Determining the principal energy pathways for allosteric communication in biomolecules, that occur as a result of thermal motion, remains challenging due to the intrinsic complexity of the systems involved. Graph theory provides an approach for making sense of such complexity, where allosteric proteins can be represented as networks of amino acids. In this work, we establish the eigenvector centrality metric in terms of the mutual information, as a mean of elucidating the allosteric mechanism that regulates the enzymatic activity of proteins. Moreover, we propose a strategy to characterize the range of the physical interactions that underlie the allosteric process. In particular, the well known enzyme, imidazol glycerol phosphate synthase (IGPS), is utilized to test the proposed methodology. The eigenvector centrality measurement successfully describes the allosteric pathways of IGPS, and allows to pinpoint key amino acids in terms of their relevance in the momentum transfer process. The resulting insight can be utilized for refining the control of IGPS activity, widening the scope for its engineering. Furthermore, we propose a new centrality metric quantifying the relevance of the surroundings of each residue. In addition, the proposed technique is validated against experimental solution NMR measurements yielding fully consistent results. Overall, the methodologies proposed in the present work constitute a powerful and cost effective strategy to gain insight on the allosteric mechanism of proteins.Allostery is a ubiquitous process of physico-chemical regulation in biological macromolecules such as enzymes. The fundamental step in the allosteric regulation is the binding of a ligand at a particular enzymatic site affecting the activity at a different and often very distant position of the protein. While allosteric processes have long been of interest, especially due to their relevance in developing potent and selective therapeutics, the mechanism for energy transfer between allosteric sites remains poorly understood. Thus, establishing a molecular level understanding of communication pathways between the physically distant enzymatic sites is crucial for the design of innovative drug therapies[1, 2] and protein engineering [3][4][5].Recently, there have been significant efforts toward the development of computational tools to support, interpret and/or predict experimental evidences for elucidation of allosteric pathways in proteins [2,[6][7][8][9][10][11][12]. Network analysis has been extensively used in this context, by incorporating concepts and methodologies from graph theory into the realm of molecular dynamics simulations [13][14][15][16][17][18][19] For instance, community network analysis (CNA) has emerged as a powerful and increasingly popular approach to analyze the dynamics of enzymes and protein/DNA (and/or RNA) complexes and to detect possible allosteric pathways [20][21][22][23][24][25][26].In these network theory-based approaches, a protein is represented as a network consisting of a set of nodes, n...
It has been suggested that the alkaline form of cytochrome c (cyt c) regulates function of this protein as an electron carrier in oxidative phosphorylation and as a peroxidase that reacts with cardiolipin (CL) during apoptosis. In this form, Met80, the native ligand to the heme iron, is replaced by a Lys. While it has become clear that the structure of cyt c changes, the extent and sequence of conformational rearrangements associated with this ligand replacement remain a subject of debate. Herein we report a high-resolution crystal structure of a Lys73-ligated cyt c conformation that reveals intricate change in the heme environment upon this switch in the heme iron ligation. The structure is surprisingly compact, and the heme coordination loop refolds into a β-hairpin with a turn formed by the highly conserved residues Pro76 and Gly77. Repositioning of residue 78 modifies the intraprotein hydrogen-bonding network and, together with adjustments of residues 52 and 74, increases the volume of the heme pocket to allow for insertion of one of the CL acyl moieties next to Asn52. Derivatization of Cys78 with maleimide creates a solution mimic of the Lys-ligated cyt c that has enhanced peroxidase activity, adding support for a role of the Lys-ligated cyt c in the apoptotic mechanism. Experiments with the heme peptide microperoxidase-8 and engineered model proteins provide a thermodynamic rationale for the switch to Lys ligation upon perturbations in the protein scaffold.
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.
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.allostery | NMR | community networks | millisecond motions T he underlying principles of allosteric regulation have been a focal point of enzymology and structural biology for decades as studies of allostery have evolved from two phenomenological models (1, 2) to recognize structural and conformational ensembles that define a broad range of enzymatic states (3-6). As a result, a great deal of emphasis has been placed on understanding dynamic contributions to allostery (7-12) and factors that enable small fluctuations in local conformations of enzymes to propagate information over large distances. A well-developed understanding of dynamic allostery opens up numerous avenues for insight into protein engineering (13,14), drug design (15), and mechanistic biochemistry (16)(17)(18)(19)(20)(21)(22). To establish universal principles for relevant enzymes, however, a link between dynamics and function must be made.The heterodimeric enzyme imidazole glycerol phosphate synthase (IGPS) plays a crucial role in amino acid and purine biosynthesis in bacteria and other microorganisms by sequentially catalyzing the hydrolysis of glutamine (Gln) in its HisH subunit and the cyclization of the substrate and allosteric effector N′-[(5′-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (PRFAR) in its HisF subunit (Scheme 1). IGPS is a V-type allosteric enzyme that is inactive unless PRFAR is bound, and communication between the active and effector sites is driven by the enhancement of conformational flexibility, namely, fluctuations taking place on the millisecond timescale (22-24). Based on NMR and computational evidence, these motions are believed to propagate from the PRFAR binding site to the glutaminase binding site, thereby disrupting a hydrogen bond between V51 and P10 of HisH. This H bond prevents formation of the oxyanion hole site necessary for glutamine hydrolysis, and its disruption allows IG...
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.
Cysteine-bound hemes are key components of many enzymes and biological sensors. Protonation (deprotonation) of the Cys ligand often accompanies redox transformations of these centers. To characterize these phenomena, we have engineered a series of Thr78Cys/Lys79Gly/Met80X mutants of yeast cytochrome c (cyt c) in which Cys78 becomes one of the axial ligands to the heme. At neutral pH, the protonation state of the coordinated Cys differs for the ferric and ferrous heme species, with Cys binding as a thiolate and a thiol, respectively. Analysis of redox-dependent stability and alkaline transitions of these model proteins, as well as comparisons to Cys binding studies with the minimalist heme peptide microperoxidase-8, demonstrate that the protein scaffold and solvent interactions play important roles in stabilizing a particular Cys-heme coordination. The increased stability of ferric thiolate compared with ferrous thiol arises mainly from entropic factors. This robust cyt c model system provides access to all four forms of Cys-bound heme, including the ferric thiol. Protein motions control the rates of heme redox reactions, and these effects are amplified at low pH, where the proteins are less stable. Thermodynamic signatures and redox reactivity of the model Cys-bound hemes highlight the critical role of the protein scaffold and its dynamics in modulating redox-linked transitions between thiols and thiolates.metalloenzyme | folding | electron transfer
1. Summary The allosteric mechanism of the heterodimeric enzyme imidazole glycerol phosphate synthase was studied in detail with solution NMR 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 – 4900 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. Further, 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 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. Graphical abstract *Corresponding Authors: ivan.rivalta@ens-lyon.fr.,
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