Interconversion of Functional Motions between Mesophilic and Thermophilic Adenylate Kinases

Dynamic properties are functionally important in many proteins, including the enzyme adenylate kinase (AK), for which the open/closed transition limits the rate of catalytic turnover. Here, we compare our previously published coarse-grained (double-well Gō) simulation of mesophilic AK from E. coli (AKmeso) to simulations of thermophilic AK from Aquifex aeolicus (AKthermo). In AKthermo, as with AKmeso, the LID domain prefers to close before the NMP domain in the presence of ligand, but LID rigid-body flexibility in the open (O) ensemble decreases significantly. Backbone foldedness in O and/or transition state (TS) ensembles increases significantly relative to AKmeso in some interdomain backbone hinges and within LID. In contact space, the TS of AKthermo has fewer contacts at the CORE-LID interface but a stronger contact network surrounding the CORE-NMP interface than the TS of AKmeso. A “heated” simulation of AKthermo at 375K slightly increases LID rigid-body flexibility in accordance with the “corresponding states” hypothesis. Furthermore, while computational mutation of 7 prolines in AKthermo to their AKmeso counterparts produces similar small perturbations, mutation of these sites, especially positions 8 and 155, to glycine is required to achieve LID rigid-body flexibility and hinge flexibilities comparable to AKmeso. Mutating the 7 sites to proline in AKmeso reduces some hinges' flexibilities, especially hinge 2, but does not reduce LID rigid-body flexibility, suggesting that these two types of motion are decoupled in AKmeso. In conclusion, our results suggest that hinge flexibility and global functional motions alike are correlated with but not exclusively determined by the hinge residues. This mutational framework can inform the rational design of functionally important flexibility and allostery in other proteins toward engineering novel biochemical pathways

Substrate inhibition imposes fitness penalty at high protein stability

Proteins are only moderately stable. It has long been debated whether this narrow range of stabilities is solely a result of neutral drift towards lower stability or purifying selection against excess stability is also at work - for which no experimental evidence was found so far. Here we show that mutations outside the active site in the essential E. coli enzyme adenylate kinase result in stability-dependent increase in substrate inhibition by AMP, thereby impairing overall enzyme activity at high stability. Such inhibition caused substantial fitness defects not only in the presence of excess substrate but also under physiological conditions. In the latter case, substrate inhibition caused differential accumulation of AMP in the stationary phase for the inhibition prone mutants. Further, we show that changes in flux through Adk could accurately describe the variation in fitness effects. Taken together, these data suggest that selection against substrate inhibition and hence excess stability may have resulted in a narrow range of optimal stability observed for modern proteins.Comment: 30 pages, 6 figures, 1 table, Supplementary figures and tables - 6 page

Mining electron density for functionally relevant protein polysterism in crystal structures.

This review focuses on conceptual and methodological advances in our understanding and characterization of the conformational heterogeneity of proteins. Focusing on X-ray crystallography, we describe how polysterism, the interconversion of pre-existing conformational substates, has traditionally been analyzed by comparing independent crystal structures or multiple chains within a single crystal asymmetric unit. In contrast, recent studies have focused on mining electron density maps to reveal previously 'hidden' minor conformational substates. Functional tests of the importance of minor states suggest that evolutionary selection shapes the entire conformational landscape, including uniquely configured conformational substates, the relative distribution of these substates, and the speed at which the protein can interconvert between them. An increased focus on polysterism may shape the way protein structure and function is studied in the coming years

Thermal adaptation of conformational dynamics in ribonuclease H

Structural changes are critical to the ability of proteins, particularly enzymes, to carry out their biological function. However, flexibility also leaves proteins vulnerable to denaturation and degradation; thus a balance must be struck between the dynamics required for function and the rigidity required for maintaining a globular protein's characteristic folded structure. These relationships have been studied in detail through comparison of homologous proteins from organisms adapted to varying properties of the bulk environment. In particular, organisms adapted to temperature extremes offer fruitful platforms for the investigation of adaptive changes in protein stability as a function of environmental pressures. Thermostable proteins are widely reported to be more rigid than their homologs from mesophilic organisms, and those from psychrophiles more flexible; this suggests the possibility of evolutionary conservation of the balance between dynamics and stability. Thus specifically functional aspects of protein dynamics may be isolable through the comparative analysis of members of protein families from organisms adapted to different thermal environments. The best experimental tool for characterizing internal conformational dynamics of proteins on a range of timescales and at site-specific resolution is nuclear magnetic resonance (NMR) spectroscopy, which has found widespread use in the study of protein flexibility and dynamics. However, it is often difficult to provide a detailed structural interpretation of NMR observations. This gap can be bridged using molecular dynamics (MD) simulations, which can directly simulate motional processes that have been observed experimentally. The potential for deep synergy between these two complementary tools has been recognized since MD methods were first applied to biological macromolecules, and recent technological developments have reinforced the mutually beneficial relationship between the two techniques. Ribonuclease HI (RNase H), an 18 kD globular protein that hydrolyzes the RNA strand of RNA:DNA hybrid substrates, has been extensively studied by NMR to characterize the differences in dynamics between homologs from the mesophilic organism \textit{E. coli} and the thermophilic organism \textit{T. thermophilus}. However, these dynamic differences are subtle and difficult to interpret structurally. The series of studies described in the present work was conceived in the pursuit of an improved understanding of the complex relationships between protein dynamics, activity, and thermostability in the RNase H protein family. The organizing principle of the work presented herein has been the close coupling between molecular dynamics simulations and NMR observations, permitting both validation of the MD trajectories by rigorous comparison to experiment and improved interpretation of the dynamics observed by NMR. Previous NMR observations of E. coli and T. thermophilus are integrated into an interpretive framework derived from simulations of the larger RNase H family. First, comparative analysis of molecular dynamics simulations of a total of five homologous RNase H families from organisms of varying preferred growth temperature reveals systematic differences in the conformational dynamics of the handle region, a loop previously identified as contributing to substrate binding. Second, analysis of the effects of activating mutations on the dynamics of ttRNH identifies rotamer dynamics whose contributions to increased catalytic activity can be rationalized in the context of observed differences in sidechain orientation in the wild-type ecRNH and ttRNH simulations. Third, a combined MD-NMR study finds that the active site residues of ecRNH, and likely of the entire RNase H family, are rigid on the ps-ns timescale while undergoing substantial conformational exchange upon Mg2+ binding; this suggests that the active site is electrostatically preorganized for binding the first metal ion, which in turn induces dynamic reorganization at longer timescales. Finally, long-timescale simulations of the RNase H family, despite unexpected local unfolding for some family members, identify handle-loop and rotamer preferences for the C. tepidum RNase H (ctRNH) homolog that unexpectedly differ from those observed for ecRNH and ttRNH, and which can be experimentally tested by NMR spectroscopy of this recently characterized and less well-studied example of an RNase H homolog from a thermophilic organism

Linkage between fitness of yeast cells and adenylate kinase catalysis

Enzymes have evolved with highly specific values of their catalytic parameters kcat and KM. This poses fundamental biological questions about the selection pressures responsible for evolutionary tuning of these parameters. Here we are address these questions for the enzyme adenylate kinase (Adk) in eukaryotic yeast cells. A plasmid shuffling system was developed to allow quantification of relative fitness (calculated from growth rates) of yeast in response to perturbations of Adk activity introduced through mutations. Biophysical characterization verified that all variants studied were properly folded and that the mutations did not cause any substantial differences to thermal stability. We found that cytosolic Adk is essential for yeast viability in our strain background and that viability could not be restored with a catalytically dead, although properly folded Adk variant. There exist a massive overcapacity of Adk catalytic activity and only 12% of the wild type kcat is required for optimal growth at the stress condition 20°C. In summary, the approach developed here has provided new insights into the evolutionary tuning of kcat for Adk in a eukaryotic organism. The developed methodology may also become useful for uncovering new aspects of active site dynamics and also in enzyme design since a large library of enzyme variants can be screened rapidly by identifying viable colonies

Exploring the Conformational Transitions of Biomolecular Systems Using a Simple Two-State Anisotropic Network Model

Biomolecular conformational transitions are essential to biological functions. Most experimental methods report on the long-lived functional states of biomolecules, but information about the transition pathways between these stable states is generally scarce. Such transitions involve short-lived conformational states that are difficult to detect experimentally. For this reason, computational methods are needed to produce plausible hypothetical transition pathways that can then be probed experimentally. Here we propose a simple and computationally efficient method, called ANMPathway, for constructing a physically reasonable pathway between two endpoints of a conformational transition. We adopt a coarse-grained representation of the protein and construct a two-state potential by combining two elastic network models (ENMs) representative of the experimental structures resolved for the endpoints. The two-state potential has a cusp hypersurface in the configuration space where the energies from both the ENMs are equal. We first search for the minimum energy structure on the cusp hypersurface and then treat it as the transition state. The continuous pathway is subsequently constructed by following the steepest descent energy minimization trajectories starting from the transition state on each side of the cusp hypersurface. Application to several systems of broad biological interest such as adenylate kinase, ATP-driven calcium pump SERCA, leucine transporter and glutamate transporter shows that ANMPathway yields results in good agreement with those from other similar methods and with data obtained from all-atom molecular dynamics simulations, in support of the utility of this simple and efficient approach. Notably the method provides experimentally testable predictions, including the formation of non-native contacts during the transition which we were able to detect in two of the systems we studied. An open-access web server has been created to deliver ANMPathway results. © 2014 Das et al

Thermophilicity and catalytic efficiency in dihydrofolate reductase

This thesis presents an investigation of the hydrogen transfer reactions between dihydrofolate (H2F) and NADPH that are catalysed by the dihydrofolate reductase (DHFR) isolated from Geobacillus stearothermophilus (BsDHFR) as well as an artificial hybrid originating from the DHFRs from mesophilic Escherichia coli (EcDHFR) and hyperthermophilic Thermotoga maritima (TmDHFR). A broad spectrum of studies, focusing on the relationship between structure, thermostability and kinetics, showed that the catalytic behaviour of BsDHFR is generally similar to other monomeric DHFRs, including ones found in the mesophile Escherichia coli and the psychrophile Moritella profunda, but significantly different from the dimeric TmDHFR. The fact that all monomeric DHFRs display similar catalytic behaviour, regardless of their widely different optimal temperatures, suggests that thermostability does not directly relate to catalytic efficiency. The biophysical differences between monomeric DHFRs and TmDHFR are likely derived from the dimeric nature of the hyperthermophilic enzyme. An artificial dimeric variant of EcDHFR, Xet-3, was prepared by introducing residues at the dimer interface of TmDHFR. While thermostability of this variant is enhanced, it showed a great decrease in its steady-state and pre-steady-state rate constants. Given that the corresponding rate constants did not increase when the loops are released in the monomeric variant of TmDHFR, the lowered catalytic ability in Xet-3 is likely a consequence of geometric distortion of the active site and loss of loop flexibility that is catalytically important in EcDHFR. In contrast, the relatively poor activity of TmDHFR is not simply a consequence of reduced loop flexibility; the dimer interface of TmDHFR plays a rather complicated role in catalysis