Roman Fedorov

Publications of Roman Fedorov

• Structural basis for the allosteric interference of myosin function by reactive thiol region mutations G680A and G680V.

  Authors: Matthias Preller, Stefanie Bauer, Nancy Adamek, Setsuko Fujita-Becker, Roman Fedorov, Michael A Geeves, Dietmar J Manstein

  The Journal of biological chemistry. 08/2011; 286(40):35051-60.

  The cold-sensitive single-residue mutation of glycine 680 in the reactive thiol region of Dictyostelium discoideum myosin-2 or the corresponding conserved glycine in other myosin isoforms has beenThe cold-sensitive single-residue mutation of glycine 680 in the reactive thiol region of Dictyostelium discoideum myosin-2 or the corresponding conserved glycine in other myosin isoforms has been reported to interfere with motor function. Here we present the x-ray structures of myosin motor domain mutants G680A in the absence and presence of nucleotide as well as the apo structure of mutant G680V. Our results show that the Gly-680 mutations lead to uncoupling of the reactive thiol region from the surrounding structural elements. Structural and functional data indicate that the mutations induce the preferential population of a state that resembles the ADP-bound state. Moreover, the Gly-680 mutants display greatly reduced dynamic properties, which appear to be related to the recovery of myosin motor function at elevated temperatures.

• Mechanism and specificity of pentachloropseudilin-mediated inhibition of myosin motor activity.

  Authors: Krishna Chinthalapudi, Manuel H Taft, René Martin, Sarah M Heissler, Matthias Preller, Falk K Hartmann, Hemma Brandstaetter, John Kendrick-Jones, Georgios Tsiavaliaris, Herwig O Gutzeit, Roman Fedorov, Folma Buss, Hans-Joachim Knölker, Lynne M Coluccio, Dietmar J Manstein

  The Journal of biological chemistry. 06/2011; 286(34):29700-8.

  Here, we report that the natural compound pentachloropseudilin (PClP) acts as a reversible and allosteric inhibitor of myosin ATPase and motor activity. IC(50) values are in the range from 1 to 5 μmHere, we report that the natural compound pentachloropseudilin (PClP) acts as a reversible and allosteric inhibitor of myosin ATPase and motor activity. IC(50) values are in the range from 1 to 5 μm for mammalian class-1 myosins and greater than 90 μm for class-2 and class-5 myosins, and no inhibition was observed with class-6 and class-7 myosins. We show that in mammalian cells, PClP selectively inhibits myosin-1c function. To elucidate the structural basis for PClP-induced allosteric coupling and isoform-specific differences in the inhibitory potency of the compound, we used a multifaceted approach combining direct functional, crystallographic, and in silico modeling studies. Our results indicate that allosteric inhibition by PClP is mediated by the combined effects of global changes in protein dynamics and direct communication between the catalytic and allosteric sites via a cascade of small conformational changes along a conserved communication pathway.

• Site-directed mutagenesis of the χ subunit of DNA polymerase III and single-stranded DNA-binding protein of E. coli reveals key residues for their interaction.

  Authors: Natalie Naue, Roman Fedorov, Andreas Pich, Dietmar J Manstein, Ute Curth

  Nucleic acids research. 10/2010; 39(4):1398-407.

  During DNA replication in Escherichia coli, single-stranded DNA-binding protein (SSB) protects single-stranded DNA from nuclease action and hairpin formation. It is known that the highly conservedDuring DNA replication in Escherichia coli, single-stranded DNA-binding protein (SSB) protects single-stranded DNA from nuclease action and hairpin formation. It is known that the highly conserved C-terminus of SSB contacts the χ subunit of DNA polymerase III. However, there only exists a theoretical model in which the 11 C-terminal amino acids of SSB have been docked onto the surface of χ. In order to refine this model of SSB/χ interaction, we exchanged amino acids in χ and SSB by site-directed mutagenesis that are predicted to be of key importance. Detailed characterization of the interaction of these mutants by analytical ultracentrifugation shows that the interaction area is correctly predicted by the model; however, the SSB C-terminus binds in a different orientation to the χ surface. We show that evolutionary conserved residues of χ form a hydrophobic pocket to accommodate the ultimate two amino acids of SSB, P176 and F177. This pocket is surrounded by conserved basic residues, important for the SSB/χ interaction. Mass spectrometric analysis of χ protein cross-linked to a C-terminal peptide of SSB reveals that K132 of χ and D172 of SSB are in close contact. The proposed SSB-binding site resembles those described for RecQ and exonuclease I.

• Targeted Optimization of a Protein Nanomachine for Operation in Biohybrid Devices.

  Authors: Mamta Amrute-Nayak, Ralph P Diensthuber, Walter Steffen, Daniela Kathmann, Falk K Hartmann, Roman Fedorov, Claus Urbanke, Dietmar J Manstein, Bernhard Brenner, Georgios Tsiavaliaris

  Angewandte Chemie (International ed. in English). 11/2009;

• Total Synthesis of Pentabromo- and Pentachloropseudilin, and Synthetic Analogues-Allosteric Inhibitors of Myosin ATPase.

  Authors: René Martin, Anne Jäger, Markus Böhl, Sabine Richter, Roman Fedorov, Dietmar J Manstein, Herwig O Gutzeit, Hans-Joachim Knölker

  Angewandte Chemie (International ed. in English). 10/2009;

• The mechanism of pentabromopseudilin inhibition of myosin motor activity.

  Authors: Roman Fedorov, Markus Böhl, Georgios Tsiavaliaris, Falk K Hartmann, Manuel H Taft, Petra Baruch, Bernhard Brenner, René Martin, Hans-Joachim Knölker, Herwig O Gutzeit, Dietmar J Manstein

  Nature structural & molecular biology. 02/2009; 16(1):80-8.

  We have identified pentabromopseudilin (PBP) as a potent inhibitor of myosin-dependent processes such as isometric tension development and unloaded shortening velocity. PBP-induced reductions in theWe have identified pentabromopseudilin (PBP) as a potent inhibitor of myosin-dependent processes such as isometric tension development and unloaded shortening velocity. PBP-induced reductions in the rate constants for ATP binding, ATP hydrolysis and ADP dissociation extend the time required per myosin ATPase cycle in the absence and presence of actin. Additionally, coupling between the actin and nucleotide binding sites is reduced in the presence of the inhibitor. The selectivity of PBP differs from that observed with other myosin inhibitors. To elucidate the binding mode of PBP, we crystallized the Dictyostelium myosin-2 motor domain in the presence of Mg(2+)-ADP-meta-vanadate and PBP. The electron density for PBP is unambiguous and shows PBP to bind at a previously unknown allosteric site near the tip of the 50-kDa domain, at a distance of 16 A from the nucleotide binding site and 7.5 A away from the blebbistatin binding pocket.

• Crystal structure of the intact archaeal translation initiation factor 2 demonstrates very high conformational flexibility in the alpha- and beta-subunits.

  Authors: Elena Stolboushkina, Stanislav Nikonov, Alexei Nikulin, Udo Bläsi, Dietmar J Manstein, Roman Fedorov, Maria Garber, Oleg Nikonov

  Journal of molecular biology. 11/2008; 382(3):680-91.

  In Eukarya and Archaea, translation initiation factor 2 (eIF2/aIF2), which contains three subunits (alpha, beta, and gamma), is pivotal for binding of charged initiator tRNA to the small ribosomalIn Eukarya and Archaea, translation initiation factor 2 (eIF2/aIF2), which contains three subunits (alpha, beta, and gamma), is pivotal for binding of charged initiator tRNA to the small ribosomal subunit. The crystal structure of the full-sized heterotrimeric aIF2 from Sulfolobus solfataricus in the nucleotide-free form has been determined at 2.8-A resolution. Superposition of four molecules in the asymmetric unit of the crystal and the comparison of the obtained structures with the known structures of the aIF2alphagamma and aIF2betagamma heterodimers revealed high conformational flexibility in the alpha- and beta-subunits. In fact, the full-sized aIF2 consists of a rigid central part, formed by the gamma-subunit, domain 3 of the alpha-subunit, and the N-terminal alpha-helix of the beta-subunit, and two mobile "wings," formed by domains 1 and 2 of the alpha-subunit, the central part, and the zinc-binding domain of the beta-subunit. High structural flexibility of the wings is probably required for interaction of aIF2 with the small ribosomal subunit. Comparative analysis of all known structures of the gamma-subunit alone and within the heterodimers and heterotrimers in nucleotide-bound and nucleotide-free states shows that the conformations of switch 1 and switch 2 do not correlate with the assembly or nucleotide states of the protein.

• Biophysical analysis of Thermus aquaticus single-stranded DNA binding protein.

  Authors: Gregor Witte, Roman Fedorov, Ute Curth

  Biophysical journal. 04/2008; 94(6):2269-79.

  Due to their involvement in processes such as DNA replication, repair, and recombination, bacterial single-stranded DNA binding (SSB) proteins are essential for the survival of the bacterial cell.Due to their involvement in processes such as DNA replication, repair, and recombination, bacterial single-stranded DNA binding (SSB) proteins are essential for the survival of the bacterial cell. Whereas most bacterial SSB proteins form homotetramers in solution, dimeric SSB proteins were recently discovered in the Thermus/Deinococcus group. In this work we characterize the biophysical properties of the SSB protein from Thermus aquaticus (TaqSSB), which is structurally quite similar to the tetrameric SSB protein from Escherichia coli (EcoSSB). The binding of TaqSSB and EcoSSB to single-stranded nucleic acids was found to be very similar in affinity and kinetics. Mediated by its highly conserved C-terminal region, TaqSSB interacts with the chi-subunit of E. coli DNA polymerase III with an affinity that is similar to that of EcoSSB. Using analytical ultracentrifugation, we show that TaqSSB mutants are able to form tetramers in solution via arginine-mediated hydrogen-bond interactions that we identified in the crystal packing of wild-type TaqSSB. In EcoSSB, we identified a homologous arginine residue involved in the formation of higher aggregates and metastable highly cooperative single-stranded DNA binding under low salt conditions.

• New insights into the interactions of the translation initiation factor 2 from archaea with guanine nucleotides and initiator tRNA.

  Authors: Oleg Nikonov, Elena Stolboushkina, Alexei Nikulin, David Hasenöhrl, Udo Bläsi, Dietmar J Manstein, Roman Fedorov, Maria Garber, Stanislav Nikonov

  Journal of molecular biology. 11/2007; 373(2):328-36.

  Heterotrimeric a/eIF2alphabetagamma (archaeal homologue of the eukaryotic translation initiation factor 2 with alpha, beta and gamma subunits) delivers charged initiator tRNA (tRNAi) to the smallHeterotrimeric a/eIF2alphabetagamma (archaeal homologue of the eukaryotic translation initiation factor 2 with alpha, beta and gamma subunits) delivers charged initiator tRNA (tRNAi) to the small ribosomal subunit. In this work, we determined the structures of aIF2gamma from the archaeon Sulfolobus solfataricus in the nucleotide-free and GDP-bound forms. Comparison of the free, GDP and Gpp(NH)p-Mg2+ forms of aIF2gamma revealed a sequence of conformational changes upon GDP and GTP binding. Our results show that the affinity of GDP to the G domain of the gamma subunit is higher than that of Gpp(NH)p. In analyzing a pyrophosphate molecule binding to domain II of the gamma subunit, we found a cleft that is very suitable for the acceptor stem of tRNA accommodation. It allows the suggestion of an alternative position for Met-tRNA i Met on the alphagamma intersubunit dimer, at variance with a recently published one. In the model reported here, the acceptor stem of the tRNAi is approximately perpendicular to that of tRNA in the ternary complex elongation factor Tu-Gpp(NH)p-tRNA. According to our analysis, the elbow and T stem of Met-tRNA i Met in this position should make extensive contact with the alpha subunit of aIF2. Thus, this model is in good agreement with experimental data showing that the alpha subunit of aIF2 is necessary for the stable interaction of aIF2gamma with Met-tRNA i Met.

• 3D structure of Thermus aquaticus single-stranded DNA-binding protein gives insight into the functioning of SSB proteins.

  Authors: Roman Fedorov, Gregor Witte, Claus Urbanke, Dietmar J Manstein, Ute Curth

  Nucleic acids research. 02/2006; 34(22):6708-17.

  In contrast to the majority of tetrameric SSB proteins, the recently discovered SSB proteins from the Thermus/Deinoccus group form dimers. We solved the crystal structures of the SSB protein fromIn contrast to the majority of tetrameric SSB proteins, the recently discovered SSB proteins from the Thermus/Deinoccus group form dimers. We solved the crystal structures of the SSB protein from Thermus aquaticus (TaqSSB) and a deletion mutant of the protein and show the structure of their ssDNA binding domains to be similar to the structure of tetrameric SSBs. Two conformations accompanied by proline cis-trans isomerization are observed in the flexible C-terminal region. For the first time, we were able to trace 6 out of 10 amino acids at the C-terminus of an SSB protein. This highly conserved region is essential for interaction with other proteins and we show it to adopt an extended conformation devoid of secondary structure. A model for binding this region to the chi subunit of DNA polymerase III is proposed. It explains at a molecular level the reason for the ssb113 phenotype observed in Escherichia coli.

• Structural analysis of isoform-specific inhibitors targeting the tetrahydrobiopterin binding site of human nitric oxide synthases.

  Authors: Hans Matter, H S Arun Kumar, Roman Fedorov, Armin Frey, Peter Kotsonis, Elisabeth Hartmann, Lothar G Fröhlich, Andreas Reif, Wolfgang Pfleiderer, Peter Scheurer, Dipak K Ghosh, Ilme Schlichting, Harald H H W Schmidt

  Journal of medicinal chemistry. 08/2005; 48(15):4783-92.

  Nitric oxide synthesized from l-arginine by nitric oxide synthase isoforms (NOS-I-III) is physiologically important but also can be deleterious when overproduced. Selective NOS inhibitors are ofNitric oxide synthesized from l-arginine by nitric oxide synthase isoforms (NOS-I-III) is physiologically important but also can be deleterious when overproduced. Selective NOS inhibitors are of clinical interest, given their differing pathophysiological roles. Here we describe our approach to target the unique NOS (6R,1'R,2'S)-5,6,7,8-tetrahydrobiopterin (H(4)Bip) binding site. By a combination of ligand- and structure-based design, the structure-activity relationship (SAR) for a focused set of 41 pteridine analogues on four scaffolds was developed, revealing selective NOS-I inhibitors. The X-ray crystal structure of rat NOS-I dimeric-oxygenase domain with H(4)Bip and l-arginine was determined and used for human isoform homology modeling. All available NOS structural information was subjected to comparative analysis of favorable protein-ligand interactions using the GRID/concensus principal component analysis (CPCA) approach to identify the isoform-specific interaction site. Our interpretation, based on protein structures, is in good agreement with the ligand SAR and thus permits the rational design of next-generation inhibitors targeting the H(4)Bip binding site with enhanced isoform selectivity for therapeutics in pathology with NO overproduction.

• Structures of nitric oxide synthase isoforms complexed with the inhibitor AR-R17477 suggest a rational basis for specificity and inhibitor design.

  Authors: Roman Fedorov, Ryan Vasan, Dipak K Ghosh, Ilme Schlichting

  Proceedings of the National Academy of Sciences of the United States of America. 05/2004; 101(16):5892-7.

  The high level of amino acid conservation and structural similarity of the substrate-binding sites of the oxygenase domains of the nitric oxide synthase (NOS) isoforms (eNOSoxy, iNOSoxy, nNOSoxy)The high level of amino acid conservation and structural similarity of the substrate-binding sites of the oxygenase domains of the nitric oxide synthase (NOS) isoforms (eNOSoxy, iNOSoxy, nNOSoxy) make the interpretation of the structural basis of inhibitor isoform specificity a challenge, and provide few clues for the design of new selective compounds. Crystal structures of iNOSoxy and nNOSoxy complexed with the neuronal NOS-specific inhibitor AR-R17447 suggest that specificity is provided by the interaction of the chlorophenyl group with an isoform-unique substrate access channel residue (L337 in rat neuronal NOS, N115 in mouse inducible NOS). This is confirmed by biochemical analysis of site-directed mutants. Inhibitors combining guanidinium-like structural motifs with long chains specifically targeting this residue are good candidates for rational isoform-specific drug design. Based on this finding, modifications of AR-R17447 to improve the specificity for the human isoforms are suggested.

• Structural basis for the specificity of the nitric-oxide synthase inhibitors W1400 and Nomega-propyl-L-Arg for the inducible and neuronal isoforms.

  Authors: Roman Fedorov, Elisabeth Hartmann, Dipak K Ghosh, Ilme Schlichting

  The Journal of biological chemistry. 12/2003; 278(46):45818-25.

  The high level of amino acid conservation and structural similarity in the immediate vicinity of the substrate binding sites of the oxygenase domains of the nitric-oxide synthase (NOS) isoformsThe high level of amino acid conservation and structural similarity in the immediate vicinity of the substrate binding sites of the oxygenase domains of the nitric-oxide synthase (NOS) isoforms (eNOSoxy, iNOSoxy, and nNOSoxy) make the interpretation of the structural basis of inhibitor isoform specificity a challenge and provide few clues for the design of new selective compounds. Crystal structures of iNOSoxy and nNOSoxy complexed with the inhibitors W1400 and Nomega-propyl-l-arginine provide a rationale for their isoform specificity. It involves differences outside the immediate active site as well as a conformational flexibility in the active site that allows the adoption of distinct conformations in response to interactions with the inhibitors. This flexibility is determined by isoform-specific residues outside the active site.

• Irreversible photoreduction of flavin in a mutated Phot-LOV1 domain.

  Authors: Tilman Kottke, Bernhard Dick, Roman Fedorov, Ilme Schlichting, Rainer Deutzmann, Peter Hegemann

  Biochemistry. 09/2003; 42(33):9854-62.

  Phot photoreceptors make up an important protein family regulating biological processes in response to blue light. They contain two light, oxygen, and voltage sensitive (LOV) domains and aPhot photoreceptors make up an important protein family regulating biological processes in response to blue light. They contain two light, oxygen, and voltage sensitive (LOV) domains and a serine/threonine kinase domain. Both LOV domains noncovalently bind a flavin mononucleotide (FMN). Upon absorption of blue light, the LOV domains undergo a photocycle, transiently forming a covalent adduct of a cysteine residue and the FMN (LOV-390). The mechanism of formation of this flavin-thiol adduct is still unclear. We studied a mutant of the LOV1 domain from the green alga Chlamydomonas reinhardtii with a methionine replacing the reactive cysteine 57 (C57M). As in the wild type, irradiation leads to formation of a photoadduct, which, however, is irreversibly converted into a red absorbing species, C57M-675. On the basis of spectroscopic results and the 2.1 A resolution crystal structure, this highly unusual FMN species was assigned to a neutral flavin radical covalently attached to the apoprotein at the N(5) position. In contrast to other flavoprotein neutral radicals, C57M-675 is stable even under aerobic or denaturing conditions. Pathways for the photoinduced formation of the adduct are discussed for the C57M mutant as well as the wild-type LOV1 domain.

• Crystal structures and molecular mechanism of a light-induced signaling switch: The Phot-LOV1 domain from Chlamydomonas reinhardtii.

  Authors: Roman Fedorov, Ilme Schlichting, Elisabeth Hartmann, Tatjana Domratcheva, Markus Fuhrmann, Peter Hegemann

  Biophysical journal. 05/2003; 84(4):2474-82.

  Phot proteins (phototropins and homologs) are blue-light photoreceptors that control mechanical processes like phototropism, chloroplast relocation, or guard-cell opening in plants. Phot receptorsPhot proteins (phototropins and homologs) are blue-light photoreceptors that control mechanical processes like phototropism, chloroplast relocation, or guard-cell opening in plants. Phot receptors consist of two flavin mononucleotide (FMN)-binding light, oxygen, or voltage (LOV) domains and a C-terminal serine/threonine kinase domain. We determined crystal structures of the LOV1 domain of Phot1 from the green alga Chlamydomonas reinhardtii in the dark and illuminated state to 1.9 A and 2.8 A resolution, respectively. The structure resembles that of LOV2 from Adiantum (Crosson, S. and K. Moffat. 2001. PROC: Natl. Acad. Sci. USA. 98:2995-3000). In the resting dark state of LOV1, the reactive Cys-57 is present in two conformations. Blue-light absorption causes formation of a proposed active signaling state that is characterized by a covalent bond between the flavin C4a and the thiol of Cys-57. There are differences around the FMN chromophore but no large overall conformational changes. Quantum chemical calculations based on the crystal structures revealed the electronic distribution in the active site during the photocycle. The results suggest trajectories for electrons, protons, and the active site cysteine and offer an interpretation of the reaction mechanism.

• Crystal structures of cyanide complexes of P450cam and the oxygenase domain of inducible nitric oxide synthase-structural models of the short-lived oxygen complexes.

  Authors: Roman Fedorov, Dipak K Ghosh, Ilme Schlichting

  Archives of biochemistry and biophysics. 02/2003; 409(1):25-31.

  The crystal structure of the ternary cyanide complex of P450cam and camphor was determined to 1.8A resolution and found to be identical with the structure of the active oxygen complex [I. SchlichtingThe crystal structure of the ternary cyanide complex of P450cam and camphor was determined to 1.8A resolution and found to be identical with the structure of the active oxygen complex [I. Schlichting et al., 2000, Science 287, 1615]. Notably, cyanide binds in a bent mode and induces the active conformation that is characterized by the presence of two water molecules and a flip of the carbonyl of the conserved Asp251. The structure of the ternary complex of cyanide, L-arginine, and the oxygenase domain of inducible nitric oxide synthase was determined to 2.4A resolution. Cyanide binds essentially linearly, interacts with L-Arg, and induces the binding of a water molecule at the active site. This water is positioned by backbone interactions, located 2.8A from the nitrogen atom of cyanide, and could provide a proton required for O-O bond scission in the hydroxylation reaction of nitric oxide synthase.

• Structure of ribosomal protein L1 from Methanococcus thermolithotrophicus. Functionally important structural invariants on the L1 surface.

  Authors: Natalia Nevskaya, Svetlana Tishchenko, Mikhail Paveliev, Yulia Smolinskaya, Roman Fedorov, Wolfgang Piendl, Yoshikazu Nakamura, Tomohiko Toyoda, Maria Garber, Stanislav Nikonov

  Acta crystallographica. Section D, Biological crystallography. 07/2002; 58(Pt 6 Pt 2):1023-9.

  The crystal structure of ribosomal protein L1 from the archaeon Methanococcus thermolithotrophicus has been determined at 2.7 A resolution. The crystals belong to space group P2(1)2(1)2(1), withThe crystal structure of ribosomal protein L1 from the archaeon Methanococcus thermolithotrophicus has been determined at 2.7 A resolution. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 67.0, b = 70.1, c = 106.3 A and two molecules per asymmetric unit. The structure was solved by the molecular-replacement method with AMoRe and refined with CNS to an R value of 18.9% and an R(free) of 25.4% in the resolution range 30-2.7 A. Comparison of this structure with those obtained previously for two L1 proteins from other sources (the bacterium Thermus thermophilus and the archaeon M. jannaschii) as well as detailed analysis of intermolecular contacts in the corresponding L1 crystals reveal structural invariants on the molecular surface which are probably important for binding the 23S ribosomal RNA and protein function within the ribosome.

• Structures of nitric oxide synthase isoforms complexed with the inhibitor AR−R17477 suggest a rational basis for specificity and inhibitor design

  Authors: Roman Fedorov, Ryan Vasan, Dipak K. Gosh, Ilme Schlichting

  Proceedings of the National Academy of Sciences of the USA, v.101, 5892-5897 (2004).

  The high level of amino acid conservation and structural similarity of the substrate−binding sites of the oxygenase domains of the nitric oxide synthase (NOS) isoforms (eNOSoxy, iNOSoxy, nNOSoxy)The high level of amino acid conservation and structural similarity of the substrate−binding sites of the oxygenase domains of the nitric oxide synthase (NOS) isoforms (eNOSoxy, iNOSoxy, nNOSoxy) make the interpretation of the structural basis of inhibitor isoform specificity a challenge, and provide few clues for the design of new selective compounds. Crystal structures of iNOSoxy and nNOSoxy complexed with the neuronal NOS−specific inhibitor AR−R17447 suggest that specificity is provided by the interaction of the chlorophenyl group with an isoform−unique substrate access channel residue (L337 in rat neuronal NOS, N115 in mouse inducible NOS). This is confirmed by biochemical analysis of site−directed mutants. Inhibitors combining guanidinium−like structural motifs with long chains specifically targeting this residue are good candidates for rational isoform−specific drug design. Based on this finding, modifications of AR−R17447 to improve the specificity for the human isoforms are suggested.