Chemical shift imprint of intersubunit communication in a symmetric homodimer

Regulatory signals between protein subunits depend on communication between sequential binding events. How such long-range communication, or allostery, operates at the subdomain level has been elusive, especially for homooligomeric proteins. To address this problem, homodimers of thymidylate synthase were generated for optimized study of individual protomers of the singly bound dimer. Mixed 15N-labeled dimers were created with a single functional active site, allowing site-specific, protomer-specific chemical shifts to report on step-wise binding effects. Long-range intersubunit communication was observed although this communication was apparent only in the second ligand-binding step, in which changes were in the first ligand-bound region. Visualization of up to four peaks for each residue amide provides a unique way to assess the allosteric mechanism

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) that are sensitive to the physical nature of the chemical step, and protein mass-modulation that slows down fast dynamics (femto- to picosecond timescale) 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 5–45 °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 13C, 15N, and 2H at nonexchangeable hydrogens yield 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

Thermal performance of external renders applied to concrete blockwork

The aim of this study was to investigate and analyse different external renders, available locally, and to study how their use may enhance the overall thermal performance of local concrete blockwork. This study provides an insight into how the various types of renders available improve the U-value of a concrete block wall. Three main types of external renders were used as the basis of this study. Results demonstrate that the ‘glass fibre additive’ type of render helped to greatly improve the U-value of a bare concrete block wall. This enhanced U-value, however, is achieved using more expensive render systems. Thus, from this study, the energy conscious designer can assess how, with the help of specific external renders, a more energy efficient building could be achieved, or how an existing building’s thermal efficiency could be improved. There are three aspects of performance that inform the selection of an external finish, namely: Aesthetic quality (colour & texture); Cost effectiveness, as compared to other types of finishes; Resilience to adverse weather conditions, particularly thermal performance. This paper investigates the issues relating to thermal performance, but also highlights the cost associated with choosing alternative but similar renders.peer-reviewe

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 (dTMP) 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 (NMR) 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

Using NMR to study fast dynamics in proteins: methods and applications

Proteins exist not as singular structures with precise coordinates, but rather, as fluctuating bodies that move rapidly through an enormous number of conformational substates. These dynamics have important implications for understanding protein function and for structure-based drug design. NMR spectroscopy is particularly well-suited to characterize dynamics of proteins and other molecules in solution at atomic resolution. Here, NMR relaxation methods for characterizing thermal motions on the picosecond-nanosecond (ps-ns) timescale are reviewed. Motion on this timescale can be conveniently captured by the Lipari-Szabo order parameter, S2, a bond-specific measure of restriction of motion. Approaches for determining order parameters are discussed, as are recent examples from the literature that link ps-ns dynamics with conformational entropy, allostery, and protein function 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 phenome non but the literature contains conflicting reports. Here we Contrary to these reports are the x-ray models of TSase, which for the case of the E. coli enzyme, have yet to capture detailed thermodynamic dissection of multi-site 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 inter-subunit 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

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 depends on inferences drawn from the crystal structures of the full-length enzymes. As aaRS Urzymes lack much of the mass of modern aaRS, 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 15N 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 1H 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 alpha helix 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

Crystallographic and Nuclear Magnetic Resonance Evaluation of the Impact of Peptide Binding to the Second PDZ Domain of Protein Tyrosine Phosphatase 1E

PDZ (PSD95/Discs large/ZO-1) domains are ubiquitous protein interaction motifs found in scaffolding proteins involved in signal transduction. Despite the fact that many PDZs show a limited tendency to undergo structural change, the PDZ family has been associated with long-range communication and allostery. One of the PDZ domains studied most in terms of structure and biophysical properties is the second PDZ (“PDZ2”) domain from protein tyrosine phophatase 1E (PTP1E, also known as PTPL1). Previously we showed through NMR relaxation studies that binding of the RA-GEF2 C-terminal peptide substrate results in long-range propagation of side-chain dynamic changes in human PDZ2 [Fuentes, et al., J. Mol. Biol. (2004), 335, 1105-1115]. Here, we present the first X-ray crystal structures of PDZ2 in the absence and presence of RA-GEF2 ligand, solved to resolutions of 1.65 and 1.3 Å, respectively. These structures deviate somewhat from previously determined NMR structures, and indicate that very minor structural changes in PDZ2 accompany peptide binding. NMR residual dipolar couplings confirm the crystal structures to be accurate models of the time-averaged atomic coordinates of PDZ2. The impact on side-chain dynamics was further tested with a C-terminal peptide from APC, which showed near-identical results to that of RA-GEF2. Thus, allosteric transmission in PDZ2 induced by peptide binding is conveyed purely and robustly by dynamics. 15N relaxation dispersion measurements did not detect appreciable populations of a kinetic structural intermediate. Collectively, for ligand binding to PDZ2, these data support a lock-and-key binding model from a structural perspective and an allosteric model from a dynamical perspective, which together suggest a complex energy landscape for functional transitions within the ensemble

Mg 2+ Binds to the Surface of Thymidylate Synthase and Affects Hydride Transfer at the Interior Active Site

Thymidylate synthase (TSase) produces the sole intracellular de novo source of thymidine (i.e. the DNA base T) and thus is a common target for antibiotic and anticancer drugs. Mg2+ has been reported to affect TSase activity, but the mechanism of this interaction has not been investigated. Here we show that Mg2+ binds to the surface of Escherichia coli TSase and affects the kinetics of hydride transfer at the interior active site (16 Å away). Examination of the crystal structures identifies a Mg2+ near the glutamyl moiety of the folate cofactor, providing the first structural evidence for Mg2+ binding to TSase. The kinetics and NMR relaxation experiments suggest that the weak binding of Mg2+ to the protein surface stabilizes the closed conformation of the ternary enzyme complex and reduces the entropy of activation on the hydride transfer step. Mg2+ accelerates the hydride transfer by ca. 7-fold but does not affect the magnitude or temperature-dependence of the intrinsic kinetic isotope effect. These results suggest that Mg2+ facilitates the protein motions that bring the hydride donor and acceptor together, but it does not change the tunneling ready state of the hydride transfer. These findings highlight how variations in cellular Mg2+ concentration can modulate enzyme activity through long-range interactions in the protein, rather than binding at the active site. The interaction of Mg2+ with the glutamyl-tail of the folate cofactor and nonconserved residues of bacterial TSase may assist in designing antifolates with poly-glutamyl substitutes as species-specific antibiotic drugs

Structure and Dynamics of the G121V Dihydrofolate Reductase Mutant: Lessons from a Transition-State Inhibitor Complex

It is well known that enzyme flexibility is critical for function. This is due to the observation that the rates of intramolecular enzyme motions are often matched to the rates of intermolecular events such as substrate binding and product release. Beyond this role in progression through the reaction cycle, it has been suggested that enzyme dynamics may also promote the chemical step itself. Dihydrofolate reductase (DHFR) is a model enzyme for which dynamics have been proposed to aid in both substrate flux and catalysis. The G121V mutant of DHFR is a well studied form that exhibits a severe reduction in the rate of hydride transfer yet there remains dispute as to whether this defect is caused by altered structure, dynamics, or both. Here we address this by presenting an NMR study of the G121V mutant bound to reduced cofactor and the transition state inhibitor, methotrexate. NMR chemical shift markers demonstrate that this form predominantly adopts the closed conformation thereby allowing us to provide the first glimpse into the dynamics of a catalytically relevant complex. Based on 15N and 2H NMR spin relaxation, we find that the mutant complex has modest changes in ps-ns flexibility with most affected residues residing in the distal adenosine binding domain rather than the active site. Thus, aberrant ps-ns dynamics are likely not the main contributor to the decreased catalytic rate. The most dramatic effect of the mutation involves changes in µs-ms dynamics of the F-G and Met20 loops. Whereas loop motion is quenched in the wild type transition state inhibitor complex, the F-G and Met20 loops undergo excursions from the closed conformation in the mutant complex. These excursions serve to decrease the population of conformers having the correct active site configuration, thus providing an explanation for the G121V catalytic defect