Mixed, nonclassical behavior in a classic allosteric protein.

Allostery is a major driver of biological processes requiring coordination. Thus, it is one of the most fundamental and remarkable phenomena in nature, and there is motivation to understand and manipulate it to a multitude of ends. Today, it is often described in terms of two phenomenological models proposed more than a half-century ago involving only T(tense) or R(relaxed) conformations. Here, methyl-based NMR provides extensive detail on a dynamic T to R switch in the classical dimeric allosteric protein, yeast chorismate mutase (CM), that occurs in the absence of substrate, but only with the activator bound. Switching of individual subunits is uncoupled based on direct observation of mixed TR states in the dimer. This unique finding excludes both classic models and solves the paradox of a coexisting hyperbolic binding curve and highly skewed substrate-free T-R equilibrium. Surprisingly, structures of the activator-bound and effector-free forms of CM appear the same by NMR, providing another example of the need to account for dynamic ensembles. The apo enzyme, which has a sigmoidal activity profile, is shown to switch, not to R, but to a related high-energy state. Thus, the conformational repertoire of CM does not just change as a matter of degree depending on the allosteric input, be it effector and/or substrate. Rather, the allosteric model appears to completely change in different contexts, which is only consistent with modern ensemble-based frameworks.

Allosteric inactivation of an engineered optogenetic GTPase.

Optogenetics is a technique for establishing direct spatiotemporal control over molecular function within living cells using light. Light application induces conformational changes within targeted proteins that produce changes in function. One of the applications of optogenetic tools is an allosteric control of proteins via light-sensing domain (LOV2), which allows direct and robust control of protein function. Computational studies supported by cellular imaging demonstrated that application of light allosterically inhibited signaling proteins Vav2, ITSN, and Rac1, but the structural and dynamic basis of such control has yet to be elucidated by experiment. Here, using NMR spectroscopy, we discover principles of action of allosteric control of cell division control protein 42 (CDC42), a small GTPase involved in cell signaling. Both LOV2 and Cdc42 employ flexibility in their function to switch between "dark"/"lit" or active/inactive states, respectively. By conjoining Cdc42 and phototropin1 LOV2 domains into the bi-switchable fusion Cdc42Lov, application of light-or alternatively, mutation in LOV2 to mimic light absorption-allosterically inhibits Cdc42 downstream signaling. The flow and patterning of allosteric transduction in this flexible system are well suited to observation by NMR. Close monitoring of the structural and dynamic properties of dark versus "lit" states of Cdc42Lov revealed lit-induced allosteric perturbations that extend to Cdc42's downstream effector binding site. Chemical shift perturbations for lit mimic, I539E, have distinct regions of sensitivity, and both the domains are coupled together, leading to bidirectional interdomain signaling. Insights gained from this optoallosteric design will increase our ability to control response sensitivity in future designs.

Dynamic allostery in substrate binding by human thymidylate synthase.

Human thymidylate synthase (hTS) is essential for DNA replication and therefore a therapeutic target for cancer. Effective targeting requires knowledge of the mechanism(s) of regulation of this 72 kDa homodimeric enzyme. Here, we investigate the mechanism of binding cooperativity of the nucleotide substrate. We have employed exquisitely sensitive methyl-based CPMG and CEST NMR experiments enabling us to identify residues undergoing bifurcated linear 3-state exchange, including concerted switching between active and inactive conformations in the apo enzyme. The inactive state is populated to only ~1.3%, indicating that conformational selection contributes negligibly to the cooperativity. Instead, methyl rotation axis order parameters, determined by 2H transverse relaxation rates, suggest that rigidification of the enzyme upon substrate binding is responsible for the entropically-driven cooperativity. Lack of the rigidification in product binding and substrate binding to an N-terminally truncated enzyme, both non-cooperative, support this idea. In addition, the lack of this rigidification in the N-terminal truncation indicates that interactions between the flexible N-terminus and the rest of the protein, which are perturbed by substrate binding, play a significant role in the cooperativity-a novel mechanism of dynamic allostery. Together, these findings yield a rare depth of insight into the substrate binding cooperativity of an essential enzyme.

Visualizing an Allosteric Intermediate Using CuAAC Stabilization of an NMR Mixed Labeled Dimer.

Homodimers are the most abundant type of enzyme in cells, and as such, they represent the most elemental system for studying the phenomenon of allostery. In these systems, in which the allosteric features are manifest by the effect of the first binding event on a similar event at the second site, the most informative state is the asymmetric singly bound (lig1) form, yet it tends to be thermodynamically elusive. Here we obtain milligram quantities of lig1 of the allosteric homodimer, chorismate mutase, in the form of a mixed isotopically labeled dimer stabilized by Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) between the subunits. Below, we outline several critical steps required to generate high yields of both types of unnatural amino acid-containing proteins and overcome multiple pitfalls intrinsic to CuAAC to obtain high yields of a highly purified, fully intact, active mixed labeled dimer, which provides the first glimpse of the lig1 intermediate. These data not only will make possible NMR-based investigations of allostery envisioned by us but also should facilitate other structural applications in which specific linkage of proteins is helpful.

Backbone and ILVM methyl resonance assignments of human thymidylate synthase in apo and substrate bound forms.

Human thymidylate synthase (hTS) is a 72 kDa homodimeric enzyme responsible for the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP), making it the sole source of de novo dTMP in human cells. As a result, hTS is an attractive anti-cancer therapeutic target. Additionally, hTS is known to possess a number of interesting biophysical features, including adoption of active and inactive conformations, positively cooperative substrate binding, half-the-sites activity, and interacting with its own mRNA. The physical mechanisms underlying these properties, and how they may be leveraged to guide therapeutic development, are yet to be fully explored. Here, as a preface to detailed NMR characterization, we present backbone amide and ILVM methyl resonance assignments for hTS in apo and dUMP bound forms. In addition, we present backbone amide resonance assignments for hTS bound to a substrate analog and the native cofactor.

Mapping allosteric communications within individual proteins.

Allostery in proteins influences various biological processes such as regulation of gene transcription and activities of enzymes and cell signaling. Computational approaches for analysis of allosteric coupling provide inexpensive opportunities to predict mutations and to design small-molecule agents to control protein function and cellular activity. We develop a computationally efficient network-based method, Ohm, to identify and characterize allosteric communication networks within proteins. Unlike previously developed simulation-based approaches, Ohm relies solely on the structure of the protein of interest. We use Ohm to map allosteric networks in a dataset composed of 20 proteins experimentally identified to be allosterically regulated. Further, the Ohm allostery prediction for the protein CheY correlates well with NMR CHESCA studies. Our webserver, Ohm.dokhlab.org, automatically determines allosteric network architecture and identifies critical coupled residues within this network.

Isotopic Labeling of Formate Dehydrogenase Perturbs the Protein Dynamics.

Isotope substitution of enzymes has become a means of addressing the participation of protein motions in enzyme-catalyzed reactions. The idea is that only the enzyme mass will be altered and not the electrostatics, so that the protein dynamics are essentially the same but at lower frequencies because of the mass change. In this study, we variably label all carbon atoms in formate dehydrogenase (FDH) with 13C, all nitrogen atoms with 15N, and all nonexchangeable hydrogen atoms with deuterium and investigate the impact that isotopic substitution has on the dynamics at the active site by two-dimensional infrared spectroscopy and compare with the measurements of the temperature dependence of the intrinsic kinetic isotope effects (KIEs). We show that 15N labeling of FDH has the largest effect and makes the active site more heterogeneous, whereas the addition of nonexchangeable deuterium appears to have the opposite effect of 15N on active-site dynamics, resulting in a behavior similar to that of native FDH. Nevertheless, the temperature dependence of the KIEs shows a monotonic trend with protein mass that does not correspond with the changes in dynamics. These results suggest that isotope labeling has more than just a mass effect on enzyme dynamics and may influence electrostatics in ways that complicate the interpretation of the protein isotope effect.

Positive Cooperativity in Substrate Binding by Human Thymidylate Synthase.

Thymidylate synthase (TS) catalyzes the production of the nucleotide dTMP from deoxyuridine monophosphate (dUMP), making the enzyme necessary for DNA replication and consequently a target for cancer therapeutics. TSs are homodimers with active sites separated by ∼30 Å. Reports of half-the-sites activity in TSs from multiple species demonstrate the presence of allosteric communication between the active sites of this enzyme. A simple explanation for the negative allosteric regulation occurring in half-the-sites activity would be that the two substrates bind with negative cooperativity. However, previous work on Escherichia coli TS revealed that dUMP substrate binds without cooperativity. To gain further insight into TS allosteric function, binding cooperativity in human TS is examined here. Isothermal titration calorimetry and two-dimensional lineshape analysis of NMR titration spectra are used to characterize the thermodynamics of dUMP binding, with a focus on quantification of cooperativity between the two substrate binding events. We find that human TS binds dUMP with ∼9-fold entropically driven positive cooperativity (ρITC = 9 ± 1, ρNMR = 7 ± 1), in contrast to the apparent strong negative cooperativity reported previously. Our work further demonstrates the necessity of globally fitting isotherms collected under various conditions, as well as accurate determination of binding competent protein concentration, for calorimetric characterization of homotropic cooperative binding. Notably, an initial curvature of the isotherm is found to be indicative of positively cooperative binding. Two-dimensional lineshape analysis NMR is also found to be an informative tool for quantifying binding cooperativity, particularly in cases in which bound intermediates yield unique resonances.

Inter-Active Site Communication Mediated by the Dimer Interface ß-Sheet in the Half-the-Sites Enzyme, Thymidylate Synthase.

Thymidylate synthase (TS) is a dimeric enzyme conserved in all life forms that exhibits the allosteric feature of half-the-sites activity. Neither the reason for nor the mechanism of this phenomenon is understood. We used a combined nuclear magnetic resonance (NMR) and molecular dynamics approach to study a stable intermediate preceding hydride transfer, which is the rate-limiting and half-the-sites step. In NMR titrations with ligands leading to this intermediate, we measured chemical shifts of the apoenzyme (lig0), the saturated holoenzyme (lig2), and the typically elusive singly bound (lig1) states. Approximately 40 amides showed quartet patterns providing direct NMR evidence of coupling between the active site and probes >30 Å away in the distal subunit. Quartet peak patterns have symmetrical character, indicating reciprocity in communicating the first and second binding events to the distal protomer. Quartets include key catalytic residues and map to the dimer interface ß-sheet, which also represents the shortest path between the two active sites. Simulations corroborate the coupling observed in solution in that there is excellent overlap between quartet residues and main-chain atoms having intersubunit cross-correlated motions. Simulations identify five hot spot residues, three of which lie at the kink in the unique ß-bulge abutting the active sites on either end of the sheet. Interstrand cross-correlated motions become more organized and pronounced as the enzyme progresses from lig0 to lig1 and ultimately lig2. Coupling in the apparently symmetrical complex has implications for half-the-sites reactivity and potentially resolves the paradox of inequivalent TS active sites despite the vast majority of X-ray structures appearing to be symmetrical.

Thermodynamic and NMR Assessment of Ligand Cooperativity and Intersubunit Communication in Symmetric Dimers: Application to Thymidylate Synthase.

Thymidylate synthase (TS) is a homodimeric enzyme with evidence for negative regulation of one protomer while the other protomer acts on substrate, so called half-the-sites reactivity. The mechanisms by which multisubunit allosteric proteins communicate between protomers is not well understood, and the simplicity of dimeric systems has advantages for observing conformational and dynamic processes that functionally connect distance-separated active sites. This review considers progress in overcoming the inherent challenges of accurate thermodynamic and atomic-resolution characterization of interprotomer communication mechanisms in symmetric protein dimers, with TS used as an example. Isothermal titration calorimetry (ITC) is used to measure ligand binding cooperativity, even in cases where the two binding enthalpies are similar, and NMR spectroscopy is used to detect site-specific changes occurring in the two protomers. The NMR approach makes use of mixed-labeled dimers, enabling protomer-specific detection of signals in the singly ligated state. The rich informational content of the NMR signals from the singly ligated state, relative to the apo and saturated states, requires new considerations that do not arise in simple cases of 1:1 protein-ligand interactions.

Protein Mass Effects on Formate Dehydrogenase.

Isotopically labeled enzymes (denoted as "heavy" or "Born-Oppenheimer" enzymes) have been used to test the role of protein dynamics in catalysis. The original idea was that the protein's higher mass would reduce the frequency of its normal-modes without altering its electrostatics. Heavy enzymes have been used to test if the vibrations in the native enzyme are coupled to the chemistry it catalyzes, and different studies have resulted in ambiguous findings. Here the temperature-dependence of intrinsic kinetic isotope effects of the enzyme formate dehydrogenase is used to examine the distribution of H-donor to H-acceptor distance as a function of the protein's mass. The protein dynamics are altered in the heavy enzyme to diminish motions that determine the transition state sampling in the native enzyme, in accordance with a Born-Oppenheimer-like effect on bond activation. Findings of this work suggest components related to fast frequencies that can be explained by Born-Oppenheimer enzyme hypothesis (vibrational) and also slower time scale events that are non-Born-Oppenheimer in nature (electrostatic), based on evaluations of protein mass dependence of donor-acceptor distance and forward commitment to catalysis along with steady state and single turnover measurements. Together, the findings suggest that the mass modulation affected both local, fast, protein vibrations associated with the catalyzed chemistry and the protein's macromolecular electrostatics at slower time scales; that is, both Born-Oppenheimer and non-Born-Oppenheimer effects are observed. Comparison to previous studies leads to the conclusion that isotopic labeling of the protein may have different effects on different systems, however, making heavy enzyme studies a very exciting technique for exploring the dynamics link to catalysis in proteins.

Widespread Perturbation of Function, Structure, and Dynamics by a Conservative Single-Atom Substitution in Thymidylate Synthase.

Thymidylate synthase (TSase) is responsible for synthesizing the sole de novo source of dTMP in all organisms. TSase is a drug target, and as such, it has been well studied in terms of both structure and reaction mechanism. Cysteine 146 in Escherichia coli TSase is universally conserved because it serves as the nucleophile in the enzyme mechanism. Here we use the C146S mutation to probe the role of the sulfur atom in early events in the catalytic cycle beyond serving as the nucleophile. Surprisingly, the single-atom substitution severely decreases substrate binding affinity, and the unfavorable ΔΔG°bind is comprised of roughly equal enthalpic and entropic components at 25 °C. Chemical shifts in the free and dUMP-bound states show the mutation causes perturbations throughout TSase, including regions important for complex stability, in agreement with a less favorable enthalpy change. We measured the nuclear magnetic resonance methyl symmetry axis order parameter (S2axis), a proxy for conformational entropy, for TSase at all vertices of the dUMP binding/C146S mutation thermodynamic cycle and found that the calculated TΔΔS°conf is similar in sign and magnitude to the calorimetric TΔΔS°. Further, we ascribed minor resonances in wild-type-dUMP spectra to a state with a covalent bond between Sγ of C146 and C6 of dUMP and find S2axis values are unaffected by covalent bond formation, indicating this reaction step is neutral with respect to ΔS°conf. Lastly, the C146S mutation allowed us to measure cofactor analog binding by isothermal titration calorimetry without the confounding heat signature of covalent bond formation. Raltitrexed binds free and singly bound TSase with similar affinities, yet the two binding events have different enthalpy changes, providing further evidence of communication between the two active sites.

Chemical shift imprint of intersubunit communication in a symmetric homodimer.

Allosteric communication is critical for protein function and cellular homeostasis, and it can be exploited as a strategy for drug design. However, unlike many protein-ligand interactions, the structural basis for the long-range communication that underlies allostery is not well understood. This lack of understanding is most evident in the case of classical allostery, in which a binding event in one protomer is sensed by a second symmetric protomer. A primary reason why study of interdomain signaling is challenging in oligomeric proteins is the difficulty in characterizing intermediate, singly bound species. Here, we use an NMR approach to isolate and characterize a singly ligated state ("lig1") of a homodimeric enzyme that is otherwise obscured by rapid exchange with apo and saturated forms. Mixed labeled dimers were prepared that simultaneously permit full population of the lig1 state and isotopic labeling of either protomer. Direct visualization of peaks from lig1 yielded site-specific ligand-state multiplets that provide a convenient format for assessing mechanisms of intersubunit communication from a variety of NMR measurements. We demonstrate this approach on thymidylate synthase from Escherichia coli, a homodimeric enzyme known to be half-the-sites reactive. Resolving the dUMP1 state shows that active site communication occurs not upon the first dUMP binding, but upon the second. Surprisingly, for many sites, dUMP1 peaks are found beyond the limits set by apo and dUMP2 peaks, indicating that binding the first dUMP pushes the enzyme ensemble to further conformational extremes than the apo or saturated forms. The approach used here should be generally applicable to homodimers.

An Ancestral Tryptophanyl-tRNA Synthetase Precursor Achieves High Catalytic Rate Enhancement without Ordered Ground-State Tertiary Structures.

Urzymes-short, active core modules derived from enzyme superfamilies-prepared from the two aminoacyl-tRNA synthetase (aaRS) classes contain only the modules shared by all related family members. They have been described as models for ancestral forms. Understanding them currently depends on inferences drawn from the crystal structures of the full-length enzymes. As aaRS Urzymes lack much of the mass of modern aaRS's, retaining only a small portion of the hydrophobic cores of the full-length enzymes, it is desirable to characterize their structures. We report preliminary characterization of (15)N tryptophanyl-tRNA synthetase Urzyme by heteronuclear single quantum coherence (HSQC) NMR spectroscopy supplemented by circular dichroism, thermal melting, and induced fluorescence of bound dye. The limited dispersion of (1)H chemical shifts (0.5 ppm) is inconsistent with a narrow ensemble of well-packed structures in either free or substrate-bound forms, although the number of resonances from the bound state increases, indicating a modest, ligand-dependent gain in structure. Circular dichroism spectroscopy shows the presence of helices and evidence of cold denaturation, and all ligation states induce Sypro Orange fluorescence at ambient temperatures. Although the term "molten globule" is difficult to define precisely, these characteristics are consistent with most such definitions. Active-site titration shows that a majority of molecules retain ∼60% of the transition state stabilization free energy observed in modern synthetases. In contrast to the conventional view that enzymes require stable tertiary structures, we conclude that a highly flexible ground-state ensemble can nevertheless bind tightly to the transition state for amino acid activation.

The Effect of Protein Mass Modulation on Human Dihydrofolate Reductase.

Dihydrofolate reductase (DHFR) from Escherichia coli has long served as a model enzyme with which to elucidate possible links between protein dynamics and the catalyzed reaction. Such physical properties of its human counterpart have not been rigorously studied so far, but recent computer-based simulations suggest that these two DHFRs differ significantly in how closely coupled the protein dynamics and the catalyzed C-H → C hydride transfer step are. To test this prediction, two contemporary probes for studying the effect of protein dynamics on catalysis were combined here: temperature dependence of intrinsic kinetic isotope effects (KIEs), which are sensitive to the physical nature of the chemical step, and protein mass modulation, which slows down fast dynamics (femto- to picosecond time scale) throughout the protein. The intrinsic H/T KIEs of human DHFR, like those of E. coli DHFR, are shown to be temperature-independent in the range from 5 to 45 °C, indicating fast sampling of donor and acceptor distances (DADs) at the reaction's transition state (or tunneling ready state, TRS). Mass modulation of these enzymes through isotopic labeling with (13)C, (15)N, and (2)H at nonexchangeable hydrogens yields an 11% heavier enzyme. The additional mass has no effect on the intrinsic KIEs of the human enzyme. This finding indicates that the mass modulation of the human DHFR affects neither DAD distribution nor the DAD's conformational sampling dynamics. Furthermore, reduction in the enzymatic turnover number and the dissociation rate constant for the product indicate that the isotopic substitution affects kinetic steps that are not the catalyzed C-H → C hydride transfer. The findings are discussed in terms of fast dynamics and their role in catalysis, the comparison of calculations and experiments, and the interpretation of isotopically modulated heavy enzymes in general.

Bacterial Thymidylate Synthase Binds Two Molecules of Substrate and Cofactor without Cooperativity.

Thymidylate synthase (TSase) is a clinically important enzyme because it catalyzes synthesis of the sole de novo source of deoxy-thymidylate. Without this enzyme, cells die a "thymineless death" since they are starved of a crucial DNA synthesis precursor. As a drug target, TSase is well studied in terms of its structure and reaction mechanism. An interesting mechanistic feature of dimeric TSase is that it is "half-the-sites reactive", which is a form of negative cooperativity. Yet, the basis for this is not well-understood. Some experiments point to cooperativity at the binding steps of the reaction cycle as being responsible for the phenomenon, but the literature contains conflicting reports. Here we use ITC and NMR to resolve these inconsistencies. This first detailed thermodynamic dissection of multisite binding of dUMP to E. coli TSase shows the nucleotide binds to the free and singly bound forms of the enzyme with nearly equal affinity over a broad range of temperatures and in multiple buffers. While small but significant differences in ΔC°P for the two binding events show that the active sites are not formally equivalent, there is little-to-no allostery at the level of ΔG°bind. In addition NMR titration data reveal that there is minor intersubunit cooperativity in formation of a ternary complex with the mechanism based inhibitor, 5F-dUMP, and cofactor. Taken together, the data show that functional communication between subunits is minimal for both binding steps of the reaction coordinate.

Contrasting roles of dynamics in protein allostery: NMR and structural studies of CheY and the third PDZ domain from PSD-95.

Allosteric regulation is a ubiquitous phenomenon exploited in biological processes to control cells in a myriad of ways. It is also of emerging interest in the design of functional proteins and therapeutics. Even though allostery was proposed over 50 years ago and has been studied intensively from a structural perspective, many key details of allosteric mechanisms remain mysterious. Over the last decade significant attention has been paid to the "dynamic component" of allostery, as opposed to the analysis of rigid structures. Nuclear magnetic resonance spectroscopy and its ability to detect conformationally dynamic processes at atomic resolution have played an important role in expanding our understanding of allosteric mechanisms and opening up new questions. This article focuses on work that highlights how protein dynamics can factor into allosteric processes in distinct ways. Two cases are contrasted. The first considers the "traditionally allosteric" protein CheY, which undergoes a conformational change as a key element of its allostery. The second considers the more rarely observed "dynamic allostery" in a PDZ domain, in which allosteric behavior arises from changes in internal structural dynamics. Interestingly, the dynamic processes in these two contrasting examples occur on different timescales. In the case of the PDZ domain, subsequent experimental and computational work is reviewed to reveal a more complete picture of this interesting case of allostery.

Backbone and ILV methyl resonance assignments of E. coli thymidylate synthase bound to cofactor and a nucleotide analogue.

Thymidylate synthase (TSase) is a 62 kDa homodimeric enzyme required for de novo synthesis of thymidine monophosphate in most organisms. This makes the enzyme an excellent target for anticancer and microbial antibiotic drugs. In addition, TSase has been shown to exhibit negative cooperativity and half-the-sites reactivity. For these collective reasons, TSase is widely studied, and much is known about its kinetics and structure as it progresses through a multi-step catalytic cycle. Recently, nuclear magnetic resonance spin relaxation has been instrumental in demonstrating the critical role of dynamics in enzyme function in small model systems. These studies raise questions about how dynamics affect function in larger enzymes with more complex reaction coordinates. TSase is an ideal candidate given its size, oligomeric state, cooperativity, and status as a drug target. Here, as a pre-requisite to spin relaxation studies, we present the backbone and ILV methyl resonance assignments of TSase from Escherichia coli bound to a substrate analogue and cofactor.

Monitoring side-chain dynamics of proteins using (2)H relaxation.

Nuclear magnetic resonance (NMR) is a powerful technique capable of monitoring a wide range of motions in proteins on a per residue basis. A variety of (2)H relaxation experiments have been developed for monitoring side-chain methyl group motions on the picosecond-nanosecond timescale. These experiments enable determination of the order parameter, S (2) axis, which reports on the rigidity of the C-CH3 bond for side-chain methyl groups. The application of a commonly used subset of these experiments is described in this chapter. It is intended to serve as a practical guide to investigators interested in monitoring side-chain motions.