Temperature dependence of the flexibility of thermophilic and mesophilic flavoenzymes of the nitroreductase fold

A widely held hypothesis regarding the thermostability ofthermophilic proteins states asserts that, at any giventemperature, thermophilic proteins are more rigid thantheir mesophilic counterparts. Many experimental andcomputational studies have addressed this question withconﬂicting results. Here, we compare two homologousenzymes, one mesophilic (Escherichia coli FMN-dependentnitroreductase; NTR) and one thermophilic (Thermusthermophilus NADH oxidase; NOX), by multiple molecu-lar dynamics simulations at temperatures from 5 to1008C. We ﬁnd that the global rigidity/ﬂexibility of thetwo proteins, assessed by a variety of metrics, is similaron the time scale of our simulations. However, the ther-mophilic enzyme retains its native conformation to amuch greater degree at high temperature than does themesophilic enzyme, both globally and within the activesite. The simulations identify the helix F–helix G ‘arm’as the region with the greatest difference in loss of nativecontacts between the two proteins with increasing temp-erature. In particular, a network of electrostatic inter-actions holds helix F to the body of the protein in thethermophilic protein, and this network is absent in themesophilic counterpart.Keywords: corresponding states/ﬂavoproteins/moleculardynamics simulations/nitroreductase/thermophilic proteinsIntroductionProteins derived from thermophilic organisms are moreresistant to thermal denaturation than homologous proteinsfrom organisms adapted to moderate temperatures (meso-philes). The differences in stability, structure and amino acidcomposition between thermophilic and mesophilic proteinshave been the subject of intense research (reviewed in Vieilleand Zeikus, 2001). In general, thermophilic enzymes areoptimally active at high temperatures at which their hostorganisms thrive, and they are relatively inactive at tempera-tures where mesophilic enzyme activity is optimal. It hasbeen proposed that the low activity of thermophilic proteinsat ‘mesophilic’ temperatures is due to their greater confor-mational rigidity at low temperatures relative to mesophilicproteins (Vihinen, 1987;Zavodsky et al., 1998).An early articulation of this idea, known as the ‘corre-sponding states hypothesis’, comes from Vihinen (1987):‘Flexibilities of proteins performing the same catalyticactivity seem to be about the same at their temperatureoptima, but the more rigid thermostable proteins reach theﬂexibility of the thermolabile proteins at higher tempera-tures’. Vihinen reached this conclusion based on the com-parison of normalized B-factors from a small number ofcrystal structures of proteins for which thermostability datawere also available. Although the validity of his analysismay be called into question, given the sensitivity ofB-factors to the details of the reﬁnement process and tocrystal contacts, the underlying hypothesis has spawnednumerous studies of homologous mesophilic and thermophi-lic proteins.The results of both experimental and computationalstudies of matched mesophilic and thermophilic protein pairshave been mixed. For instance, Fourier-transform infraredspectroscopic (FTIR) hydrogen–deuterium exchange exper-iments on thermophilic and mesophilic isopropylmalatedehydrogenases (Zavodsky et al., 1998) and glyceraldehyde-3-phosphate dehydrogenases (Wrba et al., 1990) showed thatamide hydrogen exchange was less extensive in the thermo-philic protein than in the mesophilic protein at 258C, but thisdifference was greatly reduced when the measurements weremade at the respective optimal activity temperatures of thetwo enzymes. In contrast, a hyperthermophilic zinc-containing rubredoxin (from Pyrococcus furiosus) was foundto have hydrogen exchange rates similar to those of manymesophilic proteins (Hernandez et al., 2000). Neutron scat-tering experiments with live whole cells between 5 and 378C(Tehei et al., 2004) suggest that the overall population ofmacromolecules (predominantly proteins) from hyperthermo-philes and thermophiles have slightly lower mean-squareﬂuctuations on the 0.1 ns time scale than those from meso-philes. However, neutron scattering studies on dihydrofolatereductase found that the thermophilic protein had higheratomic mean-square ﬂuctuations than the mesophilic homol-ogue (Meinhold et al., 2008). Nuclear magnetic resonance(NMR) spectroscopy studies on the ribonuclease H fromEscherichia coli and Thermus thermophilus indicated localdifferences in dynamics that may be related to thermal stab-ility and/or catalysis, but no global differences. In contrast,Kern and coworkers (Wolf-Watz et al., 2004) used NMR tocharacterize a lid closing/opening conformational change inadenylate kinase from E.coli and the hyperthermophileAquifex aeolicus. This hyperthermophilic enzyme is 9-foldless active than the mesophilic form at 208C. Each protein’slid-opening rate was found to be equal to its k