Cations play key roles in regulating G-protein-coupled receptors (GPCRs), although their mechanis... more Cations play key roles in regulating G-protein-coupled receptors (GPCRs), although their mechanisms are poorly understood. Here, 19 F NMR is used to delineate the effects of cations on functional states of the adenosine A 2A GPCR. While Na + reinforces an inactive ensemble and a partial-agonist stabilized state, Ca 2+ and Mg 2+ shift the equilibrium toward active states. Positive allosteric effects of divalent cations are more pronounced with agonist and a G-protein-derived peptide. In cell membranes, divalent cations enhance both the affinity and fraction of the high affinity agonist-bound state. Molecular dynamics simulations suggest high concentrations of divalent cations bridge specific extracellular acidic residues, bringing TM5 and TM6 together at the extracellular surface and allosterically driving open the G-protein-binding cleft as shown by rigidity-transmission allostery theory. An understanding of cation allostery should enable the design of allosteric agents and enhance our understanding of GPCR regulation in the cellular milieu.
Within the microbial rhodopsin family, heliorhodopsins (HeRs) form a phylogenetically distinct gr... more Within the microbial rhodopsin family, heliorhodopsins (HeRs) form a phylogenetically distinct group of light-harvesting retinal proteins with largely unknown functions. We have determined the 1.97 Å resolution X-ray crystal structure of Thermoplasmatales archaeon SG8-52-1 heliorhodopsin (TaHeR) in the presence of NaCl under acidic conditions (pH 4.5), which complements the known 2.4 Å TaHeR structure acquired at pH 8.0. The low pH structure revealed that the hydrophilic Schiff base cavity (SBC) accommodates a chloride anion to stabilize the protonated retinal Schiff base when its primary counterion (Glu-108) is neutralized. Comparison of the two structures at different pH revealed conformational changes connecting the SBC and the extracellular loop linking helices A–B. We corroborated this intramolecular signaling transduction pathway with computational studies, which revealed allosteric network changes propagating from the perturbed SBC to the intracellular and extracellular space...
Heterotrimeric G proteins serve as key membrane-associated signaling hubs, in concert with their ... more Heterotrimeric G proteins serve as key membrane-associated signaling hubs, in concert with their cognate G protein-coupled receptors (GPCRs). Using site-directed labels located at four key allosteric sites within the Ras-homology domain of the stimulatory G protein α-subunit, Gsα, fluorine nuclear magnetic resonance spectroscopy (19F NMR) was employed to monitor the conformational equilibria of Gsα by itself, in the intact Gsαβγ heterotrimer, or in complex with either membrane alone or membrane-embedded adenosine A2A receptor (A2AR). The results reveal a concerted equilibrium which is strongly affected by nucleotide and interactions with the βγ-subunit, the lipid bilayer, and A2AR. 19F NMR spectra of the α1 helix of Gsα exhibit significant intermediate timescale dynamics, while those associated with the α4β6 loop and the α5 helix reflect respective membrane/receptor interactions and order/disorder transitions associated with G protein activation. The αN helix adopts a key functional...
Loops are small secondary structural elements that play a crucial role in the emergence of new en... more Loops are small secondary structural elements that play a crucial role in the emergence of new enzyme functions. However, our understanding of loop functions is mainly limited to the catalytic loops. To understand the function of remote loops in enzymes, we studied Glycoside hydrolase family 19 (GH19) chitinase - an essential enzyme family for pathogen degradation in plants. By revealing the evolutionary history and loops appearance of GH19 chitinase, we discovered that one loop which is remote from the catalytic site, is necessary to acquire the new antifungal activity. We demonstrated that this remote loop directly accesses the fungal cell wall, and surprisingly, it needs to adopt a defined structure supported by long-range intramolecular interactions to perform its function. Our findings prove that Nature applies this new strategy at the molecular level to achieve a complex biological function while maintaining the original activity one in the catalytic pocket, suggesting an alte...
Volume 5B: 41st Mechanisms and Robotics Conference, 2017
Assur groups and correspondingly Assur graphs have key attributes that make them important for th... more Assur groups and correspondingly Assur graphs have key attributes that make them important for the analysis and synthesis of linkages. The topology of linkages (more precisely the existence of constraints between links) in general can be represented by various types of constraint graphs. The concept of Assur graphs applies to any such graph and thus their peculiar features can be exploited. Despite of these potential benefits the relevant methods and tools are scattered in the literature. The aim of this paper is to provide an overview of the use of constraint graphs including recent results on the construction of Assur graphs.
The entry of the SARS-CoV-2, a causative agent of COVID-19, into human host cells is mediated by ... more The entry of the SARS-CoV-2, a causative agent of COVID-19, into human host cells is mediated by the SARS-CoV-2 spike (S) glycoprotein, which critically depends on the formation of complexes involving the spike protein receptor-binding domain (RBD) and the human cellular membrane receptor angiotensin-converting enzyme 2 (hACE2). Using classical site density functional theory (SDFT) and structural bioinformatics methods, we investigate binding and conformational properties of these complexes and study the overlooked role of water-mediated interactions. Analysis of the three-dimensional reference interaction site model (3DRISM) of SDFT indicates that water mediated interactions in the form of additional water bridges strongly increases the binding between SARS-CoV-2 spike protein and hACE2 compared to SARS-CoV-1-hACE2 complex. By analyzing structures of SARS-CoV-2 and SARS-CoV-1, we find that the homotrimer SARS-CoV-2 S receptor-binding domain (RBD) has expanded in size, indicating la...
Tau is an intrinsically disordered protein (IDP) whose primary physiological role is to stabi-liz... more Tau is an intrinsically disordered protein (IDP) whose primary physiological role is to stabi-lize microtubules in neuronal axons at all stages of development. In Alzheimer's and other tauopathies, tau forms intracellular insoluble amyloid aggregates known as neurofibrillary tangles, a process that appears in many cases to be preceded by hyperphosphorylation of tau monomers. Understanding the shift in conformational bias induced by hyperphosphory-lation is key to elucidating the structural factors that drive tau pathology, however, as an IDP, tau is not amenable to conventional structural characterization. In this work, we employ a straightforward technique based on Time-Resolved ElectroSpray Ionization Mass Spec-trometry (TRESI-MS) and Hydrogen/Deuterium Exchange (HDX) to provide a detailed pic-ture of residual structure in tau, and the shifts in conformational bias induced by hyperphosphorylation. By comparing the native and hyperphosphorylated ensembles, we are able to defin...
The tourism industry has generated research and studies, into government agencies, the private se... more The tourism industry has generated research and studies, into government agencies, the private sector, and academic institutions worldwide. In Brazil, the growth of eventbased tourism, in which million direct and indirect jobs have been created, has gained prominence in this profitable industry. Brasília, the seat of the federal government, and a modern, well-developed city of cultural and international diversity, is ideally suited for the hosting of special events. For this reason, it is important that attention be given to the training of tourism professionals in Brasília, particularly as it relates to creating and delivering quality services. This research demonstrates the difficulties that event coordinators in Brasília experience in going "by the book." These professionals opt for using their own manuals, which often do not cover all the steps and details necessary for good risk management, or they prefer to use only a checklist. Checklists are important, but they are not sufficient to guarantee the success of an event. This study also reveals the chief discrepancies that arise between recommended, theoretical procedures and the work that is actually done, underscoring the need to identify each potential problem in the planning of events, in order to better manage-and minimize-risks. Consequently, we have developed a hands-on, risk-management manual based on trial and error for the benefit of event coordinators. This innovative, step-by-step manual includes the PDCA Cycle and SWOT Analysis which, applied simultaneously, will better prepare tourism professionals for planning mega events in Brasília. 1. Event-based tourism 2. Risks 3. Risk Management
To understand how proteins function it is crucial to understand the connection between their stru... more To understand how proteins function it is crucial to understand the connection between their structure, flexibility and dynamics. In the field of bioinformatics and computational biology, there is a strong interest to develop software and computational tools that analyzes various properties of protein structures. The software we are using for protein flexibility and dynamics analysis generally assumes a single task, single thread environment. To more efficiently elucidate the function of proteins, we need to perform large-scale calculations on many structures. To improve computational speed of such large scale analysis, we decided to perform parallel distributed computation with the conventional software. We designed a simple protocol dedicated to this software over http and achieved a speedup of 550 times with 600 CPU cores. With such speed ups, we are able to perform faster high-throughput computations on large number of protein structures.
This chapter describes the application of constrained geometric simulations for prediction of ant... more This chapter describes the application of constrained geometric simulations for prediction of antibody structural dynamics. We utilize constrained geometric simulations method FRODAN, which is a low computational complexity alternative to Molecular Dynamics (MD) simulations that can rapidly explore flexible motions in protein structures. FRODAN is highly suited for conformational dynamics analysis of large proteins, complexes, intrinsically disordered proteins and dynamics that occurs on longer biologically relevant time scales which are normally inaccessible to classical MD simulations. This approach predicts protein dynamics at an all-atom scale while retaining realistic covalent bonding, maintaining dihedral angles in energetically good conformations while avoiding steric clashes in addition to performing other geometric and stereochemical criteria checks. In this chapter, we apply FRODAN to showcase its applicability for probing functionally relevant dynamics of IgG2a, including...
2016 3rd Asia-Pacific World Congress on Computer Science and Engineering (APWC on CSE), 2016
Advancements in the field of mathematical rigidity theory have opened up a number of exciting opp... more Advancements in the field of mathematical rigidity theory have opened up a number of exciting opportunities for computational predictions of protein flexibility and their dynamics. Starting with a 3D protein structure, several programs such as FIRST model the protein as a constraint multigraph, consisting of vertices (atoms) and edges (covalent bonds, hydrogen bonds, electrostatic interactions, and hydrophobic contacts). FIRST applies the pebble game algorithm on the resulting multigraph which rapidly decompose the protein into rigid clusters and flexible regions. Using an extension of FIRST and the pebble game algorithm we propose a computational approach for studying a biological phenomenon 'allostery'. Allostery refers to an effect of binding at one site to another, often significantly distant functional site on the protein, allowing for regulation of the protein function. Most dynamic proteins are allosteric and allostery has even been coined the 'second secret of li...
The emergence of a variety of highly transmissible SARS-CoV-2 variants, the causative agent of CO... more The emergence of a variety of highly transmissible SARS-CoV-2 variants, the causative agent of COVID-19, with multiple spike mutations poses serious challenges in overcoming the ongoing deadly pandemic. It is, therefore, essential to understand how these variants gain enhanced ability to evade immune responses with a higher rate of spreading infection. To address this question, here we have individually assessed the effects of SARS-CoV-2 variant-specific spike (S) protein receptor-binding domain (RBD) mutations E484K, K417N, L452Q, L452R, N501Y, and T478K that characterize and differentiate several emerging variants. Despite the hundreds of apparently neutral mutations observed in the domains other than the RBD, we have shown that each RBD mutation site is differentially engaged in an interdomain allosteric network involving mutation sites from a distant domain, affecting interactions with the human receptor angiotensinconverting enzyme-2 (ACE2). This allosteric network couples the residues of the N-terminal domain (NTD) and the RBD, which are modulated by the RBD-specific mutations and are capable of propagating mutation-induced perturbations between these domains through a combination of structural changes and effector-dependent modulations of dynamics. One key feature of this network is the inclusion of compensatory mutations segregated into three characteristically different clusters, where each cluster residue site is allosterically coupled with specific RBD mutation sites. Notably, each RBD mutation acted like a positive allosteric modulator; nevertheless, K417N was shown to have the largest effects among all of the mutations on the allostery and thereby holds the highest binding affinity with ACE2. This result will be useful for designing the targeted control measure and therapeutic efforts aiming at allosteric modulators.
Over the past two decades, we have witnessed an unprecedented explosion in available biological d... more Over the past two decades, we have witnessed an unprecedented explosion in available biological data. In the age of big data, large biological datasets have created an urgent need for the development of bioinformatics methods and innovative fast algorithms. Bioinformatics tools can enable data-driven hypothesis and interpretation of complex biological data that can advance biological and medicinal knowledge discovery. Advances in structural biology and computational modelling have led to the characterization of atomistic structures of many biomolecular components of cells. Proteins in particular are the most fundamental biomolecules and the key constituent elements of all living organisms, as they are necessary for cellular functions. Proteins play crucial roles in immunity, catalysis, metabolism and the majority of biological processes, and hence there is significant interest to understand how these macromolecules carry out their complex functions. The mechanical heterogeneity of p...
ABSTRACTA new coronavirus pandemic COVID-19, caused by Severe Acute Respiratory Syndrome coronavi... more ABSTRACTA new coronavirus pandemic COVID-19, caused by Severe Acute Respiratory Syndrome coronavirus (SARS-CoV-2), poses a serious threat across continents, leading the World Health Organization to declare a Public Health Emergency of International Concern. In order to block the entry of the virus into human host cells, major therapeutic and vaccine design efforts are now targeting the interactions between the SARS-CoV-2 spike (S) glycoprotein and the human cellular membrane receptor angiotensin-converting enzyme, hACE2. By analyzing cryo-EM structures of SARS-CoV-2 and SARS-CoV-1, we report here that the homotrimer SARS-CoV-2 S receptor-binding domain (RBD) that binds with hACE2 has expanded in size, undergoing a large conformational change relative to SARS-CoV-1 S protein. Protomer with the up-conformational form of RBD, which binds with hACE2, exhibits higher intermolecular interactions at the RBD-ACE2 interface, with differential distributions and the inclusion of specific H-bon...
Cholesterol is a major component of the cell membrane and commonly regulates membrane protein fun... more Cholesterol is a major component of the cell membrane and commonly regulates membrane protein function. Here, we investigate how cholesterol modulates the conformational equilibria and signaling of the adenosine A2A receptor (A2AR) in reconstituted phospholipid bilayers. GTP hydrolysis assays show that cholesterol is a weak positive allosteric modulator of A2AR, as seen through enhanced basal signaling and a small decrease in agonist EC50. Fluorine nuclear magnetic resonance (19F NMR) spectroscopy suggests that this enhancement arises from an increase in the receptor’s active state populations and stronger G protein coupling. 19F NMR of fluorinated cholesterol analogs reveals transient and non-specific interactions with A2AR, indicating a lack of high-affinity binding sites or direct allosteric modulation. This is confirmed by computational analysis which suggests that cholesterol contacts confer a weak and possibly negative allosteric effect. The combined results suggest that the o...
Acta Crystallographica Section A Foundations and Advances, 2017
In investigating the catalytic mechanism of a bacterial homodimeric enzyme, fluoroacetate dehalog... more In investigating the catalytic mechanism of a bacterial homodimeric enzyme, fluoroacetate dehalogenase (FAcD), we applied freeze-trapping x-ray crystallography, nuclear magnetic resonance, and computational techniques to establish the distribution of conformational states and their interconversion rates along the reaction pathway. At high resolution, the crystal structure of apo-FAcD revealed constant asymmetry between the two subunits, which is dynamically averaged on a millisecond time scale. During catalysis, the rate of conformational exchange between subunits becomes faster, with the empty protomer exhibiting larger localized disorder and loosing bound water molecules. This release of water and the increase in dynamics compensate entropic losses generated during substrate binding and might facilitate sampling of the transition state. Computational studies suggest allosteric pathways for information exchange between subunits. The studies provide insights into how substrate-coupled allosteric modulation of structure and dynamics facilitates catalysis in a homodimeric enzyme.
G-protein-coupled receptors (GPCRs) represent a ubiquitous membrane protein family and are import... more G-protein-coupled receptors (GPCRs) represent a ubiquitous membrane protein family and are important drug targets. Their diverse signaling pathways are driven by complex pharmacology arising from a conformational ensemble rarely captured by structural methods. Here, fluorine nuclear magnetic resonance spectroscopy (19F NMR) is used to delineate key functional states of the adenosine A2A receptor (A2AR) complexed with heterotrimeric G protein (Gαsβ1γ2) in a phospholipid membrane milieu. Analysis of A2AR spectra as a function of ligand, G protein, and nucleotide identifies an ensemble represented by inactive states, a G-protein-bound activation intermediate, and distinct nucleotide-free states associated with either partial- or full-agonist-driven activation. The Gβγ subunit is found to be critical in facilitating ligand-dependent allosteric transmission, as shown by 19F NMR, biochemical, and computational studies. The results provide a mechanistic basis for understanding basal signaling, efficacy, precoupling, and allostery in GPCRs.
We present a method, Accuracy of NMR Structures using Random Coil Index and Rigidity (ANSURR), th... more We present a method, Accuracy of NMR Structures using Random Coil Index and Rigidity (ANSURR), that measures the accuracy of NMR protein structures. It provides a residue-by-residue comparison of two measures of local rigidity: the Random Coil Index [RCI] (a measure of the extent to which backbone chemical shifts adopt random coil values); and local rigidity predicted by mathematical rigidity theory using the computational method Floppy Inclusion and Rigid Substructure Topology [FIRST], calculated from an NMR structural model. We compare RCI and FIRST using a correlation score (which assesses the location of secondary structure), and an RMSD score (which measures overall rigidity, and mainly assesses hydrogen bond correctness). We test the performance of ANSURR using: (a) structures refined in explicit solvent, which have much better RMSD score than unrefined structures, though similar correlation; (b) decoy structures generated for 89 NMR structures. The experimental NMR structures...
Cations play key roles in regulating G-protein-coupled receptors (GPCRs), although their mechanis... more Cations play key roles in regulating G-protein-coupled receptors (GPCRs), although their mechanisms are poorly understood. Here, 19 F NMR is used to delineate the effects of cations on functional states of the adenosine A 2A GPCR. While Na + reinforces an inactive ensemble and a partial-agonist stabilized state, Ca 2+ and Mg 2+ shift the equilibrium toward active states. Positive allosteric effects of divalent cations are more pronounced with agonist and a G-protein-derived peptide. In cell membranes, divalent cations enhance both the affinity and fraction of the high affinity agonist-bound state. Molecular dynamics simulations suggest high concentrations of divalent cations bridge specific extracellular acidic residues, bringing TM5 and TM6 together at the extracellular surface and allosterically driving open the G-protein-binding cleft as shown by rigidity-transmission allostery theory. An understanding of cation allostery should enable the design of allosteric agents and enhance our understanding of GPCR regulation in the cellular milieu.
Within the microbial rhodopsin family, heliorhodopsins (HeRs) form a phylogenetically distinct gr... more Within the microbial rhodopsin family, heliorhodopsins (HeRs) form a phylogenetically distinct group of light-harvesting retinal proteins with largely unknown functions. We have determined the 1.97 Å resolution X-ray crystal structure of Thermoplasmatales archaeon SG8-52-1 heliorhodopsin (TaHeR) in the presence of NaCl under acidic conditions (pH 4.5), which complements the known 2.4 Å TaHeR structure acquired at pH 8.0. The low pH structure revealed that the hydrophilic Schiff base cavity (SBC) accommodates a chloride anion to stabilize the protonated retinal Schiff base when its primary counterion (Glu-108) is neutralized. Comparison of the two structures at different pH revealed conformational changes connecting the SBC and the extracellular loop linking helices A–B. We corroborated this intramolecular signaling transduction pathway with computational studies, which revealed allosteric network changes propagating from the perturbed SBC to the intracellular and extracellular space...
Heterotrimeric G proteins serve as key membrane-associated signaling hubs, in concert with their ... more Heterotrimeric G proteins serve as key membrane-associated signaling hubs, in concert with their cognate G protein-coupled receptors (GPCRs). Using site-directed labels located at four key allosteric sites within the Ras-homology domain of the stimulatory G protein α-subunit, Gsα, fluorine nuclear magnetic resonance spectroscopy (19F NMR) was employed to monitor the conformational equilibria of Gsα by itself, in the intact Gsαβγ heterotrimer, or in complex with either membrane alone or membrane-embedded adenosine A2A receptor (A2AR). The results reveal a concerted equilibrium which is strongly affected by nucleotide and interactions with the βγ-subunit, the lipid bilayer, and A2AR. 19F NMR spectra of the α1 helix of Gsα exhibit significant intermediate timescale dynamics, while those associated with the α4β6 loop and the α5 helix reflect respective membrane/receptor interactions and order/disorder transitions associated with G protein activation. The αN helix adopts a key functional...
Loops are small secondary structural elements that play a crucial role in the emergence of new en... more Loops are small secondary structural elements that play a crucial role in the emergence of new enzyme functions. However, our understanding of loop functions is mainly limited to the catalytic loops. To understand the function of remote loops in enzymes, we studied Glycoside hydrolase family 19 (GH19) chitinase - an essential enzyme family for pathogen degradation in plants. By revealing the evolutionary history and loops appearance of GH19 chitinase, we discovered that one loop which is remote from the catalytic site, is necessary to acquire the new antifungal activity. We demonstrated that this remote loop directly accesses the fungal cell wall, and surprisingly, it needs to adopt a defined structure supported by long-range intramolecular interactions to perform its function. Our findings prove that Nature applies this new strategy at the molecular level to achieve a complex biological function while maintaining the original activity one in the catalytic pocket, suggesting an alte...
Volume 5B: 41st Mechanisms and Robotics Conference, 2017
Assur groups and correspondingly Assur graphs have key attributes that make them important for th... more Assur groups and correspondingly Assur graphs have key attributes that make them important for the analysis and synthesis of linkages. The topology of linkages (more precisely the existence of constraints between links) in general can be represented by various types of constraint graphs. The concept of Assur graphs applies to any such graph and thus their peculiar features can be exploited. Despite of these potential benefits the relevant methods and tools are scattered in the literature. The aim of this paper is to provide an overview of the use of constraint graphs including recent results on the construction of Assur graphs.
The entry of the SARS-CoV-2, a causative agent of COVID-19, into human host cells is mediated by ... more The entry of the SARS-CoV-2, a causative agent of COVID-19, into human host cells is mediated by the SARS-CoV-2 spike (S) glycoprotein, which critically depends on the formation of complexes involving the spike protein receptor-binding domain (RBD) and the human cellular membrane receptor angiotensin-converting enzyme 2 (hACE2). Using classical site density functional theory (SDFT) and structural bioinformatics methods, we investigate binding and conformational properties of these complexes and study the overlooked role of water-mediated interactions. Analysis of the three-dimensional reference interaction site model (3DRISM) of SDFT indicates that water mediated interactions in the form of additional water bridges strongly increases the binding between SARS-CoV-2 spike protein and hACE2 compared to SARS-CoV-1-hACE2 complex. By analyzing structures of SARS-CoV-2 and SARS-CoV-1, we find that the homotrimer SARS-CoV-2 S receptor-binding domain (RBD) has expanded in size, indicating la...
Tau is an intrinsically disordered protein (IDP) whose primary physiological role is to stabi-liz... more Tau is an intrinsically disordered protein (IDP) whose primary physiological role is to stabi-lize microtubules in neuronal axons at all stages of development. In Alzheimer's and other tauopathies, tau forms intracellular insoluble amyloid aggregates known as neurofibrillary tangles, a process that appears in many cases to be preceded by hyperphosphorylation of tau monomers. Understanding the shift in conformational bias induced by hyperphosphory-lation is key to elucidating the structural factors that drive tau pathology, however, as an IDP, tau is not amenable to conventional structural characterization. In this work, we employ a straightforward technique based on Time-Resolved ElectroSpray Ionization Mass Spec-trometry (TRESI-MS) and Hydrogen/Deuterium Exchange (HDX) to provide a detailed pic-ture of residual structure in tau, and the shifts in conformational bias induced by hyperphosphorylation. By comparing the native and hyperphosphorylated ensembles, we are able to defin...
The tourism industry has generated research and studies, into government agencies, the private se... more The tourism industry has generated research and studies, into government agencies, the private sector, and academic institutions worldwide. In Brazil, the growth of eventbased tourism, in which million direct and indirect jobs have been created, has gained prominence in this profitable industry. Brasília, the seat of the federal government, and a modern, well-developed city of cultural and international diversity, is ideally suited for the hosting of special events. For this reason, it is important that attention be given to the training of tourism professionals in Brasília, particularly as it relates to creating and delivering quality services. This research demonstrates the difficulties that event coordinators in Brasília experience in going "by the book." These professionals opt for using their own manuals, which often do not cover all the steps and details necessary for good risk management, or they prefer to use only a checklist. Checklists are important, but they are not sufficient to guarantee the success of an event. This study also reveals the chief discrepancies that arise between recommended, theoretical procedures and the work that is actually done, underscoring the need to identify each potential problem in the planning of events, in order to better manage-and minimize-risks. Consequently, we have developed a hands-on, risk-management manual based on trial and error for the benefit of event coordinators. This innovative, step-by-step manual includes the PDCA Cycle and SWOT Analysis which, applied simultaneously, will better prepare tourism professionals for planning mega events in Brasília. 1. Event-based tourism 2. Risks 3. Risk Management
To understand how proteins function it is crucial to understand the connection between their stru... more To understand how proteins function it is crucial to understand the connection between their structure, flexibility and dynamics. In the field of bioinformatics and computational biology, there is a strong interest to develop software and computational tools that analyzes various properties of protein structures. The software we are using for protein flexibility and dynamics analysis generally assumes a single task, single thread environment. To more efficiently elucidate the function of proteins, we need to perform large-scale calculations on many structures. To improve computational speed of such large scale analysis, we decided to perform parallel distributed computation with the conventional software. We designed a simple protocol dedicated to this software over http and achieved a speedup of 550 times with 600 CPU cores. With such speed ups, we are able to perform faster high-throughput computations on large number of protein structures.
This chapter describes the application of constrained geometric simulations for prediction of ant... more This chapter describes the application of constrained geometric simulations for prediction of antibody structural dynamics. We utilize constrained geometric simulations method FRODAN, which is a low computational complexity alternative to Molecular Dynamics (MD) simulations that can rapidly explore flexible motions in protein structures. FRODAN is highly suited for conformational dynamics analysis of large proteins, complexes, intrinsically disordered proteins and dynamics that occurs on longer biologically relevant time scales which are normally inaccessible to classical MD simulations. This approach predicts protein dynamics at an all-atom scale while retaining realistic covalent bonding, maintaining dihedral angles in energetically good conformations while avoiding steric clashes in addition to performing other geometric and stereochemical criteria checks. In this chapter, we apply FRODAN to showcase its applicability for probing functionally relevant dynamics of IgG2a, including...
2016 3rd Asia-Pacific World Congress on Computer Science and Engineering (APWC on CSE), 2016
Advancements in the field of mathematical rigidity theory have opened up a number of exciting opp... more Advancements in the field of mathematical rigidity theory have opened up a number of exciting opportunities for computational predictions of protein flexibility and their dynamics. Starting with a 3D protein structure, several programs such as FIRST model the protein as a constraint multigraph, consisting of vertices (atoms) and edges (covalent bonds, hydrogen bonds, electrostatic interactions, and hydrophobic contacts). FIRST applies the pebble game algorithm on the resulting multigraph which rapidly decompose the protein into rigid clusters and flexible regions. Using an extension of FIRST and the pebble game algorithm we propose a computational approach for studying a biological phenomenon 'allostery'. Allostery refers to an effect of binding at one site to another, often significantly distant functional site on the protein, allowing for regulation of the protein function. Most dynamic proteins are allosteric and allostery has even been coined the 'second secret of li...
The emergence of a variety of highly transmissible SARS-CoV-2 variants, the causative agent of CO... more The emergence of a variety of highly transmissible SARS-CoV-2 variants, the causative agent of COVID-19, with multiple spike mutations poses serious challenges in overcoming the ongoing deadly pandemic. It is, therefore, essential to understand how these variants gain enhanced ability to evade immune responses with a higher rate of spreading infection. To address this question, here we have individually assessed the effects of SARS-CoV-2 variant-specific spike (S) protein receptor-binding domain (RBD) mutations E484K, K417N, L452Q, L452R, N501Y, and T478K that characterize and differentiate several emerging variants. Despite the hundreds of apparently neutral mutations observed in the domains other than the RBD, we have shown that each RBD mutation site is differentially engaged in an interdomain allosteric network involving mutation sites from a distant domain, affecting interactions with the human receptor angiotensinconverting enzyme-2 (ACE2). This allosteric network couples the residues of the N-terminal domain (NTD) and the RBD, which are modulated by the RBD-specific mutations and are capable of propagating mutation-induced perturbations between these domains through a combination of structural changes and effector-dependent modulations of dynamics. One key feature of this network is the inclusion of compensatory mutations segregated into three characteristically different clusters, where each cluster residue site is allosterically coupled with specific RBD mutation sites. Notably, each RBD mutation acted like a positive allosteric modulator; nevertheless, K417N was shown to have the largest effects among all of the mutations on the allostery and thereby holds the highest binding affinity with ACE2. This result will be useful for designing the targeted control measure and therapeutic efforts aiming at allosteric modulators.
Over the past two decades, we have witnessed an unprecedented explosion in available biological d... more Over the past two decades, we have witnessed an unprecedented explosion in available biological data. In the age of big data, large biological datasets have created an urgent need for the development of bioinformatics methods and innovative fast algorithms. Bioinformatics tools can enable data-driven hypothesis and interpretation of complex biological data that can advance biological and medicinal knowledge discovery. Advances in structural biology and computational modelling have led to the characterization of atomistic structures of many biomolecular components of cells. Proteins in particular are the most fundamental biomolecules and the key constituent elements of all living organisms, as they are necessary for cellular functions. Proteins play crucial roles in immunity, catalysis, metabolism and the majority of biological processes, and hence there is significant interest to understand how these macromolecules carry out their complex functions. The mechanical heterogeneity of p...
ABSTRACTA new coronavirus pandemic COVID-19, caused by Severe Acute Respiratory Syndrome coronavi... more ABSTRACTA new coronavirus pandemic COVID-19, caused by Severe Acute Respiratory Syndrome coronavirus (SARS-CoV-2), poses a serious threat across continents, leading the World Health Organization to declare a Public Health Emergency of International Concern. In order to block the entry of the virus into human host cells, major therapeutic and vaccine design efforts are now targeting the interactions between the SARS-CoV-2 spike (S) glycoprotein and the human cellular membrane receptor angiotensin-converting enzyme, hACE2. By analyzing cryo-EM structures of SARS-CoV-2 and SARS-CoV-1, we report here that the homotrimer SARS-CoV-2 S receptor-binding domain (RBD) that binds with hACE2 has expanded in size, undergoing a large conformational change relative to SARS-CoV-1 S protein. Protomer with the up-conformational form of RBD, which binds with hACE2, exhibits higher intermolecular interactions at the RBD-ACE2 interface, with differential distributions and the inclusion of specific H-bon...
Cholesterol is a major component of the cell membrane and commonly regulates membrane protein fun... more Cholesterol is a major component of the cell membrane and commonly regulates membrane protein function. Here, we investigate how cholesterol modulates the conformational equilibria and signaling of the adenosine A2A receptor (A2AR) in reconstituted phospholipid bilayers. GTP hydrolysis assays show that cholesterol is a weak positive allosteric modulator of A2AR, as seen through enhanced basal signaling and a small decrease in agonist EC50. Fluorine nuclear magnetic resonance (19F NMR) spectroscopy suggests that this enhancement arises from an increase in the receptor’s active state populations and stronger G protein coupling. 19F NMR of fluorinated cholesterol analogs reveals transient and non-specific interactions with A2AR, indicating a lack of high-affinity binding sites or direct allosteric modulation. This is confirmed by computational analysis which suggests that cholesterol contacts confer a weak and possibly negative allosteric effect. The combined results suggest that the o...
Acta Crystallographica Section A Foundations and Advances, 2017
In investigating the catalytic mechanism of a bacterial homodimeric enzyme, fluoroacetate dehalog... more In investigating the catalytic mechanism of a bacterial homodimeric enzyme, fluoroacetate dehalogenase (FAcD), we applied freeze-trapping x-ray crystallography, nuclear magnetic resonance, and computational techniques to establish the distribution of conformational states and their interconversion rates along the reaction pathway. At high resolution, the crystal structure of apo-FAcD revealed constant asymmetry between the two subunits, which is dynamically averaged on a millisecond time scale. During catalysis, the rate of conformational exchange between subunits becomes faster, with the empty protomer exhibiting larger localized disorder and loosing bound water molecules. This release of water and the increase in dynamics compensate entropic losses generated during substrate binding and might facilitate sampling of the transition state. Computational studies suggest allosteric pathways for information exchange between subunits. The studies provide insights into how substrate-coupled allosteric modulation of structure and dynamics facilitates catalysis in a homodimeric enzyme.
G-protein-coupled receptors (GPCRs) represent a ubiquitous membrane protein family and are import... more G-protein-coupled receptors (GPCRs) represent a ubiquitous membrane protein family and are important drug targets. Their diverse signaling pathways are driven by complex pharmacology arising from a conformational ensemble rarely captured by structural methods. Here, fluorine nuclear magnetic resonance spectroscopy (19F NMR) is used to delineate key functional states of the adenosine A2A receptor (A2AR) complexed with heterotrimeric G protein (Gαsβ1γ2) in a phospholipid membrane milieu. Analysis of A2AR spectra as a function of ligand, G protein, and nucleotide identifies an ensemble represented by inactive states, a G-protein-bound activation intermediate, and distinct nucleotide-free states associated with either partial- or full-agonist-driven activation. The Gβγ subunit is found to be critical in facilitating ligand-dependent allosteric transmission, as shown by 19F NMR, biochemical, and computational studies. The results provide a mechanistic basis for understanding basal signaling, efficacy, precoupling, and allostery in GPCRs.
We present a method, Accuracy of NMR Structures using Random Coil Index and Rigidity (ANSURR), th... more We present a method, Accuracy of NMR Structures using Random Coil Index and Rigidity (ANSURR), that measures the accuracy of NMR protein structures. It provides a residue-by-residue comparison of two measures of local rigidity: the Random Coil Index [RCI] (a measure of the extent to which backbone chemical shifts adopt random coil values); and local rigidity predicted by mathematical rigidity theory using the computational method Floppy Inclusion and Rigid Substructure Topology [FIRST], calculated from an NMR structural model. We compare RCI and FIRST using a correlation score (which assesses the location of secondary structure), and an RMSD score (which measures overall rigidity, and mainly assesses hydrogen bond correctness). We test the performance of ANSURR using: (a) structures refined in explicit solvent, which have much better RMSD score than unrefined structures, though similar correlation; (b) decoy structures generated for 89 NMR structures. The experimental NMR structures...
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Papers by Adnan Sljoka