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 15N 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.

Validating Molecular Dynamics Simulations against Experimental Observables in Light of Underlying Conformational Ensembles

“…Experimental order parameters for EnHD were obtained from BMRB entry 15336. 81 Experimental order parameters for RNase H were obtained from the Supporting Information of Stafford et al 92 MD-derived NH bond order parameters were calculated using the method described by Wong and Daggett using a 250 ps window for EnHD and a 10 000 ps window for RNase H given its ∼9.7 ns tumbling time. 93 Final order parameters are reported by averaging the results from the three replicate simulations.…”

Detection of Chemical Exchange in Methyl Groups of Macromolecules

“…Each of these residues has been demonstrated to be directly involved in or located within the substrate-binding handle region associated with conformational transitions from open to closed states by 15 N amide spin relaxation experiments and/or molecular dynamics simulations [54][55][56] .…”

Detection of chemical exchange in methyl groups of macromolecules

“…Each of these residues has been demonstrated to be directly involved in or located within the substrate-binding handle region associated with conformational transitions from open to closed states by 15 N amide spin relaxation experiments and/or molecular dynamics simulations [54][55][56] . Figure 5 shows a graph of ΔR MQ versus η HH based on data previously reported (see Results are shown for the four Met residues in AlkB: Met 49, Met 57, and Met 61 are located in the active site or nucleotide recognition lid (NRL) and Met 92 is located at a hinge between the between the two states (∆ω C >= 2.1 ppm) for this residue 21,57,58 .…”