosomes that is identical to the universally conserved motif of release factors implicated in promoting peptidyl-tRNA hydrolysis, and 5) the large number of modifications, in which some of the residues were methylated to about50%,might indicatethat protein RPL36AL is apreferential target for regulation. (10.1002/cbic.201200208)
  DOI : 10.1002/cbic.201200208