During transitions proteins make fleeting bonds.

How do proteins efficiently and precisely shift from one conformation to another? Gardino et al. (2009) show that transient hydrogen bonds are critical to the conformational transition of the nitrogen regulatory protein NtrC between its native state and its active state.

Driving Protein Conformational Cycles in Physiology and Disease: "Frustrated" Amino Acid Interaction Networks Define Dynamic Energy Landscapes: Amino Acid Interaction Networks Change Progressively Along Alpha Tryptophan Synthase's Catalytic Cycle.

A general framework by which dynamic interactions within a protein will promote the necessary series of structural changes, or "conformational cycle," required for function is proposed. It is suggested that the free-energy landscape of a protein is biased toward this conformational cycle. Fluctuations into higher energy, although thermally accessible, conformations drive the conformational cycle forward. The amino acid interaction network is defined as those intraprotein interactions that contribute most to the free-energy landscape. Some network connections are consistent in every structural state, while others periodically change their interaction strength according to the conformational cycle. It is reviewed here that structural transitions change these periodic network connections, which then predisposes the protein toward the next set of network changes, and hence the next structural change. These concepts are illustrated by recent work on tryptophan synthase. Disruption of these dynamic connections may lead to aberrant protein function and disease states.

Different Solvent and Conformational Entropy Contributions to the Allosteric Activation and Inhibition Mechanisms of Yeast Chorismate Mutase.

Allosteric regulation is important in many biological processes, including cell signaling, gene regulation, and metabolism. Saccharomyces cerevisiae chorismate mutase (ScCM) is a key homodimeric enzyme in the shikimate pathway responsible for the generation of aromatic amino acids, where it is allosterically inhibited and activated by Tyr and Trp, respectively. Our previous studies indicated that binding of both allosteric effectors is negatively cooperative, that is binding at one allosteric binding site discourages binding at the other, due to the entropic penalty of binding the second allosteric effector. We utilized variable temperature isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) experiments to better understand the entropic contributions to allosteric effector binding, including changes to solvent entropy and protein conformational entropy. Upon binding either Tyr or Trp, ScCM experiences a quenching of motions on the picosecond-to-nanosecond time scale, which we could relate to a loss of protein conformational entropy. Further ITC and NMR studies were consistent with the Tyr-bound form of ScCM being associated with more water molecules compared to the Trp-bound form and Tyr binding being associated with a less positive solvent entropy change. These studies provide insight into the role of structural dynamics in ScCM function and highlight the importance of solvent entropy changes in allosteric regulation, a historically underappreciated concept.

2'-C-methylated nucleotides terminate virus RNA synthesis by preventing active site closure of the viral RNA-dependent RNA polymerase.

The 2'-C-methyl ribonucleosides are nucleoside analogs representing an important class of antiviral agents, especially against positive-strand RNA viruses. Their value is highlighted by the highly successful anti-hepatitis C drug sofosbuvir. When appropriately phosphorylated, these nucleotides are successfully incorporated into RNA by the virally encoded RNA-dependent RNA polymerase (RdRp). This activity prevents further RNA extension, but the mechanism is poorly characterized. Previously, we had identified NMR signatures characteristic of formation of RdRp-RNA binary and RdRp-RNA-NTP ternary complexes for the poliovirus RdRp, including an open-to-closed conformational change necessary to prepare the active site for catalysis of phosphoryl transfer. Here we used these observations as a framework for interpreting the effects of 2'-C-methyl adenosine analogs on RNA chain extension in solution-state NMR spectroscopy experiments, enabling us to gain additional mechanistic insights into 2'-C-methyl ribonucleoside-mediated RNA chain termination. Contrary to what has been proposed previously, poliovirus RdRp that was bound to RNA with an incorporated 2'-C-methyl nucleotide could still bind to the next incoming NTP. Our results also indicated that incorporation of the 2'-C-methyl nucleotide does not disrupt RdRp-RNA interactions and does not prevent translocation. Instead, incorporation of the 2'-C-methyl nucleotide blocked closure of the RdRp active site upon binding of the next correct incoming NTP, which prevented further nucleotide addition. We propose that other nucleotide analogs that act as nonobligate chain terminators may operate through a similar mechanism.

Energy and Enzyme Activity Landscapes of Yeast Chorismate Mutase at Cellular Concentrations of Allosteric Effectors.

In solution, proteins fluctuate among many conformational substates, with their relative free energies determining substate populations and energy barriers determining conformational exchange kinetics. It has been suggested that members of the conformational ensemble may be responsible for different protein functions, although it is generally difficult to test such a proposal in most systems. A model protein for deciphering individual substate contributions is the homodimeric Saccharomyces cerevisiae chorismate mutase (ScCM) enzyme, which is negatively and positively regulated by tyrosine and tryptophan, respectively. Previous X-ray crystallography structures revealed two equivalent allosteric binding pockets that can be occupied by either tryptophan or tyrosine. We proposed that under cellular conditions there are six potential states of ScCM: no allosteric effector bound, a single tyrosine bound, a single tryptophan bound, two tyrosines bound, two tryptophans bound, and a mixed bound state in which tyrosine and tryptophan occupy different allosteric sites. We used isothermal titration calorimetry and solution-state nuclear magnetic resonance spectroscopy to confirm the existence of all six states and construct the complete six-state equilibrium binding profile. We were also able to assign enzyme activities to each state, which allowed us to derive the enzyme activity landscape across the range of cellular concentrations of tyrosine and tryptophan. Surprisingly, the mixed bound state had the highest enzyme activity, which suggested that the shikimate pathway is shunted toward tyrosine production under most conditions.

Rational Control of Poliovirus RNA-Dependent RNA Polymerase Fidelity by Modulating Motif-D Loop Conformational Dynamics.

The conserved structural motif D is an important determinant of the speed and fidelity of viral RNA-dependent RNA polymerases (RdRps). Structural and computational studies have suggested that conformational changes in the motif-D loop that help to reposition the catalytic lysine represent critical steps in nucleotide selection and incorporation. Conformations of the motif-D loop in the poliovirus RdRp are likely controlled in part by noncovalent interactions involving the motif-D residue Glu364. This residue swivels between making interactions with Lys228 and Asn370 to stabilize the open and closed loop conformations, respectively. We show here that we can rationally control the motif-D loop conformation by breaking these interactions. The K228A variant favors a more active closed conformation, leading to increased nucleotide incorporation rates and decreased nucleotide selectivity, and the N370A variant favors a less active open conformation, leading to decreased nucleotide incorporation rates and increased nucleotide selectivity. Similar competing interactions likely control nucleotide incorporation rates and fidelity in other viral RdRps. Rational engineering of these interactions may be important in the generation of live, attenuated vaccine strains, considering the established relationships between RdRp function and viral pathogenesis.

Assigning methyl resonances for protein solution-state NMR studies.

Solution-state NMR is an important tool for studying protein structure and function. The ability to probe methyl groups has substantially expanded the scope of proteins accessible by NMR spectroscopy, including facilitating study of proteins and complexes greater than 100 kDa in size. While the toolset for studying protein structure and dynamics by NMR continues to grow, a major rate-limiting step in these studies is the initial resonance assignments, especially for larger (>50 kDa) proteins. In this practical review, we present strategies to efficiently isotopically label proteins, delineate NMR pulse sequences that can be used to determine methyl resonance assignments in the presence and absence of backbone assignments, and outline computational methods for NMR data analysis. We use our experiences from assigning methyl resonances for the aromatic biosynthetic enzymes tryptophan synthase and chorismate mutase to provide advice for all stages of experimental set-up and data analysis.

Engineering Allostery into Proteins.

Our ability to engineer protein structure and function has grown dramatically over recent years. Perhaps the next level in protein design is to develop proteins whose function can be regulated in response to various stimuli, including ligand binding, pH changes, and light. Endeavors toward these goals have tested and expanded on our understanding of protein function and allosteric regulation. In this chapter, we provide examples from different methods for developing new allosterically regulated proteins. These methods range from whole insertion of regulatory domains into new host proteins, to covalent attachment of photoswitches to generate light-responsive proteins, and to targeted changes to specific amino acid residues, especially to residues identified to be important for relaying allosteric information across the protein framework. Many of the examples we discuss have already found practical use in medical and biotechnology applications.

Triphosphate Reorientation of the Incoming Nucleotide as a Fidelity Checkpoint in Viral RNA-dependent RNA Polymerases.

The nucleotide incorporation fidelity of the viral RNA-dependent RNA polymerase (RdRp) is important for maintaining functional genetic information but, at the same time, is also important for generating sufficient genetic diversity to escape the bottlenecks of the host's antiviral response. We have previously shown that the structural dynamics of the motif D loop are closely related to nucleotide discrimination. Previous studies have also suggested that there is a reorientation of the triphosphate of the incoming nucleotide, which is essential before nucleophilic attack from the primer RNA 3'-hydroxyl. Here, we have used 31P NMR with poliovirus RdRp to show that the binding environment of the triphosphate is different when correct versus incorrect nucleotide binds. We also show that amino acid substitutions at residues known to interact with the triphosphate can alter the binding orientation/environment of the nucleotide, sometimes lead to protein conformational changes, and lead to substantial changes in RdRp fidelity. The analyses of other fidelity variants also show that changes in the triphosphate binding environment are not always accompanied by changes in the structural dynamics of the motif D loop or other regions known to be important for RdRp fidelity, including motif B. Altogether, our studies suggest that the conformational changes in motifs B and D, and the nucleoside triphosphate reorientation represent separable, "tunable" fidelity checkpoints.

The RNA template channel of the RNA-dependent RNA polymerase as a target for development of antiviral therapy of multiple genera within a virus family.

The genus Enterovirus of the family Picornaviridae contains many important human pathogens (e.g., poliovirus, coxsackievirus, rhinovirus, and enterovirus 71) for which no antiviral drugs are available. The viral RNA-dependent RNA polymerase is an attractive target for antiviral therapy. Nucleoside-based inhibitors have broad-spectrum activity but often exhibit off-target effects. Most non-nucleoside inhibitors (NNIs) target surface cavities, which are structurally more flexible than the nucleotide-binding pocket, and hence have a more narrow spectrum of activity and are more prone to resistance development. Here, we report a novel NNI, GPC-N114 (2,2'-[(4-chloro-1,2-phenylene)bis(oxy)]bis(5-nitro-benzonitrile)) with broad-spectrum activity against enteroviruses and cardioviruses (another genus in the picornavirus family). Surprisingly, coxsackievirus B3 (CVB3) and poliovirus displayed a high genetic barrier to resistance against GPC-N114. By contrast, EMCV, a cardiovirus, rapidly acquired resistance due to mutations in 3Dpol. In vitro polymerase activity assays showed that GPC-N114 i) inhibited the elongation activity of recombinant CVB3 and EMCV 3Dpol, (ii) had reduced activity against EMCV 3Dpol with the resistance mutations, and (iii) was most efficient in inhibiting 3Dpol when added before the RNA template-primer duplex. Elucidation of a crystal structure of the inhibitor bound to CVB3 3Dpol confirmed the RNA-binding channel as the target for GPC-N114. Docking studies of the compound into the crystal structures of the compound-resistant EMCV 3Dpol mutants suggested that the resistant phenotype is due to subtle changes that interfere with the binding of GPC-N114 but not of the RNA template-primer. In conclusion, this study presents the first NNI that targets the RNA template channel of the picornavirus polymerase and identifies a new pocket that can be used for the design of broad-spectrum inhibitors. Moreover, this study provides important new insight into the plasticity of picornavirus polymerases at the template binding site.

Structural dynamics as a contributor to error-prone replication by an RNA-dependent RNA polymerase.

RNA viruses encoding high- or low-fidelity RNA-dependent RNA polymerases (RdRp) are attenuated. The ability to predict residues of the RdRp required for faithful incorporation of nucleotides represents an essential step in any pipeline intended to exploit perturbed fidelity as the basis for rational design of vaccine candidates. We used x-ray crystallography, molecular dynamics simulations, NMR spectroscopy, and pre-steady-state kinetics to compare a mutator (H273R) RdRp from poliovirus to the wild-type (WT) enzyme. We show that the nucleotide-binding site toggles between the nucleotide binding-occluded and nucleotide binding-competent states. The conformational dynamics between these states were enhanced by binding to primed template RNA. For the WT, the occluded conformation was favored; for H273R, the competent conformation was favored. The resonance for Met-187 in our NMR spectra reported on the ability of the enzyme to check the correctness of the bound nucleotide. Kinetic experiments were consistent with the conformational dynamics contributing to the established pre-incorporation conformational change and fidelity checkpoint. For H273R, residues comprising the active site spent more time in the catalytically competent conformation and were more positively correlated than the WT. We propose that by linking the equilibrium between the binding-occluded and binding-competent conformations of the nucleotide-binding pocket and other active-site dynamics to the correctness of the bound nucleotide, faithful nucleotide incorporation is achieved. These studies underscore the need to apply multiple biophysical and biochemical approaches to the elucidation of the physical basis for polymerase fidelity.

Functional identification of the general acid and base in the dehydration step of indole-3-glycerol phosphate synthase catalysis.

The tryptophan biosynthetic enzyme indole-3-glycerol phosphate synthase is a proposed target for new antimicrobials and is a favored starting framework in enzyme engineering studies. Forty years ago, Parry proposed that the enzyme mechanism proceeds through two intermediates in a series of condensation, decarboxylation, and dehydration steps. X-ray crystal structures have suggested that Lys-110 (numbering according to the Sulfolobus solfataricus enzyme) behaves as a general acid both in the condensation and dehydration steps, but did not reveal an efficient pathway for the reprotonation of this critical residue. Our mutagenesis and kinetic experiments suggest an alternative mechanism whereby Lys-110 acts as a general acid in the condensation step, but another invariant residue, Lys-53, acts as the general acid in the dehydration step. These studies also indicate that the conserved residue Glu-51 acts as the general base in the dehydration step. The revised mechanism effectively divides the active site into discrete regions where the catalytic surfaces containing Lys-110 and Lys-53/Glu-51 catalyze the ring closure (i.e. condensation and decarboxylation) and dehydration steps, respectively. These results can be leveraged toward the development of novel inhibitors against this validated antimicrobial target and toward the rational engineering of the enzyme to produce indole derivatives that are highly prized by the pharmaceutical and agricultural industries.

Vaccine-derived mutation in motif D of poliovirus RNA-dependent RNA polymerase lowers nucleotide incorporation fidelity.

All viral RNA-dependent RNA polymerases (RdRps) have a conserved structural element termed motif D. Studies of the RdRp from poliovirus (PV) have shown that a conformational change of motif D leads to efficient and faithful nucleotide addition by bringing Lys-359 into the active site where it serves as a general acid. The RdRp of the Sabin I vaccine strain has Thr-362 changed to Ile. Such a drastic change so close to Lys-359 might alter RdRp function and contribute in some way to the attenuated phenotype of Sabin type I. Here we present our characterization of the T362I RdRp. We find that the T362I RdRp exhibits a mutator phenotype in biochemical experiments in vitro. Using NMR, we show that this change in nucleotide incorporation fidelity correlates with a change in the structural dynamics of motif D. A recombinant PV expressing the T362I RdRp exhibits normal growth properties in cell culture but expresses a mutator phenotype in cells. For example, the T362I-containing PV is more sensitive to the mutagenic activity of ribavirin than wild-type PV. Interestingly, the T362I change was sufficient to cause a statistically significant reduction in viral virulence. Collectively, these studies suggest that residues of motif D can be targeted when changes in nucleotide incorporation fidelity are desired. Given the observation that fidelity mutants can serve as vaccine candidates, it may be possible to use engineering of motif D for this purpose.

Amino acid networks in a (ß/α)8 barrel enzyme change during catalytic turnover.

Proteins can be viewed as small-world networks of amino acid residues connected through noncovalent interactions. Nuclear magnetic resonance chemical shift covariance analyses were used to identify long-range amino acid networks in the α subunit of tryptophan synthase both for the resting state (in the absence of substrate and product) and for the working state (during catalytic turnover). The amino acid networks observed stretch from the surface of the protein into the active site and are different between the resting and working states. Modification of surface residues on the network alters the structural dynamics of active-site residues over 25 Å away and leads to changes in catalytic rates. These findings demonstrate that amino acid networks, similar to those studied here, are likely important for coordinating structural changes necessary for enzyme function and regulation.

Promiscuity in protein-RNA interactions: conformational ensembles facilitate molecular recognition in the spliceosome: conformational diversity in U2AF65 facilitates binding to diverse RNA sequences.

Here I discuss findings that suggest a universal mechanism for proteins (and RNA) to recognize and interact with various binding partners by selectively binding to different conformations that pre-exist in the free protein's conformational ensemble. The tandem RNA recognition motif domains of splicing factor U2AF65 fluctuate in solution between a predominately closed conformation in which the RNA binding site of one of the domains is blocked, and a lowly populated open conformation in which both RNA binding pockets are accessible. RNA binding to U2AF65 may thus occur through the weakly populated open conformation, and the binding interaction stabilizes the open conformation. The conformational diversity observed in U2AF65 might also facilitate binding to diverse RNA sequences as found in the polypyrimidine tracts that help define 3' splice sites. Similar binding pathways in other systems have important consequences in biological regulation, molecular evolution, and information storage.

A distal mutation perturbs dynamic amino acid networks in dihydrofolate reductase.

Correlated networks of amino acids have been proposed to play a fundamental role in allostery and enzyme catalysis. These networks of amino acids can be traced from surface-exposed residues all the way into the active site, and disruption of these networks can decrease enzyme activity. Substitution of the distal Gly121 residue in Escherichia coli dihydrofolate reductase results in an up to 200-fold decrease in the hydride transfer rate despite the fact that the residue is located 15 Å from the active-site center. In this study, nuclear magnetic resonance relaxation experiments are used to demonstrate that dynamics on the picosecond to nanosecond and microsecond to millisecond time scales are changed significantly in the G121V mutant of dihydrofolate reductase. In particular, picosecond to nanosecond time scale dynamics are decreased in the FG loop (containing the mutated residue at position 121) and the neighboring active-site loop (the Met20 loop) in the mutant compared to those of the wild-type enzyme, suggesting that these loops are dynamically coupled. Changes in methyl order parameters reveal a pathway by which dynamic perturbations can be propagated more than 25 Å across the protein from the site of mutation. All of the enzyme complexes, including the model Michaelis complex with folate and nicotinamide adenine dinucleotide phosphate bound, assume an occluded ground-state conformation, and we do not observe sampling of a higher-energy closed conformation by (15)N R2 relaxation dispersion experiments. This is highly significant, because it is only in the closed conformation that the cofactor and substrate reactive centers are positioned for reaction. The mutation also impairs microsecond to millisecond time scale fluctuations that have been implicated in the release of product from the wild-type enzyme. Our results are consistent with an important role for Gly121 in controlling protein dynamics critical for enzyme function and further validate the dynamic energy landscape hypothesis of enzyme catalysis.

Millisecond timescale fluctuations in dihydrofolate reductase are exquisitely sensitive to the bound ligands.

Enzyme catalysis can be described as progress over a multi-dimensional energy landscape where ensembles of interconverting conformational substates channel the enzyme through its catalytic cycle. We applied NMR relaxation dispersion to investigate the role of bound ligands in modulating the dynamics and energy landscape of Escherichia coli dihydrofolate reductase to obtain insights into the mechanism by which the enzyme efficiently samples functional conformations as it traverses its reaction pathway. Although the structural differences between the occluded substrate binary complexes and product ternary complexes are very small, there are substantial differences in protein dynamics. Backbone fluctuations on the micros-ms timescale in the cofactor binding cleft are similar for the substrate and product binary complexes, but fluctuations on this timescale in the active site loops are observed only for complexes with substrate or substrate analog and are not observed for the binary product complex. The dynamics in the substrate and product binary complexes are governed by quite different kinetic and thermodynamic parameters. Analogous dynamic differences in the E:THF:NADPH and E:THF:NADP(+) product ternary complexes are difficult to rationalize from ground-state structures. For both of these complexes, the nicotinamide ring resides outside the active site pocket in the ground state. However, they differ in the structure, energetics, and dynamics of accessible higher energy substates where the nicotinamide ring transiently occupies the active site. Overall, our results suggest that dynamics in dihydrofolate reductase are exquisitely "tuned" for every intermediate in the catalytic cycle; structural fluctuations efficiently channel the enzyme through functionally relevant conformational space.

Regulatory mechanisms triggered by enzyme interactions with lipid membrane surfaces.

Recruitment of enzymes to intracellular membranes often modulates their catalytic activity, which can be important in cell signaling and membrane trafficking. Thus, re-localization is not only important for these enzymes to gain access to their substrates, but membrane interactions often allosterically regulate enzyme function by inducing conformational changes across different time and amplitude scales. Recent structural, biophysical and computational studies have revealed how key enzymes interact with lipid membrane surfaces, and how this membrane binding regulates protein structure and function. This review summarizes the recent progress in understanding regulatory mechanisms involved in enzyme-membrane interactions.

Allostery, engineering and inhibition of tryptophan synthase.

The final two steps of tryptophan biosynthesis are catalyzed by the enzyme tryptophan synthase (TS), composed of alpha (αTS) and beta (ßTS) subunits. Recently, experimental and computational methods have mapped "allosteric networks" that connect the αTS and ßTS active sites. In αTS, allosteric networks change across the catalytic cycle, which might help drive the conformational changes associated with its function. Directed evolution studies to increase catalytic function and expand the substrate profile of stand-alone ßTS have also revealed the importance of αTS in modulating the conformational changes in ßTS. These studies also serve as a foundation for the development of TS inhibitors, which can find utility against Mycobacterium tuberculosis and other bacterial pathogens.