Molecular Mechanisms Of Allosteric Regulation Research Articles

Insights into Conformational Dynamics and Allostery in DNMT1-H3Ub/USP7 Interactions.

DNA methyltransferases (DNMTs) including DNMT1 are a conserved family of cytosine methylases that play crucial roles in epigenetic regulation. The versatile functions of DNMT1 rely on allosteric networks between its different interacting partners, emerging as novel therapeutic targets. In this work, based on the modeling structures of DNMT1-ubiquitylated H3 (H3Ub)/ubiquitin specific peptidase 7 (USP7) complexes, we have used a combination of elastic network models, molecular dynamics simulations, structural residue perturbation, network modeling, and pocket pathway analysis to examine their molecular mechanisms of allosteric regulation. The comparative intrinsic and conformational dynamics analysis of three DNMT1 systems has highlighted the pivotal role of the RFTS domain as the dynamics hub in both intra- and inter-molecular interactions. The site perturbation and network modeling approaches have revealed the different and more complex allosteric interaction landscape in both DNMT1 complexes, involving the events caused by mutational hotspots and post-translation modification sites through protein-protein interactions (PPIs). Furthermore, communication pathway analysis and pocket detection have provided new mechanistic insights into molecular mechanisms underlying quaternary structures of DNMT1 complexes, suggesting potential targeting pockets for PPI-based allosteric drug design.

Allosteric regulation of histone lysine methyltransferases: from context-specific regulation to selective drugs.

Histone lysine methyltransferases (HKMTs) are key regulators of many cellular processes. By definition, HKMTs catalyse the methylation of lysine residues in histone proteins. The enzymatic activities of HKMTs are under precise control, with their allosteric regulation emerging as a prevalent paradigm. We review the molecular mechanisms of allosteric regulation of HKMTs using well-studied histone H3 (K4, K9, K27 and K36) methyltransferases as examples. We discuss the current advances and future potential in targeting allosteric sites of HKMTs for drug development.

Structure and function of crocodilian hemoglobins and allosteric regulation by chloride, ATP, and CO2.

Hemoglobins (Hbs) of crocodilians are reportedly characterized by unique mechanisms of allosteric regulatory control, but there are conflicting reports regarding the importance of different effectors, such as chloride ions, organic phosphates, and CO2. Progress in understanding the unusual properties of crocodilian Hbs has also been hindered by a dearth of structural information. Here, we present the first comparative analysis of blood properties and Hb structure and function in a phylogenetically diverse set of crocodilian species. We examine mechanisms of allosteric regulation in the Hbs of 13 crocodilian species belonging to the families Crocodylidae and Alligatoridae. We also report new amino acid sequences for the α- and β-globins of these taxa, which, in combination with structural analyses, provide insights into molecular mechanisms of allosteric regulation. All crocodilian Hbs exhibited a remarkably strong sensitivity to CO2, which would permit effective O2 unloading to tissues in response to an increase in metabolism during intense activity and diving. Although the Hbs of all crocodilians exhibit similar intrinsic O2-affinities, there is considerable variation in sensitivity to Cl- ions and ATP, which appears to be at least partly attributable to variation in the extent of NH2-terminal acetylation. Whereas chloride appears to be a potent allosteric effector of all crocodile Hbs, ATP has a strong, chloride-independent effect on Hb-O2 affinity only in caimans. Modeling suggests that allosteric ATP binding has a somewhat different structural basis in crocodilian and mammalian Hbs.

Mechanism of allosteric activation of human mRNA cap methyltransferase (RNMT) by RAM: insights from accelerated molecular dynamics simulations.

The RNA guanine-N7 methyltransferase (RNMT) in complex with RNMT-activating miniprotein (RAM) catalyses the formation of a N7-methylated guanosine cap structure on the 5′ end of nascent RNA polymerase II transcripts. The mRNA cap protects the primary transcript from exonucleases and recruits cap-binding complexes that mediate RNA processing, export and translation. By using microsecond standard and accelerated molecular dynamics simulations, we provide for the first time a detailed molecular mechanism of allosteric regulation of RNMT by RAM. We show that RAM selects the RNMT active site conformations that are optimal for binding of substrates (AdoMet and the cap), thus enhancing their affinity. Furthermore, our results strongly suggest the likely scenario in which the cap binding promotes the subsequent AdoMet binding, consistent with the previously suggested cooperative binding model. By employing the network community analyses, we revealed the underlying long-range allosteric networks and paths that are crucial for allosteric regulation by RAM. Our findings complement and explain previous experimental data on RNMT activity. Moreover, this study provides the most complete description of the cap and AdoMet binding poses and interactions within the enzyme’s active site. This information is critical for the drug discovery efforts that consider RNMT as a promising anti-cancer target.

Atomistic Modeling of the ABL Kinase Regulation by Allosteric Modulators Using Structural Perturbation Analysis and Community-Based Network Reconstruction of Allosteric Communications.

In this work, we have examined the molecular mechanisms of allosteric regulation of the ABL tyrosine kinase at the atomic level. Atomistic modeling of the ABL complexes with a panel of allosteric modulators has been performed using a combination of molecular dynamics simulations, structural residue perturbation scanning, and a novel community analysis of the residue interaction networks. Our results have indicated that allosteric inhibitors and activators may exert a differential control on allosteric signaling between the kinase binding sites and functional regions. While the inhibitor binding can strengthen the closed ABL state and induce allosteric communications directed from the allosteric pocket to the ATP binding site, the DPH activator may induce a more dynamic open form and activate allosteric couplings between the ATP and substrate binding sites. By leveraging a network-centric theoretical framework, we have introduced a novel community analysis method and global topological parameters that have unveiled the hierarchical modularity and the intercommunity bridging sites in the residue interaction network. We have found that allosteric functional hotspots responsible for the kinase regulation may serve the intermodular bridges in the global interaction network. The central conclusion from this analysis is that the regulatory switch centers play a fundamental role in the modular network organization of ABL as the unique intercommunity bridges that connect the SH2 and SH3 domains with the catalytic core into a functional kinase assembly. The hierarchy of network organization in the ABL regulatory complexes may allow for the synergistic action of dense intercommunity links required for the robust signal transfer in the catalytic core and sparse network bridges acting as the regulatory control points that orchestrate allosteric transitions between the inhibited and active kinase forms.

Microsecond molecular dynamics simulations provide insight into the ATP-competitive inhibitor-induced allosteric protection of Akt kinase phosphorylation.

Akt is a serine/threonine protein kinase, a critical mediator of growth factor-induced survival in key cellular pathways. Allosteric signaling between protein intramolecular domains requires long-range communication mediated by hotspot residues, often triggered by ligand binding. Here, based on extensive 3μs explicit solvent molecular dynamics (MD) simulations of Akt1 kinase domain in the unbound (apo) and ATP-competitive inhibitor, GDC-0068-bound states, we propose a molecular mechanism for allosteric regulation of Akt1 kinase phosphorylation by GDC-0068 binding to the ATP-binding site. MD simulations revealed that the apo Akt1 is flexible with two disengaged N- and C-lobes, equilibrated between the open and closed conformations. GDC-0068 occupancy of the ATP-binding site shifts the conformational equilibrium of Akt1 from the open conformation toward the closed conformation and stabilizes the closed state. This effect enables allosteric signal propagation from the GDC-0068 to the phosphorylated T308 (pT308) in the activation loop and restrains phosphatase access to pT308, thereby protecting the pT308 in the GDC-0068-bound Akt1. Importantly, functional hotspots involved in the allosteric communication from the GDC-0068 to the pT308 are identified. Our analysis of GDC-0068-induced allosteric protection of Akt kinase phosphorylation yields important new insights into the molecular mechanism of allosteric regulation of Akt kinase activity.

Probing Allosteric Inhibition Mechanisms of the Hsp70 Chaperone Proteins Using Molecular Dynamics Simulations and Analysis of the Residue Interaction Networks.

Although molecular mechanisms of allosteric regulation in the Hsp70 chaperones have been extensively studied at both structural and functional levels, the current understanding of allosteric inhibition of chaperone activities by small molecules is still lacking. In the current study, using a battery of computational approaches, we probed allosteric inhibition mechanisms of E. coli Hsp70 (DnaK) and human Hsp70 proteins by small molecule inhibitors PET-16 and novolactone. Molecular dynamics simulations and binding free energy analysis were combined with network-based modeling of residue interactions and allosteric communications to systematically characterize and compare molecular signatures of the apo form, substrate-bound, and inhibitor-bound chaperone complexes. The results suggested a mechanism by which the allosteric inhibitors may leverage binding energy hotspots in the interaction networks to stabilize a specific conformational state and impair the interdomain allosteric control. Using the network-based centrality analysis and community detection, we demonstrated that substrate binding may strengthen the connectivity of local interaction communities, leading to a dense interaction network that can promote an efficient allosteric communication. In contrast, binding of PET-16 to DnaK may induce significant dynamic changes and lead to a fractured interaction network and impaired allosteric communications in the DnaK complex. By using a mechanistic-based analysis of distance fluctuation maps and allosteric propensities of protein residues, we determined that the allosteric network in the PET-16 complex may be small and localized due to the reduced communication and low cooperativity of the substrate binding loops, which may promote the higher rates of substrate dissociation and the decreased substrate affinity. In comparison with the significant effect of PET-16, binding of novolactone to HSPA1A may cause only moderate network changes and preserve allosteric coupling between the allosteric pocket and the substrate binding region. The impact of novolactone on the conformational dynamics and allosteric communications in the HSPA1A complex was comparable to the substrate effect, which is consistent with the experimental evidence that PET-16, but not novolactone binding, can significantly decrease substrate affinity. We argue that the unique dynamic and network signatures of PET-16 and novolactone may be linked with the experimentally observed functional effects of these inhibitors on allosteric regulation and substrate binding.

Study of Functional and Allosteric Sites in Protein Superfamilies

The interaction of proteins (enzymes) with a variety of low-molecular-weight compounds, as well as protein-protein interactions, is the most important factor in the regulation of their functional properties. To date, research effort has routinely focused on studying ligand binding to the functional sites of proteins (active sites of enzymes), whereas the molecular mechanisms of allosteric regulation, as well as binding to other pockets and cavities in protein structures, remained poorly understood. Recent studies have shown that allostery may be an intrinsic property of virtually all proteins. Novel approaches are needed to systematically analyze the architecture and role of various binding sites and establish the relationship between structure, function, and regulation. Computational biology, bioinformatics, and molecular modeling can be used to search for new regulatory centers, characterize their structural peculiarities, as well as compare different pockets in homologous proteins, study the molecular mechanisms of allostery, and understand the communication between topologically independent binding sites in protein structures. The establishment of an evolutionary relationship between different binding centers within protein superfamilies and the discovery of new functional and allosteric (regulatory) sites using computational approaches can improve our understanding of the structure-function relationship in proteins and provide new opportunities for drug design and enzyme engineering.

Understanding the Molecular Mechanisms by which Allosteric Ligands Inhibit the RNA Polymerase from the Hepatitis C Virus

The Hepatitis C Virus (HCV) infects roughly 170 million individuals worldwide. Currently, there is no cure and available treatments have limited efficacy and sever side-effects. Therefore, new HCV treatments are in high demand. The RNA polymerase (gene product NS5B) from HCV is a validated drug target because of its importance for viral replication. Currently, there are five known allosteric binding sites to which diverse inhibitors can bind. However, the molecular mechanisms that underlie allosteric inhibition are unclear from the available structural and biochemical data. Previously, we employed molecular dynamics (MD) simulations and other computational analyses to characterize the dynamics of free and ligand-bound NS5B to better understand how allosteric inhibitors prevent polymerase function. This information was used to predict which amino acids might be necessary for functional dynamics and/or mediating allosteric inhibition. Mutating residues necessary for functional dynamics should yield a non-functional protein, while mutating residues necessary for allosteric inhibition should yield a functional enzyme even in the presence of inhibitor. Here, we report the results of simulations performed on mutant enzymes to test our predictions. Results from this study will provide a better understanding of the molecular mechanisms of allosteric regulation of NS5B and related polymerases. In addition, this knowledge may facilitate the development of novel inhibitors for NS5B, thus leading to more effective treatments for HCV.

Surface dynamics in allosteric regulation of protein-protein interactions: modulation of calmodulin functions by Ca2+.

Knowledge of the structural basis of protein-protein interactions (PPI) is of fundamental importance for understanding the organization and functioning of biological networks and advancing the design of therapeutics which target PPI. Allosteric modulators play an important role in regulating such interactions by binding at site(s) orthogonal to the complex interface and altering the protein's propensity for complex formation. In this work, we apply an approach recently developed by us for analyzing protein surfaces based on steered molecular dynamics simulation (SMD) to the study of the dynamic properties of functionally distinct conformations of a model protein, calmodulin (CaM), whose ability to interact with target proteins is regulated by the presence of the allosteric modulator Ca2+. Calmodulin is a regulatory protein that acts as an intracellular Ca2+ sensor to control a wide variety of cellular processes. We demonstrate that SMD analysis is capable of pinpointing CaM surfaces implicated in the recognition of both the allosteric modulator Ca2+ and target proteins. Our analysis of changes in the dynamic properties of the CaM backbone elicited by Ca2+ binding yielded new insights into the molecular mechanism of allosteric regulation of CaM-target interactions.

Interfacial communications in recombinant rabbit kidney pyruvate kinase.

Tissue-specific isozymes of pyruvate kinase are particularly attractive systems to elucidate the molecular mechanism(s) of conferring allostery. The muscle- and kidney-type isozymes are coded by the same gene. As a consequence of alternative message RNA splicing, the two primary sequences differ by a small number of residues. However, they exhibit very different regulatory behavior. In an effort to identify the roles of specific residues in conferring allostery, the gene encoding rabbit kidney-type pyruvate kinase was cloned and expressed in Escherichia coli. The primary structure of recombinant rabbit kidney-type pyruvate kinase (rRKPK) and recombinant rabbit muscle-type pyruvate kinase (rRMPK) differ at 22 positions, which are located in a region that forms important intersubunit contacts in the RMPK structure. Velocity sedimentation and analytical gel chromatographic studies show that rRKPK undergoes reversible dimer left and right arrow tetramer assembly with an equilibrium constant of 28 +/- 3 mL/mg. This subunit assembly process provides the opportunity to elucidate the role of this dimer interface in transmission of signal upon binding of substrates and allosteric effectors. The assembly to tetrameric rRKPK is favored by the binding of phosphoenolpyruvate (PEP), one of the two substrates, or fructose 1,6-bisphosphate (FBP), an activator. In contrast, the equilibrium is shifted toward dimeric rRKPK upon binding of adenosine diphosphate (ADP), the other substrate, or l-phenylalanine (Phe), the inhibitor. These observations provide significant new insights to the molecular mechanism of allosteric regulation in the pyruvate kinase system. First, all substrates and effectors communicate through this particular dimer-dimer interface. Second, the thermodynamic signatures of these communications are qualitatively different for the two substrates and between the activator, FBP, and inhibitor, Phe.

The allosteric regulation of pyruvate kinase

Crystallographic and mutagenesis studies have unravelled the general features of the allosteric transition mechanism in pyruvate kinase. The enzyme displays a dramatic conformational change in going from the T- to the R-state. All three domains forming each subunit of the tetrameric enzyme undergo simultaneous and concerted rotations, in such a way that all subunit and domain interfaces are modified. This mechanism is unpreceDAnted since in all tetrameric allosteric enzymes, characterised at atomic resolution, at least one of the domain or subunit interfaces remains unchanged on the T- to R-state transition. The molecular mechanism of allosteric regulation here proposed proviDAs a rationale for the effect of single site mutations observed in the human erythrocyte pyruvate kinase associated with a congenital anaemia.