title: Conformational preferences underlying reduced activity of a thermophilic ribonuclease H.
abstract: The conformational basis for reduced activity of the thermophilic ribonuclease HI enzyme from Thermus thermophilus, compared to its mesophilic homolog from Escherichia coli, is elucidated using a combination of NMR spectroscopy and molecular dynamics (MD) simulations. Explicit-solvent all-atom MD simulations of the two wild-type proteins and an E. coli mutant in which a glycine residue is inserted after position 80 to mimic the T. thermophilus protein reproduce the differences in conformational dynamics determined from (15)N spin-relaxation NMR spectroscopy of three loop regions that surround the active site and contain functionally important residues: the glycine-rich region, the handle region, and the β5/αE loop. Examination of the MD trajectories indicates that the thermophilic protein samples conformations productive for substrate binding and activity less frequently than the mesophilic enzyme, although these differences may manifest as either increased or decreased relative flexibility of the different regions. Additional MD simulations indicate that mutations increasing activity of the T. thermophilus enzyme at mesophilic temperatures do so by reconfiguring the local environments of the mutated sites to more closely resemble active conformations. Taken together, the results show that both locally increased and decreased flexibility contribute to an overall reduction in activity of T. thermophilus ribonuclease H compared to its mesophilic E. coli homolog.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA.
authors.forename: Kate A
authors.initials: KA
authors.lastname: Stafford
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA.
authors.forename: Nikola
authors.initials: N
authors.lastname: Trbovic
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA.
authors.forename: Joel A
authors.initials: JA
authors.lastname: Butterwick
authors.affiliation: Department of Chemistry, Columbia University, New York, NY 10027, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Abel
authors.affiliation: Department of Chemistry, Columbia University, New York, NY 10027, USA.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Friesner
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA. Electronic address: agp6@columbia.edu.
authors.forename: Arthur G
authors.initials: AG
authors.lastname: Palmer
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology*; D006358:Hot Temperature; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D025341:Principal Component Analysis; D011485:Protein Binding; D011487:Protein Conformation; D016914:Ribonuclease H / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure*; D013816:Thermodynamics; D016963:Thermus thermophilus / Q000201:enzymology*
chemical_list: D016914:Ribonuclease H
keywords: Michaelis–Menten enzyme kinetics; chemical exchange; molecular dynamics simulation; order parameter; relaxation dispersion