Allosteric Signal Research Articles

A Fold-Independent Interface Residue Is Crucial for Complex Formation and Allosteric Signaling in Class I Glutamine Amidotransferases.

The members of the glutamine amidotransferase (GATase) family catalyze the incorporation of ammonia within numerous metabolic pathways and can be categorized in two classes. Here, we concentrated on class I GATases, which are heteromeric enzyme complexes consisting of synthase subunits and glutaminase subunits with a catalytic Cys-His-Glu triad. Glutamine hydrolysis at the glutaminase subunit is (i) dependent on the formation of tight synthase-glutaminase complexes and (ii) allosterically coupled to the presence of the substrate at the synthase subunit. The structural basis of both complex formation and allostery is poorly understood. However, previous work on 4-amino-4-deoxychorismate synthase and imidazole glycerol phosphate synthase suggested that a conserved aspartate residue in their synthase subunits, which is located at the subunit interface close to the glutaminase catalytic triad, might be important for both features. We performed a computational screen of class I GATases from the Protein Data Bank and identified conserved and similarly located aspartate residues. We then generated alanine and glutamate mutants of these residues and characterized them by analytical gel filtration and steady-state enzyme kinetics. The results confirmed the important role of the wild-type aspartate residues for the formation of stable synthase-glutaminase complexes (in three of four cases) and the stimulation of glutaminase activity in the analyzed GATases (in all four cases). We present a model for rationalizing the dual role of the conserved aspartate residue toward a unifying regulation mechanism in the entire class I GATase family.

Generalizable Protein Biosensors Based on Synthetic Switch Modules.

Allosteric protein switches are key controllers of information and energy processing in living organisms and are desirable engineered control tools in synthetic systems. Here we present a generally applicable strategy for construction of allosteric signaling systems with inputs and outputs of choice. We demonstrate conversion of constitutively active enzymes into peptide-operated synthetic allosteric ON switches by insertion of a calmodulin domain into rationally selected sites. Switches based on EGFP, glucose dehydrogenase, NanoLuciferase, and dehydrofolate reductase required minimal optimization and demonstrated a dynamic response ranging from 1.8-fold in the former case to over 200-fold in the latter case. The peptidic nature of the calmodulin ligand enables incorporation of such synthetic switch modules into higher order sensory architectures. Here, a ligand-mediated increase in proximity of the allosteric switch and the engineered activator peptide modulates biosensor's activity. Created biosensors were used to measure concentrations of clinically relevant drugs and biomarkers in plasma, saliva, and urine with accuracy comparable to that of the currently used clinical diagnostic assays. The approach presented is generalizable as it allows rapid construction of efficient protein switches that convert binding of a broad range of analytes into a biochemical activity of choice enabling construction of artificial signaling and metabolic circuits of potentially unlimited complexity.

Citrate-Induced p85α⁻PTEN Complex Formation Causes G2/M Phase Arrest in Human Pharyngeal Squamous Carcinoma Cell Lines.

Citrate is a key intermediate of the tricarboxylic acid cycle and acts as an allosteric signal to regulate the production of cellular ATP. An elevated cytosolic citrate concentration inhibits growth in several types of human cancer cells; however, the underlying mechanism by which citrate induces the growth arrest of cancer cells remains unclear. The results of this study showed that treatment of human pharyngeal squamous carcinoma (PSC) cells with a growth-suppressive concentration of citrate caused cell cycle arrest at the G2/M phase. A coimmunoprecipitation study demonstrated that citrate-induced cell cycle arrest in the G2/M phase was associated with stabilizing the formation of cyclin B1–phospho (p)-cyclin-dependent kinase 1 (CDK1) (Thr 161) complexes. The citrate-induced increased levels of cyclin B1 and G2/M phase arrest were suppressed by the caspase-3 inhibitor Ac-DEVD-CMK and caspase-3 cleavage of mutant p21 (D112N). Ectopic expression of the constitutively active form of protein kinase B (Akt1) could overcome the induction of p21 cleavage, cyclin B1–p-CDK1 (Thr 161) complexes, and G2/M phase arrest by citrate. p85α–phosphatase and tensin homolog deleted from chromosome 10 (PTEN) complex-mediated inactivation of Akt was required for citrate-induced G2/M phase cell cycle arrest because PTEN short hairpin RNA or a PTEN inhibitor (SF1670) blocked the suppression of Akt Ser 473 phosphorylation and the induction of cyclin B1–p-CDK1 (Thr 161) complexes and G2/M phase arrest by citrate. In conclusion, citrate induces G2/M phase arrest in PSC cells by inducing the formation of p85α–PTEN complexes to attenuate Akt-mediated signaling, thereby causing the formation of cyclin B1–p-CDK1 (Thr 161) complexes.

Symmetry, Rigidity, and Allosteric Signaling: From Monomeric Proteins to Molecular Machines.

Allosteric signaling in biological molecules, which may be viewed as specific action at a distance due to localized perturbation upon binding of ligands or changes in environmental cues, is pervasive in biology. Insightful phenomenological Monod, Wyman, and Changeux (MWC) and Koshland, Nemethy, and Filmer (KNF) models galvanized research in describing allosteric transitions for over five decades, and these models continue to be the basis for describing the mechanisms of allostery in a bewildering array of systems. However, understanding allosteric signaling and the associated dynamics between distinct allosteric states at the molecular level is challenging and requires novel experiments complemented by computational studies. In this review, we first describe symmetry and rigidity as essential requirements for allosteric proteins or multisubunit structures. The general features, with MWC and KNF as two extreme scenarios, emerge when allosteric signaling is viewed from an energy landscape perspective. To go beyond the general theories, we describe computational tools that are either based solely on multiple sequences or their structures to predict the allostery wiring diagram. These methods could be used to predict the network of residues that carry allosteric signals. Methods to obtain molecular insights into the dynamics of allosteric transitions are briefly mentioned. The utility of the methods is illustrated by applications to systems ranging from monomeric proteins in which there is little conformational change in the transition between two allosteric states to membrane bound G-protein coupled receptors and multisubunit proteins. Finally, the role allostery plays in the functions of ATP-consuming molecular machines, bacterial chaperonin GroEL and molecular motors, is described. Although universal molecular principles governing allosteric signaling do not exist, we can draw the following general conclusions from a survey of different systems. (1) Multiple pathways connecting allosteric states are highly heterogeneous. (2) Allosteric signaling is exquisitely sensitive to the specific architecture of the system, which implies that the capacity for allostery is encoded in the structure itself. (3) The mechanical modes that connect distinct allosteric states are robust to sequence variations. (4) Extensive investigations of allostery in Hemoglobin and, more recently GroEL, show that to a large extent a network of salt bridge rearrangements serves as allosteric switches. In both these examples the dynamical changes in the allosteric switches are related to function.

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.

Ligand-Binding-Site Structure Shapes Allosteric Signal Transduction and the Evolution of Allostery in Protein Complexes.

The structure of ligand-binding sites has been shown to profoundly influence the evolution of function in homomeric protein complexes. Complexes with multichain binding sites (MBSs) have more conserved quaternary structure, more similar binding sites and ligands between homologs, and evolve new functions slower than homomers with single-chain binding sites (SBSs). Here, using in silico analyses of protein dynamics, we investigate whether ligand-binding-site structure shapes allosteric signal transduction pathways, and whether the structural similarity of binding sites influences the evolution of allostery. Our analyses show that: 1) allostery is more frequent among MBS complexes than in SBS complexes, particularly in homomers; 2) in MBS homomers, semirigid communities and critical residues frequently connect interfaces and thus they are characterized by signal transduction pathways that cross protein–protein interfaces, whereas SBS homomers usually not; 3) ligand binding alters community structure differently in MBS and SBS homomers; and 4) except MBS homomers, allosteric proteins are more likely to have homologs with similar binding site than nonallosteric proteins, suggesting that binding site similarity is an important factor driving the evolution of allostery.

Hinge Twists and Population Shifts Deliver Regulated Catalysis for ATP-PRT in Histidine Biosynthesis

Allosteric regulation plays an important role in the control of metabolic flux in biosynthetic pathways. In microorganisms, many enzymes in these pathways adopt different strategies of allostery to allow the tuning of their activities in response to metabolic demand. Thus, it is important to uncover the mechanism of allosteric signal transmission to fully comprehend the complex control of enzyme function and its evolution. ATP-phosphoribosyltransferase (ATP-PRT), as the first enzyme in the histidine biosynthetic pathway, is allosterically regulated by histidine and offers a good platform for the study of allostery. Two forms of ATP-PRT, namely long and short forms, were discovered that show different arrangements of their regulatory machinery. Crystal structures of the long-form ATP-PRT have revealed overall conformational changes in the inhibited state, but the observed changes in the active state are quite subtle, making the elucidation of its allosteric mechanism difficult. Here, we combine computational methods (ligand docking, quantum mechanics/molecular mechanics optimization, and molecular dynamic simulations) with experimental studies to probe the signal transmission between remote allosteric and active sites. Our results reveal that distinct conformational ensembles of the catalytic domain with different dynamic properties exist in the ligand-free and histidine-bound enzymes. These ensembles display different capabilities in supporting the catalytic and allosteric function of ATP-PRT. The findings give insight into the underlying mechanism of allostery and allow us to propose that the hinge twisting within the catalytic domain is the key for both enhancement of catalysis and provision of regulation in ATP-PRT enzymes.

Allostery in Coagulation Factor VIIa Revealed by Ensemble Refinement of Crystallographic Structures

A critical step in injury-induced initiation of blood coagulation is the formation of the complex between the trypsin-like protease coagulation factor VIIa (FVIIa) and its cofactor tissue factor (TF), which converts FVIIa from an intrinsically poor enzyme to an active protease capable of activating zymogens of downstream coagulation proteases. Unlike its constitutively active ancestor trypsin, FVIIa is allosterically activated (by TF). Here, ensemble refinement of crystallographic structures, which uses multiple copies of the entire structure as a means of representing structural flexibility, is applied to explore the impacts of inhibitor binding to trypsin and FVIIa, as well as cofactor binding to FVIIa. To assess the conformational flexibility and its role in allosteric pathways in these proteases, main-chain hydrogen bond networks are analyzed by calculating the hydrogen-bond propensity. Mapping pairwise propensity differences between relevant structures shows that binding of the inhibitor benzamidine to trypsin has a minor influence on the protease flexibility. For FVIIa, in contrast, the protease domain is “locked” into the catalytically competent trypsin-like configuration upon benzamidine binding as indicated by the stabilization of key structural features: the nonprime binding cleft and the oxyanion hole are stabilized, and the effect propagates from the active site region to the calcium-binding site and to the vicinity of the disulphide bridge connecting with the light chain. TF binding to FVIIa furthermore results in stabilization of the 170 loop, which in turn propagates an allosteric signal from the TF-binding region to the active site. Analyses of disulphide bridge energy and flexibility reflect the striking stability difference between the unregulated enzyme and the allosterically activated form after inhibitor or cofactor binding. The ensemble refinement analyses show directly, for the first time to our knowledge, whole-domain structural footprints of TF-induced allosteric networks present in x-ray crystallographic structures of FVIIa, which previously only have been hypothesized or indirectly inferred.

Molecular basis for feedback inhibition of tyrosine-regulated 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase from Escherichia coli

3-Deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAHPS) is responsible for the biosynthesis of essential aromatic compounds in microorganisms and plants. It plays a crucial role in the regulation of the carbon flow into the shikimate pathway. Until now, the crystal structures and regulatory mechanisms of dimeric DAHPS enzymes from type Iα subclass have not been reported. Here, we reported dimeric structures of the tyrosine-regulated DAHPS from Escherichia coli, both in its apo form and complex with the inhibitor tyrosine at 2.5 and 2.0 Å resolutions, respectively. DAHPS(Tyr) has a typical (β/α)8 TIM barrel, which is decorated with an N-terminal extension and an antiparallel β sheet, β6a/β6b. Inhibitor tyrosine binds at a cavity formed by residues of helices α3, α4, strands β6a, β6b and the adjacent loops, and directly interacts with residues P148, Q152, S181, I213 and N8*. Although the small angle X-ray scattering profiles from DAHPS(Tyr) with and without tyrosine shows that tyrosine binding leaves most of DAHPS(Tyr) structures unaffected. The comparison of the liganded and unliganded crystal structures reveals that conformational changes of residues P148, Q152 and I213 initiate a transmission pathway to propagate the allosteric signal from the tyrosine-binding site to the active site, which is different from DAHPS(Phe), a phenylalanine-regulated isozyme from E. coli. In addition, mutations of five tyrosine-binding residues P148, Q152, S181, I213 and N8* leads to tyrosine-resistant DAHPS(Tyr) enzymes. These findings provide a new insight into the regulatory mechanism of DAHPS enzymes and a basis for further engineering studies.

An allosteric interaction network promotes conformation state‐dependent eviction of the Nas6 assembly chaperone from nascent 26S proteasomes

The 26S proteasome is the central ATP‐dependent protease in eukaryotes and is essential for organismal health. Proteasome assembly is mediated in part by several dedicated, evolutionarily conserved chaperone proteins. These chaperones associate transiently with assembly intermediates but are absent from mature proteasomes. Chaperone eviction upon completion of proteasome assembly is necessary for normal proteasome function, but how they are released remains unresolved. Here, we demonstrate that the Nas6 assembly chaperone, homolog of the human oncogene gankyrin, is evicted from nascent proteasomes during completion of assembly via a conformation‐specific allosteric interaction of the Rpn5 subunit with the proteasomal ATPase ring. Subsequent ATP‐binding by the ATPase subunit Rpt3 then promotes conformational remodeling of the ATPase ring that evicts Nas6 from the nascent proteasome. Our study demonstrates how assembly‐coupled allosteric signals promote chaperone eviction, and provide a framework for understanding the eviction of other chaperones from this biomedically important molecular machine.Support or Funding Information1R01GM118600 to R.J.T.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Interpreting the Dynamics of Binding Interactions of snRNA and U1A Using a Coarse-Grained Model

The binding interactions of small nuclear RNAs (snRNA) and the associated protein factors are critical to the function of spliceosomes in alternatively splicing primary RNA transcripts. Although molecular dynamics simulations are a powerful tool to interpret the mechanism of biological processes, the atomic-level simulations are, however, too expensive and with limited accuracy for the large-size systems, such as snRNA-protein complexes. We extend the coarse-grained Gaussian network model, which models the RNA-protein complexes as a harmonic chain of Cα, P, and O4′ atoms, to investigating the impact of the snRNA-binding interaction on the dynamic stability of the human U1A protein, which is a major component of the spliceosomal U1 small nuclear ribonucleoprotein particle. The results reveal that the first and third loops and the C-terminal helix regions of the U1A domain undergo a significant loss of flexibility upon the RNA binding due to the forming of mostly electrostatic and hydrogen bond interactions with RNA 5′ stem and loop. By examining the residues whose mutations significantly change the binding free energy between U1A and snRNA, the Gaussian network model-based calculations show that not only the residues at the binding sites that are traditionally considered to play a major role in U1A-RNA association but also those residues that are far away from the RNA-binding interface can participate in the long-range allosteric signal transmission; these calculations are quantitatively consistent with the data observed in the recent snRNA binding experiments. The study demonstrates a useful avenue to utilize the simplified elastic network model to investigate the dynamics characteristics of the biologically important macromolecular interactions.

Conserved Residues Control the T1R3-Specific Allosteric Signaling Pathway of the Mammalian Sweet-Taste Receptor.

Mammalian sensory systems detect sweet taste through the activation of a single heteromeric T1R2/T1R3 receptor belonging to class C G-protein-coupled receptors. Allosteric ligands are known to interact within the transmembrane domain, yet a complete view of receptor activation remains elusive. By combining site-directed mutagenesis with computational modeling, we investigate the structure and dynamics of the allosteric binding pocket of the T1R3 sweet-taste receptor in its apo form, and in the presence of an allosteric ligand, cyclamate. A novel positively charged residue at the extracellular loop 2 is shown to interact with the ligand. Molecular dynamics simulations capture significant differences in the behavior of a network of conserved residues with and without cyclamate, although they do not directly interact with the allosteric ligand. Structural models show that they adopt alternate conformations, associated with a conformational change in the transmembrane region. Site-directed mutagenesis confirms that these residues are unequivocally involved in the receptor function and the allosteric signaling mechanism of the sweet-taste receptor. Similar to a large portion of the transmembrane domain, they are highly conserved among mammals, suggesting an activation mechanism that is evolutionarily conserved. This work provides a structural basis for describing the dynamics of the receptor, and for the rational design of new sweet-taste modulators.

Asymmetric Recruitment of β-Arrestin1/2 by the Angiotensin II Type I and Prostaglandin F2α Receptor Dimer.

Initially identified as monomers, G protein-coupled receptors (GPCRs) can also form functional homo- and heterodimers that act as distinct signaling hubs for cellular signal integration. We previously found that the angiotensin II (Ang II) type 1 receptor (AT1R) and the prostaglandin F2α (PGF2α) receptor (FP), both important in the control of smooth muscle contractility, form such a functional heterodimeric complex in HEK 293 and vascular smooth muscle cells. Here, we hypothesize that both Ang II- and PGF2α-induced activation of the AT1R/FP dimer, or the parent receptors alone, differentially regulate signaling by distinct patterns of β-arrestin recruitment. Using BRET-based biosensors, we assessed the recruitment kinetics of β-arrestin1/2 to the AT1R/FP dimer, or the parent receptors alone, when stimulated by either Ang II or PGF2α. Using cell lines with CRISPR/Cas9-mediated gene deletion, we also examined the role of G proteins in such recruitment. We observed that Ang II induced a rapid, robust, and sustained recruitment of β-arrestin1/2 to AT1R and, to a lesser extent, the heterodimer, as expected, since AT1R is a strong recruiter of both β-arrestin subtypes. However, PGF2α did not induce such recruitment to FP alone, although it did when the AT1R is present as a heterodimer. β-arrestins were likely recruited to the AT1R partner of the dimer. Gαq, Gα11, Gα12, and Gα13 were all involved to some extent in PGF2α-induced β-arrestin1/2 recruitment to the dimer as their combined absence abrogated the response, and their separate re-expression was sufficient to partially restore it. Taken together, our data sheds light on a new mechanism whereby PGF2α specifically recruits and signals through β-arrestin but only in the context of the AT1R/FP dimer, suggesting that this may be a new allosteric signaling entity.

Toward Comprehensive Allosteric Control over Protein Activity

Universality of allosteric signaling in proteins, molecular machines, and receptors complemented by the great advantages of prospected allosteric drugs in the highly specific, non-competitive, and modulatory nature of their actions calls for deeper theoretical understanding of allosteric communication. We present a computational model that makes it possible to tackle the problem of modulating the energetics of protein allosteric communication. In the context of the energy landscape paradigm, allosteric signaling is always a result of perturbations, such as ligand binding, mutations, and intermolecular interactions. The calculation of local partition functions in the protein harmonic model with perturbations allows us to evaluate the energetics of allosteric communication at the single-residue level. In this framework, Allosteric Signaling Maps are proposed as a tool to exhaustively describe allosteric communication in the protein, to tune already existing signaling, and to design new elements of regulation for taking the protein activity under allosteric control.

Characterization of Differential Dynamics, Specificity, and Allostery of Lipoxygenase Family Members.

Accurate modeling of structural dynamics of proteins and their differentiation across different species can help us understand generic mechanisms of function shared by family members and the molecular basis of the specificity of individual members. We focused here on the family of lipoxygenases, enzymes that catalyze lipid oxidation, the mammalian and bacterial structures of which have been elucidated. We present a systematic method of approach for characterizing the sequence, structure, dynamics, and allosteric signaling properties of these enzymes using a combination of structure-based models and methods and bioinformatics tools applied to a data set of 88 structures. The analysis elucidates the signature dynamics of the lipoxygenase family and its differentiation among members, as well as key sites that enable its adaptation to specific substrate binding and allosteric activity.

Towards Comprehensive Control and Design of Targeted Signalling in Allosteric Regulation of Protein Activity

We developed a computational approach combining protein harmonic model with statistical mechanical formalism in a perturbation-based approach, which allows one to evaluate per-residue allosteric free energy as a result of ligand binding and/or mutations. The model was implemented in the AlloSigMA web-server that provides an interactive framework for estimating allosteric free energy, can help to find potential latent regulatory exosites and candidates for allosteric mutations. Because of the critical role of global protein dynamics in allosteric signaling, we hypothesized reversibility of allosteric communication, according to which allosteric sites can be detected via the perturbation of the functional sites. Validating the “reversibility hypothesis”, we also show that, in addition to known allosteric sites, perturbation of functional sites unravels rather extended protein regions, which can host latent regulatory exosites. These protein parts that are dynamically coupled with functional sites can also be used for inducing and tuning allosteric communication. Defining generic characteristic of the allosteric effect of mutation, the modulation range, we build Allosteric Signaling Maps (ASMs) of proteins on the basis of residue-by-residue scanning of mutations. ASMs allow one: (i) to establish a relationship between mutations allosterically modulating protein activity and those that mainly change the protein stability; (ii) to observe a role of domain/subunit in protein allosteric communication via the distance-dependent mode switching in allosteric signaling; (iii) to complement and tune already existing allosteric signaling and to design new elements of regulation.

Mechanism of Cholesterol Sensing in the Niemann Pick Protein (NPC1) using Molecular Dynamics Simulations

The accumulation of cholesterol and other lipids in lysosomes and endosomes leads to the Niemann Pick type C (NPC) disease. Cholesterol accumulation results from disrupted cholesterol transport mediated by the NPC1 and NPC2 proteins. NPC1 is a large transmembrane (TM) protein which putatively receives luminal cholesterol from the soluble NPC2, and subsequently transfers it to the lysosomal membrane. The mechanism by which the luminal N-terminal domain (NTD) of NPC1 transfers cholesterol to the lysosomal membrane nearly 8 nm away remains unknown. The allosteric signalling mechanism between the cholesterol-sensing TM domain and the spatially distant luminal domains of NPC1 are equally fascinating. We implemented large-scale Molecular Dynamics (MD) simulations of NPC1 in the presence and absence of cholesterol in the lysosomal membrane, and show that cholesterol binding to the sterol sensing domain (SSD) to the TM region of NPC1 suppresses conformational changes in the luminal domains that can potentially tilt the NTD towards the lipid bilayer. Using a structural alphabet which translates structural ensembles into strings which can then be subject to statistical analysis (GSATools), we propose a mechanism of allosteric signalling from the transmembrane SSD to the luminal NTD. We provide evidence that the NTD of NPC1 is indeed remarkably flexible, and can alternate between membrane-bound and luminal conformations to transport cholesterol from the lumen to the membrane. We also show that this conformational equilibrium is influenced by the binding of membrane cholesterol to the SSD of NPC1.

Allosteric Effects and Signal Transduction in the Peptide-MHC Binding to a Human T Cell Receptor

Allosteric Effects and Signal Transduction in the Peptide-MHC Binding to a Human T Cell Receptor

Observation of Allosteric Signaling through DNA with Single-Molecule FRET and Cryo-EM

Observation of Allosteric Signaling through DNA with Single-Molecule FRET and Cryo-EM

Discorhabdin N, a South African Natural Compound, for Hsp72 and Hsc70 Allosteric Modulation: Combined Study of Molecular Modeling and Dynamic Residue Network Analysis.

The human heat shock proteins (Hsps), predominantly Hsp72 and Hsp90, have been strongly implicated in various critical stages of oncogenesis and progression of human cancers. While drug development has extensively focused on Hsp90 as a potential anticancer target, much less effort has been put against Hsp72. This work investigated the therapeutic potential of Hsp72 and its constitutive isoform, Hsc70, via in silico-based screening against the South African Natural Compounds Database (SANCDB). A comparative modeling approach was used to obtain nearly full-length 3D structures of the closed conformation of Hsp72 and Hsc70 proteins. Molecular docking of SANCDB compounds identified one potential allosteric modulator, Discorhabdin N, binding to the allosteric β substrate binding domain (SBDβ) back pocket, with good binding affinities in both cases. This allosteric region was identified in one of our previous studies. Subsequent all-atom molecular dynamics simulations and free energy calculations exhibited promising protein–ligand association characteristics, indicative of strong binding qualities. Further, we utilised dynamic residue network analysis (DRN) to highlight protein regions actively involved in cross-domain communication. Most residues identified agreed with known allosteric signal regulators from literature, and were further investigated for the purpose of deducing meaningful insights into the allosteric modulation properties of Discorhabdin N.