Huan-Xiang Zhou - Academia.edu (original) (raw)

Papers by Huan-Xiang Zhou

Biomolecules, 2020

How sequences of intrinsically disordered proteins (IDPs) code for their conformational dynamics ... more How sequences of intrinsically disordered proteins (IDPs) code for their conformational dynamics is poorly understood. Here, we combined NMR spectroscopy, small-angle X-ray scattering (SAXS), and molecular dynamics (MD) simulations to characterize the conformations and dynamics of ChiZ1-64. MD simulations, first validated by SAXS and secondary chemical shift data, found scant α-helices or β-strands but a considerable propensity for polyproline II (PPII) torsion angles. Importantly, several blocks of residues (e.g., 11–29) emerge as “correlated segments”, identified by their frequent formation of PPII stretches, salt bridges, cation-π interactions, and sidechain-backbone hydrogen bonds. NMR relaxation experiments showed non-uniform transverse relaxation rates (R2s) and nuclear Overhauser enhancements (NOEs) along the sequence (e.g., high R2s and NOEs for residues 11–14 and 23–28). MD simulations further revealed that the extent of segmental correlation is sequence-dependent; segments...

Upon Ca2+ influx, synaptic vesicles fuse with the presynaptic plasma membrane (PM) to release neu... more Upon Ca2+ influx, synaptic vesicles fuse with the presynaptic plasma membrane (PM) to release neurotransmitters. Membrane fusion is triggered by synaptotagmin-1, a transmembrane protein in the vesicle membrane (VM), but the mechanism is under debate. Synaptotagmin-1 contains a single transmembrane helix (TM) and two tandem C2-domains (C2A and C2B). The present study aimed to use molecular dynamics simulations to elucidate how Ca2+-bound synaptotagmin-1, by simultaneously associating with VM and PM, brings them together for fusion. While C2A stably associates with VM via two Ca2+-binding loops, C2B has a propensity to partially dissociate. Importantly, an acidic motif in the TM-C2A linker competes with VM for interacting with C2B, thereby flipping its orientation to face PM. Subsequently C2B can readily associate with PM via a polybasic cluster and a Ca2+-binding loop. These results delineate the functional process of fusion triggered by synaptotagmin-1.

Chemical reviews, Jan 8, 2016

The functions of many proteins are regulated through allostery, whereby effector binding at a dis... more The functions of many proteins are regulated through allostery, whereby effector binding at a distal site changes the functional activity (e.g., substrate binding affinity or catalytic efficiency) at the active site. Most allosteric studies have focused on thermodynamic properties, in particular, substrate binding affinity. Changes in substrate binding affinity by allosteric effectors have generally been thought to be mediated by conformational transitions of the proteins or, alternatively, by changes in the broadness of the free energy basin of the protein conformational state without shifting the basin minimum position. When effector binding changes the free energy landscape of a protein in conformational space, the change affects not only thermodynamic properties but also dynamic properties, including the amplitudes of motions on different time scales and rates of conformational transitions. Here we assess the roles of conformational dynamics in allosteric regulation. Two cases a...

Structure (London, England : 1993), Jan 29, 2015

The tetrameric M2 protein from influenza A conducts protons into the virus upon acid activation o... more The tetrameric M2 protein from influenza A conducts protons into the virus upon acid activation of its His37 tetrad and is a proven drug target. Here, in studies of full-length M2 protein solubilized in native-like liquid-crystalline lipid bilayers, a pH titration monitored by solid-state nuclear magnetic resonance revealed a clustering of the first three His37 pKas (6.3, 6.3, and 5.5). When the +2 state of the tetrad accepts a third proton from the externally exposed portion of the channel pore and releases a proton to the internally exposed pore, successful proton conductance is achieved, but more frequently the tetrad accepts and returns the proton to the externally exposed pore, resulting in a futile cycle. Both dynamics and conformational heterogeneity of the His37 tetrad featuring short hydrogen bonds between imidazolium-imidazole pairs are characterized, and the heterogeneity appears to reflect oligomeric helix packing and the extent of transmembrane helical bending around Gl...

Annual review of biophysics, 2013

The number of membrane protein structures in the Protein Data Bank is becoming significant and gr... more The number of membrane protein structures in the Protein Data Bank is becoming significant and growing. Here, the transmembrane domain structures of the helical membrane proteins are evaluated to assess the influences of the membrane mimetic environments. Toward this goal, many of the biophysical properties of membranes are discussed and contrasted with those of the membrane mimetics commonly used for structure determination. Although the mimetic environments can perturb the protein structures to an extent that potentially gives rise to misinterpretation of functional mechanisms, there are also many structures that have a native-like appearance. From this assessment, an initial set of guidelines is proposed for distinguishing native-like from nonnative-like membrane protein structures. With experimental techniques for validation and computational methods for refinement and quality assessment and enhancement, there are good prospects for achieving native-like structures for these ver...

Nature communications, Jan 14, 2011

Ionotropic glutamate receptors (iGluRs) mediate fast excitatory synaptic transmission in the cent... more Ionotropic glutamate receptors (iGluRs) mediate fast excitatory synaptic transmission in the central nervous system. Upon agonist binding, an iGluR opens to allow the flow of cations and subsequently enters into a desensitized state. It remains unclear how agonist binding to the ligand-binding domain (LBD) is transmitted to the transmembrane domain (TMD) for channel activation and desensitization. Here we report molecular dynamics simulations of an AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-subtype iGluR in explicit water and membrane. Channel opening and closing were observed in simulations of the activation and desensitization processes, respectively. The motions of the LBD-TMD linkers along the central axis of the receptor and in the lateral plane contributed cooperatively to channel opening and closing. The detailed mechanism of channel activation and desensitization suggested by the simulations here is consistent with existing data and may serve as a guide for...

Biophysical Journal, 2015

Membranes in cancer cells are relatively more fluid compared to healthy cells. Higher membrane fl... more Membranes in cancer cells are relatively more fluid compared to healthy cells. Higher membrane fluidity in cancer cells closely relates to their invasive potential, proliferation, and metastatic ability. Normalization of membrane fluidity in cancer cells represents a novel therapeutic modality, however, there are no strategies currently focused on targeting this modality as cancer therapeutics.. This study describes the introduction BPM 31510, a proprietary CoQ10 based liposomal formulation that specifically targets cell membrane fluidity as one of the modalities influenced in cancer cell to effectuate a therapeutic end-point, i.e decrease in cell proliferation. First, CoQ10 concentrations was systematically varied in the liposomal formulation and membrane rigidity (Fluorescence Anisotropy) measured as function of temperature. Increasing concentrations of CoQ10 was associated with progressive and significant increase in rigidity of liposomal membranes followed by decrease at higher concentration. Interestingly, the concentration at which the local maxima in rigidity occurred matched with the composition of BPM 31510. Later, we demonstrate that BPM 31510 treatment temporarily increases cell membrane rigidity that orchestrates adaption in lipidome, proteome, and cell bioenergetics. To better understand differential response to BPM 31510, a spectrum of cancer and healthy cells were stratified based on intrinsic membrane rigidity, cell bioenergetics, and proliferation rates, and relative changes following treatment with BPM 31510 were compared. Collectively, the data provides novel insight into CoQ10 effect on cell membrane dynamics, suggesting an integration of biophysical, biochemical and molecular effects attributable to BPM 31510 mechanism of action in the treatment of cancer. Overall, the study provides compelling data in support of targeting of membrane fluidity, a biophysical characteristic of cell, as a novel target amenable to pharmacological manipulation in the treatment of cancer.

Current opinion in virology, 2012

The M2 protein from influenza A is a proton channel as a tetramer, with a single transmembrane he... more The M2 protein from influenza A is a proton channel as a tetramer, with a single transmembrane helix from each monomer lining the pore. Val27 and Trp41 form gates at either end of the pore and His37 mediates the shuttling of protons across a central barrier between the N-terminal and C-terminal aqueous pore regions. Numerous structures of this transmembrane domain and of a longer construct that includes an amphipathic helix are now in the Protein Data Bank. Many structural differences are apparent from samples obtained in a variety of membrane mimetic environments. High-resolution structural results in lipid bilayers have provided novel insights into the functional mechanism of the unique HxxxW cluster in the M2 proton channel.

Proceedings of the National Academy of Sciences, 2014

Significance Understanding the structure and function of the cell division apparatus of Mycobacte... more Significance Understanding the structure and function of the cell division apparatus of Mycobacterium tuberculosis is crucial for advancing drug development against tuberculosis. Here, we report the solid-state NMR structure of a transmembrane protein, CrgA, that is a central component of the M. tuberculosis divisome. Small helical membrane protein structures are particularly sensitive to their environment, and consequently, we characterized CrgA in an environment that models well the biophysical properties of the native membrane. To determine the structure, both oriented sample and magic-angle spinning NMR data from liquid–crystalline lipid bilayer preparations were used along with refinement by restrained molecular dynamics simulations in the same lipid environment. The structure suggests how CrgA serves as a platform for binding and recruiting other proteins of the divisome.

Trends in Biochemical Sciences, 2011

Membrane protein structures are stabilized by weak interactions and are influenced by additional ... more Membrane protein structures are stabilized by weak interactions and are influenced by additional interactions with the solubilizing environment. Structures of Influenza virus A M2 protein, a proven drug target, have been determined in three different environments, thus providing a unique opportunity to assess environmental influences. Structures determined in detergents and detergent micelles can have notable differences from those determined in lipid bilayers. These differences make it imperative to validate membrane protein structures. The importance of the membrane environment Anfinsen's thermodynamic hypothesis [1] states "that the native conformation [of a protein] [SC1]is determined by the totality of inter-atomic interactions and hence by the amino acid sequence, in a given environment." Too often these last four words are ignored. The influence of the environment on the structures of membrane proteins is especially significant (Box 1). Despite their functional importance, the structural biology of membrane proteins has been particularly challenging, as evidenced by the small number of membrane protein structures that have been determined (250 unique structures as of June, 2010; http:// blanco.biomol.uci.edu/Membrane_Proteins_xtal.html). Integral membrane proteins are present in a heterogeneous environment that poses major obstacles for existing structural methodologies (Box 2). Hence one must contend with solubilizing membrane proteins in mimetics of the native membranes. However, it is very difficult to obtain membrane mimetic environments that support the native structure(s), dynamics, and function(s) of a membrane protein. In fact, functional assays of membrane proteins might not be feasible in the chosen membrane mimetic environment, as is the case for ion channels in detergent micelles. Consequently, adequate validation by functional assays has not been carried out for many membrane protein structures. The influence of protein-membrane interactions compared to intra-protein interactions will be greater for smaller proteins and for proteins that do not have prosthetic groups to help

Nature communications, 2014

The pore-lining helix (PLH) bundles are central to the function of all ion channels, as their con... more The pore-lining helix (PLH) bundles are central to the function of all ion channels, as their conformational rearrangements dictate channel gating. Here we explore all plausible oligomeric arrangements of the PLH bundles within homomeric ion channels by building models using generic restraints. In particular, the distance between two neighbouring PLHs was bounded both below and above in order to avoid steric clash and allow proper packing. The resulting models provide a theoretical representation of the accessible space for oligomeric arrangements. While the represented space is confined, it encompasses nearly all the ion channel PLH bundles for which the structures are currently known. For a multitude of channels, gating models suggested by paths within the confined accessible space are in qualitative agreement with those established in previous structural and computational studies.

Trends in Biochemical Sciences, 2010

Protein dynamics is essential for virtually all protein functions, certainly for gating mechanism... more Protein dynamics is essential for virtually all protein functions, certainly for gating mechanisms of ion channels and regulation of enzyme catalysis. Ion channels usually feature a gate in the channel pore that prevents ion permeation in the closed state. Some bifunctional enzymes with two distant active sites use a tunnel to transport intermediate products; a gate can help prevent premature leakage. Enzymes with a buried active site also require a tunnel for substrate entrance; a gate along the tunnel can contribute to selectivity. The gates in these different contexts show distinct characteristics in sequence, structure, and dynamics, but they also have common features. In particular, aromatic residues often appear to serve as gates, probably due to their ability, through side chain rotation, to effect large changes in cross section.

Science, 2010

M2 Out of the Envelope The M2 protein from influenza A virus forms an acid-activated tetrameric p... more M2 Out of the Envelope The M2 protein from influenza A virus forms an acid-activated tetrameric proton channel in the viral envelope and is essential for viral replication. Two manuscripts shed light on the functional mechanism of this channel. Sharma et al. (p. 509 ; see the Perspective by Fiorin et al. ) determined the structure of the conductance domain in a lipid bilayer and propose that a histidine and tryptophan from each monomer form a cluster that guides protons through the channel in a mechanism that involves forming and breaking hydrogen bonds between adjacent pairs of histidines. Hu et al. (p. 505 ; see the Perspective by Fiorin et al. ) focused on the structure and dynamics of the proton-selective histidine at high and low pH, proposing that proton conduction involves histidine deprotonation and reprotonation.

Protein Science, 2013

The M2 protein, a proton channel, from Influenza A has been structurally characterized by X-ray d... more The M2 protein, a proton channel, from Influenza A has been structurally characterized by X-ray diffraction and by solution and solid-state NMR spectroscopy in a variety of membrane mimetic environments. These structures show substantial backbone differences even though they all present a left-handed tetrameric helical bundle for the transmembrane domain. Variations in the helix tilt influence drug binding and the chemistry of the histidine tetrad responsible for acid activation, proton selectivity and transport. Some of the major structural differences do not arise from the lack of precision, but instead can be traced to the influences of the membrane mimetic environments. The structure in lipid bilayers displays unique chemistry for the histidine tetrad, which binds two protons cooperatively to form a pair of imidazole-imidazolium dimers. The resulting interhistidine hydrogen bonds contribute to a three orders of magnitude enhancement in tetramer stability. Integration with computation has provided detailed understanding of the functional mechanism for proton selectivity, conductance and gating of this important drug target.

Proceedings of the National Academy of Sciences, 2006

The heart of the H + conductance mechanism in the homotetrameric M2 H + channel from influenza A ... more The heart of the H + conductance mechanism in the homotetrameric M2 H + channel from influenza A is a set of four histidine side chains. Here, we show that protonation of the third of these imidazoles coincides with acid activation of this transmembrane channel and that, at physiological pH, the channel is closed by two imidazole–imidazolium dimers, each sharing a low-barrier hydrogen bond. This unique construct succeeds in distributing a pair of charges over four rings and many atoms in a low dielectric environment to minimize charge repulsion. These dimers form with identical pK a s of 8.2 ± 0.2, suggesting cooperative H + binding and clearly illustrating high H + affinity for this channel. The protonation behavior of the histidine side chains has been characterized by using solid-state NMR spectroscopy on the M2 transmembrane domain in fully hydrated lipid bilayers where the tetrameric backbone structure is known. Furthermore, electrophysiological measurements of multichannel and...

Proceedings of the National Academy of Sciences, 2013

Significance The opening of P2X receptor channels by extracellular ATP underlies purinergic signa... more Significance The opening of P2X receptor channels by extracellular ATP underlies purinergic signaling in many tissues. Here we use computational and functional approaches to study helix interactions within the transmembrane domain of P2X receptors. Our results suggest that the intersubunit crevices observed in the X-ray structure of detergent-solubilized ATP-bound receptors are nonnative but confirm helix interactions within individual subunits observed in both apo and ATP-bound receptors and identify a hot spot within a narrow internal region where the gating and permeation properties can be readily tuned.

Journal of Molecular Biology, 2014

MgtR, a highly hydrophobic peptide expressed in Salmonella enterica serovar Typhimurium,inhibits ... more MgtR, a highly hydrophobic peptide expressed in Salmonella enterica serovar Typhimurium,inhibits growth in macrophages through binding to the membrane protein MgtC that has been identified as essential for replication in macrophages. While the Mycobacterium tuberculosis MgtC is highly homologuous to its S. Typhimurium analogue, there does not appear to be an Mtb homologue for MgtR, raising significant pharmacological interest in this system. Here, solid-state NMR and EPR spectroscopy in lipid bilayer preparations were used to demonstrate the formation of a heterodimer between S. Typhimurium MgtR and the transmembrane helix 4 of Mtb MgtC. Based on the experimental restraints, a structural model of this heterodimer was developed using computational techniques. The result is that MgtR appears to be ideally situated in the membrane to influence the functionality of MgtC.

Biophysical Journal, 2013

N-methyl-D-aspartate (NMDA) receptors are obligate heterotetrameric ligand-gated ion channels tha... more N-methyl-D-aspartate (NMDA) receptors are obligate heterotetrameric ligand-gated ion channels that play critical roles in learning and memory. Here, using targeted molecular dynamics simulations, we developed an atomistic model for the gating of the GluN1/GluN2A NMDA receptor. Upon agonist binding, lobe closure of the ligand-binding domain produced outward pulling of the M3-D2 linkers, leading to outward movements of the C-termini of the pore-lining M3 helices and opening of the channel. The GluN2A subunits, similar to the distal (B/D) subunits in the homotetrameric GluA2 a-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate receptor, had greater M3 outward movements and thus contributed more to channel gating than the GluN1 subunits. Our gating model is validated by functional studies, including cysteine modification data indicating wider accessibility to the GluN1 M3 helices than to the GluN2A M3 helices from the lumen of the open channel, and reveals why the Lurcher mutation in GluN1 has a stronger ability in maintaining channel opening than the counterpart in GluN2A. The resulting structural model for the open state provides an explanation for the Ca 2þ permeability of NMDA receptors, and the structural differences between the closed and open states form the basis for drug design.

Biophysical Journal, 2010

Plaques containing aggregated b-Amyloid (Ab) peptide in the brain are the main indication of Alzh... more Plaques containing aggregated b-Amyloid (Ab) peptide in the brain are the main indication of Alzheimers disease. These plaques consist of Ab fibrils. Oligomers of Ab have been implicated as infective agents in the disease and may also be intermediates of fibril formation. Therefore, methods to study oligomers on the timescale of aggregation are sought. We show that by EPR the dynamics of spin-labeled Ab in solutions in which fibrils are formed can be determined. The EPR experiments were performed on solutions of the Ab peptide with 42 residues (1-42 Ab) containing an N-terminal cysteine, which was spin labeled with the MTSL spin label (1-oxyl-2,2,5,5-tetramethyl-D-pyrroline-3methyl]methanethiosulfonate) (SL-Ab). For diamagnetic dilution, SLAb was mixed with unlabeled Ab. Fibril-formation in these solutions is shown by Congo-red binding and electron microscopy. Continuous wave, 9 GHz EPR reveals three fractions of different spin-label mobility, a fast one attributed to monomeric Ab, one with a mobility that corresponds to a multimer of eight to 15 monomers, and a slow one due to larger aggregates or fibrils. The approach, in principle, allows detection of oligomers on the timescale of aggregation.

Biophysical Journal, 2013

Biomolecules, 2020

How sequences of intrinsically disordered proteins (IDPs) code for their conformational dynamics ... more How sequences of intrinsically disordered proteins (IDPs) code for their conformational dynamics is poorly understood. Here, we combined NMR spectroscopy, small-angle X-ray scattering (SAXS), and molecular dynamics (MD) simulations to characterize the conformations and dynamics of ChiZ1-64. MD simulations, first validated by SAXS and secondary chemical shift data, found scant α-helices or β-strands but a considerable propensity for polyproline II (PPII) torsion angles. Importantly, several blocks of residues (e.g., 11–29) emerge as “correlated segments”, identified by their frequent formation of PPII stretches, salt bridges, cation-π interactions, and sidechain-backbone hydrogen bonds. NMR relaxation experiments showed non-uniform transverse relaxation rates (R2s) and nuclear Overhauser enhancements (NOEs) along the sequence (e.g., high R2s and NOEs for residues 11–14 and 23–28). MD simulations further revealed that the extent of segmental correlation is sequence-dependent; segments...

Upon Ca2+ influx, synaptic vesicles fuse with the presynaptic plasma membrane (PM) to release neu... more Upon Ca2+ influx, synaptic vesicles fuse with the presynaptic plasma membrane (PM) to release neurotransmitters. Membrane fusion is triggered by synaptotagmin-1, a transmembrane protein in the vesicle membrane (VM), but the mechanism is under debate. Synaptotagmin-1 contains a single transmembrane helix (TM) and two tandem C2-domains (C2A and C2B). The present study aimed to use molecular dynamics simulations to elucidate how Ca2+-bound synaptotagmin-1, by simultaneously associating with VM and PM, brings them together for fusion. While C2A stably associates with VM via two Ca2+-binding loops, C2B has a propensity to partially dissociate. Importantly, an acidic motif in the TM-C2A linker competes with VM for interacting with C2B, thereby flipping its orientation to face PM. Subsequently C2B can readily associate with PM via a polybasic cluster and a Ca2+-binding loop. These results delineate the functional process of fusion triggered by synaptotagmin-1.

Chemical reviews, Jan 8, 2016

The functions of many proteins are regulated through allostery, whereby effector binding at a dis... more The functions of many proteins are regulated through allostery, whereby effector binding at a distal site changes the functional activity (e.g., substrate binding affinity or catalytic efficiency) at the active site. Most allosteric studies have focused on thermodynamic properties, in particular, substrate binding affinity. Changes in substrate binding affinity by allosteric effectors have generally been thought to be mediated by conformational transitions of the proteins or, alternatively, by changes in the broadness of the free energy basin of the protein conformational state without shifting the basin minimum position. When effector binding changes the free energy landscape of a protein in conformational space, the change affects not only thermodynamic properties but also dynamic properties, including the amplitudes of motions on different time scales and rates of conformational transitions. Here we assess the roles of conformational dynamics in allosteric regulation. Two cases a...

Structure (London, England : 1993), Jan 29, 2015

The tetrameric M2 protein from influenza A conducts protons into the virus upon acid activation o... more The tetrameric M2 protein from influenza A conducts protons into the virus upon acid activation of its His37 tetrad and is a proven drug target. Here, in studies of full-length M2 protein solubilized in native-like liquid-crystalline lipid bilayers, a pH titration monitored by solid-state nuclear magnetic resonance revealed a clustering of the first three His37 pKas (6.3, 6.3, and 5.5). When the +2 state of the tetrad accepts a third proton from the externally exposed portion of the channel pore and releases a proton to the internally exposed pore, successful proton conductance is achieved, but more frequently the tetrad accepts and returns the proton to the externally exposed pore, resulting in a futile cycle. Both dynamics and conformational heterogeneity of the His37 tetrad featuring short hydrogen bonds between imidazolium-imidazole pairs are characterized, and the heterogeneity appears to reflect oligomeric helix packing and the extent of transmembrane helical bending around Gl...

Annual review of biophysics, 2013

The number of membrane protein structures in the Protein Data Bank is becoming significant and gr... more The number of membrane protein structures in the Protein Data Bank is becoming significant and growing. Here, the transmembrane domain structures of the helical membrane proteins are evaluated to assess the influences of the membrane mimetic environments. Toward this goal, many of the biophysical properties of membranes are discussed and contrasted with those of the membrane mimetics commonly used for structure determination. Although the mimetic environments can perturb the protein structures to an extent that potentially gives rise to misinterpretation of functional mechanisms, there are also many structures that have a native-like appearance. From this assessment, an initial set of guidelines is proposed for distinguishing native-like from nonnative-like membrane protein structures. With experimental techniques for validation and computational methods for refinement and quality assessment and enhancement, there are good prospects for achieving native-like structures for these ver...

Nature communications, Jan 14, 2011

Ionotropic glutamate receptors (iGluRs) mediate fast excitatory synaptic transmission in the cent... more Ionotropic glutamate receptors (iGluRs) mediate fast excitatory synaptic transmission in the central nervous system. Upon agonist binding, an iGluR opens to allow the flow of cations and subsequently enters into a desensitized state. It remains unclear how agonist binding to the ligand-binding domain (LBD) is transmitted to the transmembrane domain (TMD) for channel activation and desensitization. Here we report molecular dynamics simulations of an AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-subtype iGluR in explicit water and membrane. Channel opening and closing were observed in simulations of the activation and desensitization processes, respectively. The motions of the LBD-TMD linkers along the central axis of the receptor and in the lateral plane contributed cooperatively to channel opening and closing. The detailed mechanism of channel activation and desensitization suggested by the simulations here is consistent with existing data and may serve as a guide for...

Biophysical Journal, 2015

Membranes in cancer cells are relatively more fluid compared to healthy cells. Higher membrane fl... more Membranes in cancer cells are relatively more fluid compared to healthy cells. Higher membrane fluidity in cancer cells closely relates to their invasive potential, proliferation, and metastatic ability. Normalization of membrane fluidity in cancer cells represents a novel therapeutic modality, however, there are no strategies currently focused on targeting this modality as cancer therapeutics.. This study describes the introduction BPM 31510, a proprietary CoQ10 based liposomal formulation that specifically targets cell membrane fluidity as one of the modalities influenced in cancer cell to effectuate a therapeutic end-point, i.e decrease in cell proliferation. First, CoQ10 concentrations was systematically varied in the liposomal formulation and membrane rigidity (Fluorescence Anisotropy) measured as function of temperature. Increasing concentrations of CoQ10 was associated with progressive and significant increase in rigidity of liposomal membranes followed by decrease at higher concentration. Interestingly, the concentration at which the local maxima in rigidity occurred matched with the composition of BPM 31510. Later, we demonstrate that BPM 31510 treatment temporarily increases cell membrane rigidity that orchestrates adaption in lipidome, proteome, and cell bioenergetics. To better understand differential response to BPM 31510, a spectrum of cancer and healthy cells were stratified based on intrinsic membrane rigidity, cell bioenergetics, and proliferation rates, and relative changes following treatment with BPM 31510 were compared. Collectively, the data provides novel insight into CoQ10 effect on cell membrane dynamics, suggesting an integration of biophysical, biochemical and molecular effects attributable to BPM 31510 mechanism of action in the treatment of cancer. Overall, the study provides compelling data in support of targeting of membrane fluidity, a biophysical characteristic of cell, as a novel target amenable to pharmacological manipulation in the treatment of cancer.

Current opinion in virology, 2012

The M2 protein from influenza A is a proton channel as a tetramer, with a single transmembrane he... more The M2 protein from influenza A is a proton channel as a tetramer, with a single transmembrane helix from each monomer lining the pore. Val27 and Trp41 form gates at either end of the pore and His37 mediates the shuttling of protons across a central barrier between the N-terminal and C-terminal aqueous pore regions. Numerous structures of this transmembrane domain and of a longer construct that includes an amphipathic helix are now in the Protein Data Bank. Many structural differences are apparent from samples obtained in a variety of membrane mimetic environments. High-resolution structural results in lipid bilayers have provided novel insights into the functional mechanism of the unique HxxxW cluster in the M2 proton channel.

Proceedings of the National Academy of Sciences, 2014

Significance Understanding the structure and function of the cell division apparatus of Mycobacte... more Significance Understanding the structure and function of the cell division apparatus of Mycobacterium tuberculosis is crucial for advancing drug development against tuberculosis. Here, we report the solid-state NMR structure of a transmembrane protein, CrgA, that is a central component of the M. tuberculosis divisome. Small helical membrane protein structures are particularly sensitive to their environment, and consequently, we characterized CrgA in an environment that models well the biophysical properties of the native membrane. To determine the structure, both oriented sample and magic-angle spinning NMR data from liquid–crystalline lipid bilayer preparations were used along with refinement by restrained molecular dynamics simulations in the same lipid environment. The structure suggests how CrgA serves as a platform for binding and recruiting other proteins of the divisome.

Trends in Biochemical Sciences, 2011

Membrane protein structures are stabilized by weak interactions and are influenced by additional ... more Membrane protein structures are stabilized by weak interactions and are influenced by additional interactions with the solubilizing environment. Structures of Influenza virus A M2 protein, a proven drug target, have been determined in three different environments, thus providing a unique opportunity to assess environmental influences. Structures determined in detergents and detergent micelles can have notable differences from those determined in lipid bilayers. These differences make it imperative to validate membrane protein structures. The importance of the membrane environment Anfinsen's thermodynamic hypothesis [1] states "that the native conformation [of a protein] [SC1]is determined by the totality of inter-atomic interactions and hence by the amino acid sequence, in a given environment." Too often these last four words are ignored. The influence of the environment on the structures of membrane proteins is especially significant (Box 1). Despite their functional importance, the structural biology of membrane proteins has been particularly challenging, as evidenced by the small number of membrane protein structures that have been determined (250 unique structures as of June, 2010; http:// blanco.biomol.uci.edu/Membrane_Proteins_xtal.html). Integral membrane proteins are present in a heterogeneous environment that poses major obstacles for existing structural methodologies (Box 2). Hence one must contend with solubilizing membrane proteins in mimetics of the native membranes. However, it is very difficult to obtain membrane mimetic environments that support the native structure(s), dynamics, and function(s) of a membrane protein. In fact, functional assays of membrane proteins might not be feasible in the chosen membrane mimetic environment, as is the case for ion channels in detergent micelles. Consequently, adequate validation by functional assays has not been carried out for many membrane protein structures. The influence of protein-membrane interactions compared to intra-protein interactions will be greater for smaller proteins and for proteins that do not have prosthetic groups to help

Nature communications, 2014

The pore-lining helix (PLH) bundles are central to the function of all ion channels, as their con... more The pore-lining helix (PLH) bundles are central to the function of all ion channels, as their conformational rearrangements dictate channel gating. Here we explore all plausible oligomeric arrangements of the PLH bundles within homomeric ion channels by building models using generic restraints. In particular, the distance between two neighbouring PLHs was bounded both below and above in order to avoid steric clash and allow proper packing. The resulting models provide a theoretical representation of the accessible space for oligomeric arrangements. While the represented space is confined, it encompasses nearly all the ion channel PLH bundles for which the structures are currently known. For a multitude of channels, gating models suggested by paths within the confined accessible space are in qualitative agreement with those established in previous structural and computational studies.

Trends in Biochemical Sciences, 2010

Protein dynamics is essential for virtually all protein functions, certainly for gating mechanism... more Protein dynamics is essential for virtually all protein functions, certainly for gating mechanisms of ion channels and regulation of enzyme catalysis. Ion channels usually feature a gate in the channel pore that prevents ion permeation in the closed state. Some bifunctional enzymes with two distant active sites use a tunnel to transport intermediate products; a gate can help prevent premature leakage. Enzymes with a buried active site also require a tunnel for substrate entrance; a gate along the tunnel can contribute to selectivity. The gates in these different contexts show distinct characteristics in sequence, structure, and dynamics, but they also have common features. In particular, aromatic residues often appear to serve as gates, probably due to their ability, through side chain rotation, to effect large changes in cross section.

Science, 2010

M2 Out of the Envelope The M2 protein from influenza A virus forms an acid-activated tetrameric p... more M2 Out of the Envelope The M2 protein from influenza A virus forms an acid-activated tetrameric proton channel in the viral envelope and is essential for viral replication. Two manuscripts shed light on the functional mechanism of this channel. Sharma et al. (p. 509 ; see the Perspective by Fiorin et al. ) determined the structure of the conductance domain in a lipid bilayer and propose that a histidine and tryptophan from each monomer form a cluster that guides protons through the channel in a mechanism that involves forming and breaking hydrogen bonds between adjacent pairs of histidines. Hu et al. (p. 505 ; see the Perspective by Fiorin et al. ) focused on the structure and dynamics of the proton-selective histidine at high and low pH, proposing that proton conduction involves histidine deprotonation and reprotonation.

Protein Science, 2013

The M2 protein, a proton channel, from Influenza A has been structurally characterized by X-ray d... more The M2 protein, a proton channel, from Influenza A has been structurally characterized by X-ray diffraction and by solution and solid-state NMR spectroscopy in a variety of membrane mimetic environments. These structures show substantial backbone differences even though they all present a left-handed tetrameric helical bundle for the transmembrane domain. Variations in the helix tilt influence drug binding and the chemistry of the histidine tetrad responsible for acid activation, proton selectivity and transport. Some of the major structural differences do not arise from the lack of precision, but instead can be traced to the influences of the membrane mimetic environments. The structure in lipid bilayers displays unique chemistry for the histidine tetrad, which binds two protons cooperatively to form a pair of imidazole-imidazolium dimers. The resulting interhistidine hydrogen bonds contribute to a three orders of magnitude enhancement in tetramer stability. Integration with computation has provided detailed understanding of the functional mechanism for proton selectivity, conductance and gating of this important drug target.

Proceedings of the National Academy of Sciences, 2006

The heart of the H + conductance mechanism in the homotetrameric M2 H + channel from influenza A ... more The heart of the H + conductance mechanism in the homotetrameric M2 H + channel from influenza A is a set of four histidine side chains. Here, we show that protonation of the third of these imidazoles coincides with acid activation of this transmembrane channel and that, at physiological pH, the channel is closed by two imidazole–imidazolium dimers, each sharing a low-barrier hydrogen bond. This unique construct succeeds in distributing a pair of charges over four rings and many atoms in a low dielectric environment to minimize charge repulsion. These dimers form with identical pK a s of 8.2 ± 0.2, suggesting cooperative H + binding and clearly illustrating high H + affinity for this channel. The protonation behavior of the histidine side chains has been characterized by using solid-state NMR spectroscopy on the M2 transmembrane domain in fully hydrated lipid bilayers where the tetrameric backbone structure is known. Furthermore, electrophysiological measurements of multichannel and...

Proceedings of the National Academy of Sciences, 2013

Significance The opening of P2X receptor channels by extracellular ATP underlies purinergic signa... more Significance The opening of P2X receptor channels by extracellular ATP underlies purinergic signaling in many tissues. Here we use computational and functional approaches to study helix interactions within the transmembrane domain of P2X receptors. Our results suggest that the intersubunit crevices observed in the X-ray structure of detergent-solubilized ATP-bound receptors are nonnative but confirm helix interactions within individual subunits observed in both apo and ATP-bound receptors and identify a hot spot within a narrow internal region where the gating and permeation properties can be readily tuned.

Journal of Molecular Biology, 2014

MgtR, a highly hydrophobic peptide expressed in Salmonella enterica serovar Typhimurium,inhibits ... more MgtR, a highly hydrophobic peptide expressed in Salmonella enterica serovar Typhimurium,inhibits growth in macrophages through binding to the membrane protein MgtC that has been identified as essential for replication in macrophages. While the Mycobacterium tuberculosis MgtC is highly homologuous to its S. Typhimurium analogue, there does not appear to be an Mtb homologue for MgtR, raising significant pharmacological interest in this system. Here, solid-state NMR and EPR spectroscopy in lipid bilayer preparations were used to demonstrate the formation of a heterodimer between S. Typhimurium MgtR and the transmembrane helix 4 of Mtb MgtC. Based on the experimental restraints, a structural model of this heterodimer was developed using computational techniques. The result is that MgtR appears to be ideally situated in the membrane to influence the functionality of MgtC.

Biophysical Journal, 2013

N-methyl-D-aspartate (NMDA) receptors are obligate heterotetrameric ligand-gated ion channels tha... more N-methyl-D-aspartate (NMDA) receptors are obligate heterotetrameric ligand-gated ion channels that play critical roles in learning and memory. Here, using targeted molecular dynamics simulations, we developed an atomistic model for the gating of the GluN1/GluN2A NMDA receptor. Upon agonist binding, lobe closure of the ligand-binding domain produced outward pulling of the M3-D2 linkers, leading to outward movements of the C-termini of the pore-lining M3 helices and opening of the channel. The GluN2A subunits, similar to the distal (B/D) subunits in the homotetrameric GluA2 a-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate receptor, had greater M3 outward movements and thus contributed more to channel gating than the GluN1 subunits. Our gating model is validated by functional studies, including cysteine modification data indicating wider accessibility to the GluN1 M3 helices than to the GluN2A M3 helices from the lumen of the open channel, and reveals why the Lurcher mutation in GluN1 has a stronger ability in maintaining channel opening than the counterpart in GluN2A. The resulting structural model for the open state provides an explanation for the Ca 2þ permeability of NMDA receptors, and the structural differences between the closed and open states form the basis for drug design.

Biophysical Journal, 2010

Plaques containing aggregated b-Amyloid (Ab) peptide in the brain are the main indication of Alzh... more Plaques containing aggregated b-Amyloid (Ab) peptide in the brain are the main indication of Alzheimers disease. These plaques consist of Ab fibrils. Oligomers of Ab have been implicated as infective agents in the disease and may also be intermediates of fibril formation. Therefore, methods to study oligomers on the timescale of aggregation are sought. We show that by EPR the dynamics of spin-labeled Ab in solutions in which fibrils are formed can be determined. The EPR experiments were performed on solutions of the Ab peptide with 42 residues (1-42 Ab) containing an N-terminal cysteine, which was spin labeled with the MTSL spin label (1-oxyl-2,2,5,5-tetramethyl-D-pyrroline-3methyl]methanethiosulfonate) (SL-Ab). For diamagnetic dilution, SLAb was mixed with unlabeled Ab. Fibril-formation in these solutions is shown by Congo-red binding and electron microscopy. Continuous wave, 9 GHz EPR reveals three fractions of different spin-label mobility, a fast one attributed to monomeric Ab, one with a mobility that corresponds to a multimer of eight to 15 monomers, and a slow one due to larger aggregates or fibrils. The approach, in principle, allows detection of oligomers on the timescale of aggregation.

Biophysical Journal, 2013