Allostery is a ubiquitous biological regulatory process in which distant binding sites within a protein or enzyme are functionally and thermodynamically coupled. Allosteric interactions play ...essential roles in many enzymological mechanisms, often facilitating formation of enzyme–substrate complexes and/or product release. Thus, elucidating the forces that drive allostery is critical to understanding the complex transformations of biomolecules. Currently, a number of models exist to describe allosteric behavior, taking into account energetics as well as conformational rearrangements and fluctuations. In the following Review, we discuss the use of solution NMR techniques designed to probe allosteric mechanisms in enzymes. NMR spectroscopy is unequaled in its ability to detect structural and dynamical changes in biomolecules, and the case studies presented herein demonstrate the range of insights to be gained from this valuable method. We also provide a detailed technical discussion of several specialized NMR experiments that are ideally suited for the study of enzymatic allostery.
Determining the principal energy-transfer pathways responsible for allosteric communication in biomolecules remains challenging, partially due to the intrinsic complexity of the systems and the lack ...of effective characterization methods. In this work, we introduce the eigenvector centrality metric based on mutual information to elucidate allosteric mechanisms that regulate enzymatic activity. Moreover, we propose a strategy to characterize the range of correlations that underlie the allosteric processes. We use the V-type allosteric enzyme imidazole glycerol phosphate synthase (IGPS) to test the proposed methodology. The eigenvector centrality method identifies key amino acid residues of IGPS with high susceptibility to effector binding. The findings are validated by solution NMR measurements yielding important biological insights, including direct experimental evidence for interdomain motion, the central role played by helix hα 1, and the short-range nature of correlations responsible for the allosteric mechanism. Beyond insights on IGPS allosteric pathways and the nature of residues that could be targeted by therapeutic drugs or site-directed mutagenesis, the reported findings demonstrate the eigenvector centrality analysis as a general cost-effective methodology to gain fundamental understanding of allosteric mechanisms at the molecular level.
•Analyses of NMR chemical shifts are described for the elucidation of allosteric coupling within proteins.•A focus on the quintessential simple readout of NMR spectroscopy, the chemical shift, ...provides an opportunity for high levels of insight for specialists and non-specialists.•Practical considerations for various analyses are discussed, as is the complementarity between various methods.•Protein conformational equilibria are dissected on multiple exchange regimes to reveal networks of amino acids that drive functional responses.
The exquisite sensitivity of the NMR chemical shift to local environment makes it an ideal probe to assess atomic level perturbations in proteins of all sizes and structural compositions. Recent advances in solution and solid-state NMR spectroscopy of biomolecules have leveraged the chemical shift to report on short- and long-range couplings between individual amino acids to establish “networks” of residues that form the basis of allosteric pathways that transmit chemical signals through the protein matrix to induce functional responses. The simple premise that thermodynamically and functionally coupled regions of a protein (i.e. active and allosteric sites) should be reciprocally sensitive to structural or dynamic perturbations has enabled NMR spectroscopy, the premier method for molecular resolution of protein structural fluctuations, to occupy a place at the forefront of investigations into protein allostery. Here, we detail several key methods of NMR chemical shift analysis to extract mechanistic information about long-range chemical signaling in a protein, focusing on practical methodological aspects and the circumstances under which a given approach would be relevant. We also detail some of the experimental considerations that should be made when applying these methods to specific protein systems.
CRISPR–Cas9 is a widely employed genome-editing tool with functionality reliant on the ability of the Cas9 endonuclease to introduce site-specific breaks in double-stranded DNA. In this system, an ...intriguing allosteric communication has been suggested to control its DNA cleavage activity through flexibility of the catalytic HNH domain. Here, solution NMR experiments and a novel Gaussian-accelerated molecular dynamics (GaMD) simulation method are used to capture the structural and dynamic determinants of allosteric signaling within the HNH domain. We reveal the existence of a millisecond time scale dynamic pathway that spans HNH from the region interfacing the adjacent RuvC nuclease and propagates up to the DNA recognition lobe in full-length CRISPR–Cas9. These findings reveal a potential route of signal transduction within the CRISPR–Cas9 HNH nuclease, advancing our understanding of the allosteric pathway of activation. Further, considering the role of allosteric signaling in the specificity of CRISPR–Cas9, this work poses the mechanistic basis for novel engineering efforts aimed at improving its genome-editing capability.
Allostery in enzyme catalysis Lisi, George P; Loria, J Patrick
Current opinion in structural biology,
December 2017, 2017-12-00, 20171201, Letnik:
47
Journal Article
Recenzirano
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•Protein motions are critical for allostery.•Often optimal allosteric pathways exhibit synchronous motions.•Conservative mutations can dramatically disrupt motions and ...allostery.•Allosteric information transfer is sensitive to small adjustments to ligand structure.
Modern interpretations of allostery typically rely on conformational ensembles to describe enzyme function. Conformational motions controlling these ensembles are often stimulated or quenched by allosteric effectors, and are critical to optimizing ligand binding pockets and enzyme architectures. Thus, enzymes rely on dynamic allosteric pathways that transmit long-range binding information to control catalysis. In this review, we provide a brief discussion of the ever-expanding principles of allosteric regulation in enzyme catalysis and highlight in-depth studies of three enzymes that have contributed to the paradigms of dynamic allostery.
Imidazole glycerol phosphate synthase (IGPS) is a V-type allosteric enzyme, meaning that its catalytic rate is critically dependent on activation by its allosteric ligand, ...N′-(5′-phosphoribulosyl)formimino-5-aminoimidazole-4-carboxamide ribonucleotide (PRFAR). The allosteric mechanism of IGPS is reliant on millisecond conformational motions for efficient catalysis. We engineered four mutants of IGPS designed to disrupt millisecond motions and allosteric coupling to identify regions that are critical to IGPS function. Multiple-quantum Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion experiments and NMR chemical shift titrations reveal diminished enzyme flexibility and a reshaping of the allosteric connectivity in each mutant construct, respectively. The functional relevance of the observed motional quenching is confirmed by significant reductions in glutaminase kinetic activity and allosteric ligand binding affinity. This work presents relevant conclusions toward the control of protein allostery and design of unique allosteric sites for potential enzyme inhibitors with regulatory or therapeutic benefit.
The allosteric mechanism of the heterodimeric enzyme imidazole glycerol phosphate synthase was studied in detail with solution nuclear magnetic resonance spectroscopy and molecular dynamics ...simulations. We studied IGPS in complex with a series of allosteric activators corresponding to a large range of catalytic rate enhancements (26- to 4,900-fold), in which ligand binding is entropically driven. Conformational flexibility on the millisecond timescale plays a crucial role in intersubunit communication. Carr-Purcell-Meiboom-Gill relaxation dispersion experiments probing Ile, Leu, and Val methyl groups reveal that the apo- and glutamine-mimicked complexes are static on the millisecond timescale. Domain-wide motions are stimulated in the presence of the allosteric activators. These studies, in conjunction with ligand titrations, demonstrate that the allosteric network is widely dispersed and varies with the identity of the effector. Furthermore, we find that stronger allosteric ligands create more conformational flexibility on the millisecond timescale throughout HisF. This domain-wide loosening leads to maximum catalytic activity.
•Allosteric ligand binding is correlated to structural dynamics in a model enzyme•A widely dispersed allosteric network has been identified•Catalytic rate enhancement varies with the degree of millisecond flexibility•Allosteric ligand binding is communicated over a 25-Å distance
Lisi et al. use NMR and computational methods to probe the allosteric influence of small-molecule activators. Allosteric activator-induced changes in millisecond motions are responsible for enhancing the catalytic rate at a distant active site. Interestingly the most activating ligand results in the largest degree of motions.
This paper presents an innovative approach for predicting the relative populations of protein conformations using AlphaFold 2, an AI-powered method that has revolutionized biology by enabling the ...accurate prediction of protein structures. While AlphaFold 2 has shown exceptional accuracy and speed, it is designed to predict proteins' ground state conformations and is limited in its ability to predict conformational landscapes. Here, we demonstrate how AlphaFold 2 can directly predict the relative populations of different protein conformations by subsampling multiple sequence alignments. We tested our method against nuclear magnetic resonance experiments on two proteins with drastically different amounts of available sequence data, Abl1 kinase and the granulocyte-macrophage colony-stimulating factor, and predicted changes in their relative state populations with more than 80% accuracy. Our subsampling approach worked best when used to qualitatively predict the effects of mutations or evolution on the conformational landscape and well-populated states of proteins. It thus offers a fast and cost-effective way to predict the relative populations of protein conformations at even single-point mutation resolution, making it a useful tool for pharmacology, analysis of experimental results, and predicting evolution.
Allosteric enzymes regulate a wide range of catalytic transformations, including biosynthetic mechanisms of important human pathogens, upon binding of substrate molecules to an orthosteric (or ...active) site and effector ligands at distant (allosteric) sites. We find that enzymatic activity can be impaired by small molecules that bind along the allosteric pathway connecting the orthosteric and allosteric sites, without competing with endogenous ligands. Noncompetitive allosteric inhibitors disrupted allostery in the imidazole glycerol phosphate synthase (IGPS) enzyme from Thermotoga maritima as evidenced by nuclear magnetic resonance, microsecond time-scale molecular dynamics simulations, isothermal titration calorimetry, and kinetic assays. The findings are particularly relevant for the development of allosteric antibiotics, herbicides, and antifungal compounds because IGPS is absent in mammals but provides an entry point to fundamental biosynthetic pathways in plants, fungi, and bacteria.
Remdesivir is an antiviral drug initially designed against the Ebola virus. The results obtained with it both in biochemical studies in vitro and in cell line assays in vivo were very promising, but ...it proved to be ineffective in clinical trials. Remdesivir exhibited far better efficacy when repurposed against SARS-CoV-2. The chemistry that accounts for this difference is the subject of this study. Here, we examine the hypothesis that remdesivir monophosphate (RMP)-containing RNA functions as a template at the polymerase site for the second run of RNA synthesis, and as mRNA at the decoding center for protein synthesis. Our hypothesis is supported by the observation that RMP can be incorporated into RNA by the RNA-dependent RNA polymerases (RdRps) of both viruses, although some of the incorporated RMPs are subsequently removed by exoribonucleases. Furthermore, our hypothesis is consistent with the fact that RdRp of SARS-CoV-2 selects RMP for incorporation over AMP by 3-fold in vitro, and that RMP-added RNA can be rapidly extended, even though primer extension is often paused when the added RMP is translocated at the i + 3 position (with i the nascent base pair at an initial insertion site of RMP) or when the concentrations of the subsequent nucleoside triphosphates (NTPs) are below their physiological concentrations. These observations have led to the hypothesis that remdesivir might be a delayed chain terminator. However, that hypothesis is challenged under physiological concentrations of NTPs by the observation that approximately three-quarters of RNA products efficiently overrun the pause.
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