Visualization of SV2A conformations in situ by the use of Protein Tomography

The synaptic vesicle protein 2A (SV2A), the brain-binding site of the anti-epileptic drug levetiracetam (LEV), has been characterized by Protein Tomography™. We identified two major conformations of SV2A in mouse brain tissue: first, a compact, funnel-structure with a pore-like opening towards the cytoplasm; second, a more open, V-shaped structure with a cleft-like opening towards the intravesicular space. The large differences between these conformations suggest a high degree of flexibility and support a valve-like mechanism consistent with the postulated transporter role of SV2A. These two conformations are represented both in samples treated with LEV, and in saline-treated samples, which indicates that LEV binding does not cause a large-scale conformational change of SV2A, or lock a specific conformational state of the protein. This study provides the first direct structural data on SV2A, and supports a transporter function suggested by sequence homology to MFS class of transporter proteins.     

Combining modelling and mutagenesis studies of synaptic vesicle protein 2A to identify a series of residues involved in racetam binding

LEV (levetiracetam), an antiepileptic drug which possesses a unique profile in animal models of seizure and epilepsy, has as its unique binding site in brain, SV2A (synaptic vesicle protein 2A). Previous studies have used a chimaeric and site-specific mutagenesis approach to identify three residues in the putative tenth transmembrane helix of SV2A that, when mutated, alter binding of LEV and related racetam derivatives to SV2A. In the present paper, we report a combined modelling and mutagenesis study that successfully identifies another 11 residues in SV2A that appear to be involved in ligand binding. Sequence analysis and modelling of SV2A suggested residues equivalent to critical functional residues of other MFS (major facilitator superfamily) transporters. Alanine scanning of these and other SV2A residues resulted in the identification of residues affecting racetam binding, including Ile273 which differentiated between racetam analogues, when mutated to alanine. Integrating mutagenesis results with docking analysis led to the construction of a mutant in which six SV2A residues were replaced with corresponding SV2B residues. This mutant showed racetam ligand-binding affinity intermediate to the affinities observed for SV2A and SV2B.     

The Human Synaptic Vesicle Protein,SV2A,Functions as a Galactose Transporter in Saccharomyces cerevisiae

SV2A is a synaptic vesicle membrane protein expressed in neurons and endocrine cells and involved in the regulation of neurotransmitter release. Although the exact function of SV2A still remains elusive, it was identified as the specific binding site for levetiracetam, a second generation antiepileptic drug. Our sequence analysis demonstrates that SV2A has significant homology with several yeast transport proteins belonging to the major facilitator superfamily (MFS). Many of these transporters are involved in sugar transport into yeast cells. Here we present evidence showing, for the first time, that SV2A is a galactose transporter. We expressed human SV2A in hexose transport-deficient EBY.VW4000 yeast cells and demonstrated that these cells are able to grow on galactose-containing medium but not on other fermentable carbon sources. Furthermore, the addition of the SV2A-binding antiepileptic drug levetiracetam to the medium inhibited the galactose-dependent growth of hexose transport-deficient EBY.VW4000 yeast cells expressing human SV2A. Most importantly, direct measurement of galactose uptake in the same strain verified that SV2A is able to transport extracellular galactose inside the cells. The newly identified galactose transport capability of SV2A may have an important role in regulating/modulating synaptic function.     

Levetiracetam treatment ameliorates LRRK2 pathological mutant phenotype

Mutations in leucine‐rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson's disease (PD). The LRRK2 physiological and pathological function is still debated. However, different experimental evidence based on LRRK2 cellular localization and LRRK2 protein interactors suggests that LRRK2 may be part and regulate a protein network modulating vesicle dynamics/trafficking. Interestingly, the synaptic vesicle protein SV2A is part of this protein complex. Importantly, SV2A is the binding site of the levetiracetam (LEV), a compound largely used in human therapy for epilepsy treatment. The binding of LEV to SV2A reduces the neuronal firing by the modulation of vesicle trafficking although by an unclear molecular mechanism. In this short communication, we have analysed the interaction between the LRRK2 and SV2A pathways by LEV treatment. Interestingly, LEV significantly counteracts the effect of LRRK2 G2019S pathological mutant expression in three different cellular experimental models. Our data strongly suggest that LEV treatment may have a neuroprotective effect on LRRK2 pathological mutant toxicity and that LEV repositioning could be a viable compound for PD treatment.     

Understanding peptide competitive inhibition of botulinum neurotoxin a binding to SV2 protein via molecular dynamics simulations

Botulinum neurotoxins (BoNTs) are known as the most toxic natural substances. Synaptic vesicle protein 2 (SV2) has been proposed to be a protein receptor for BoNT/A. Recently, two short peptides (BoNT/A‐A2 and SV2C‐A3) were designed to inhibit complex formation between the BoNT/A receptor‐binding domain (BoNT/A‐RBD) and the synaptic vesicle protein 2C luminal domain (SV2C‐LD). In this article, the two peptide complex systems are studied by molecular dynamics (MD) simulations. The structural stability analysis indicates that BoNT/A‐A2 system is more stable than SV2C‐A3 system. The conformational analysis implies that the β‐sheet in BoNT/A‐A2 system maintains its secondary structure but the two β‐strands in SV2C‐A3 system have remarkable conformational changes. Based on the calculation of hydrogen bonds, hydrophobic interactions and cation‐π interactions, it is found that the internal hydrogen bonds play crucial roles in the structural stability of the peptides. Because of the stable secondary structure, the β‐sheet in BoNT/A‐A2 system establishes effective interactions at the interface and inhibits BoNT/A‐RBD binding to SV2C‐LD. In contrast, without other β‐strands forming internal hydrogen bonds, the two isolated β‐strands in SV2C‐A3 system become the random coil. This conformational change breaks important hydrogen bonds and weakens cation‐π interaction in the interface, so the complex formation is only partially inhibited by the two β‐strands. These results are consistent with experimental studies and may be helpful in understanding the inhibition mechanisms of peptide inhibitors. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 597–608, 2015.     

Searching the conformational complexity and binding properties of HDAC6 through docking and molecular dynamic simulations

Histone deacetylases (HDACs) are a family of proteins involved in the deacetylation of histones and other non-histones substrates. HDAC6 belongs to class II and shares similar biological functions with others of its class. Nevertheless, its three-dimensional structure that involves the catalytic site remains unknown for exploring the ligand recognition properties. Therefore, in this contribution, homology modeling, 100-ns-long Molecular Dynamics (MD) simulation and docking calculations were combined to explore the conformational complexity and binding properties of the catalytic domain 2 from HDAC6 (DD2-HDAC6), for which activity and affinity toward five different ligands have been reported. Clustering analysis allowed identifying the most populated conformers present during the MD simulation, which were used as starting models to perform docking calculations with five DD2-HDAC6 inhibitors: Cay10603 (CAY), Rocilinostat (RCT), Tubastatin A (TBA), Tubacin (TBC), and Nexturastat (NXT), and then were also submitted to 100-ns-long MD simulations. Docking calculations revealed that the five inhibitors bind at the DD2-HDAC6 binding site with the lowest binding free energy, the same binding mode is maintained along the 100-ns-long MD simulations. Overall, our results provide structural information about the molecular flexibility of apo and holo DD2-HDAC6 states as well as insight of the map of interactions between DD2-HDAC6 and five well-known DD2-HDAC6 inhibitors allowing structural details to guide the drug design. Finally, we highlight the importance of combining different theoretical approaches to provide suitable structural models for structure-based drug design.     

Structural analysis of ABC-family periplasmic zinc binding protein provides new insights into mechanism of ligand uptake and release

ATP-binding cassette superfamily of periplasmic metal transporters are known to be vital for maintaining ion homeostasis in several pathogenic and non-pathogenic bacteria. We have determined crystal structure of the high-affinity zinc transporter ZnuA from Escherichia coli at 1.8 A resolution. This structure represents the first native (non-recombinant) protein structure of a periplasmic metal binding protein. ZnuA reveals numerous conformational features, which occur either in Zn(2+) or in Mn(2+) transporters, and presents a unique conformational state. A comprehensive comparison of ZnuA with other periplasmic ligand binding protein structures suggests vital mechanistic differences between bound and release states of metal transporters. The key new attributes in ZnuA include a C-domain disulfide bond, an extra alpha-helix proximal to the highly charged metal chelating mobile loop region, alternate conformations of secondary shell stabilizing residues at the metal binding site, and domain movements potentially controlled by salt bridges. Based on in-depth structural analyses of five metal binding transporters, we present here a mechanistic model termed as "partial domain slippage" for binding and release of Zn(2+).     

Integration of Evolutionary Features for the Identification of Functionally Important Residues in Major Facilitator Superfamily Transporters

The identification of functionally important residues is an important challenge for understanding the molecular mechanisms of proteins. Membrane protein transporters operate two-state allosteric conformational changes using functionally important cooperative residues that mediate long-range communication from the substrate binding site to the translocation pathway. In this study, we identified functionally important cooperative residues of membrane protein transporters by integrating sequence conservation and co-evolutionary information. A newly derived evolutionary feature, the co-evolutionary coupling number, was introduced to measure the connectivity of co-evolving residue pairs and was integrated with the sequence conservation score. We tested this method on three Major Facilitator Superfamily (MFS) transporters, LacY, GlpT, and EmrD. MFS transporters are an important family of membrane protein transporters, which utilize diverse substrates, catalyze different modes of transport using unique combinations of functional residues, and have enough characterized functional residues to validate the performance of our method. We found that the conserved cores of evolutionarily coupled residues are involved in specific substrate recognition and translocation of MFS transporters. Furthermore, a subset of the residues forms an interaction network connecting functional sites in the protein structure. We also confirmed that our method is effective on other membrane protein transporters. Our results provide insight into the location of functional residues important for the molecular mechanisms of membrane protein transporters.     

Ligand binding and homology modelling of insect odorant‐binding proteins

This review describes the main characteristics of odorant‐binding proteins (OBPs) for homology modelling and presents a summary of structure prediction studies on insect OBPs, along with the steps involved and some limitations and improvements. The technique involves a computing approach to model protein structures and is based on a comparison between a target (unknown structure) and one or more templates (experimentally determined structures). As targets for structure prediction, OBPs are considered to play a functional role for recognition, desorption, scavenging, protection and transportation of hydrophobic molecules (odourants) across an aqueous environment (lymph) to olfactory receptor neurones (ORNs) located in sensilla, the main olfactory units of insect antennae. Lepidopteran pheromone‐binding proteins, a subgroup of OBPs, are characterized by remarkable structural features, in which high sequence identities (approximately 30%) among these OBPs and a large number of available templates can facilitate the prediction of precise homology models. Approximately 30 studies have been performed on insect OBPs using homology modelling as a tool to predict their structures. Although some of the studies have assessed ligand‐binding affinity using structural information and biochemical measurements, few have performed docking and molecular dynamic (MD) simulations as a virtual method to predict best ligands. Docking and MD simulations are discussed in the context of discovery of novel semiochemicals (super‐ligands) using homology modelling to conceive further strategies in insect management.     

Exploring the conformational and binding properties of unphosphorylated/phosphorylated monomeric and trimeric Bcl‐2 through docking and molecular dynamics simulations

B‐cell lymphoma (Bcl‐2) is commonly associated with the progression and preservation of cancer and certain lymphomas; therefore, it is considered as a biological target against cancer. Nevertheless, evidence of all its structural binding sites has been hidden because of the lack of a complete Bcl‐2 model, given the presence of a flexible loop domain (FLD), which is responsible for its complex behavior. FLD region has been implicated in phosphorylation, homotrimerization, and heterodimerization associated with Bcl‐2 antiapoptotic function. In this contribution, homology modeling, molecular dynamics (MD) simulations in the microsecond (µs) time‐scale and docking calculations were combined to explore the conformational complexity of unphosphorylated/phosphorylated monomeric and trimeric Bcl‐2 systems. Conformational ensembles generated through MD simulations allowed for identifying the most populated unphosphorylated/phosphorylated monomeric conformations, which were used as starting models to obtain trimeric complexes through protein–protein docking calculations, also submitted to µs MD simulations. Principal component analysis showed that FLD represents the main contributor to total Bcl‐2 mobility, and is affected by phosphorylation and oligomerization. Subsequently, based on the most representative unphosphorylated/phosphorylated monomeric and trimeric Bcl‐2 conformations, docking studies were initiated to identify the ligand binding site of several known Bcl‐2 inhibitors to explain their influence in homo‐complex formation and phosphorylation. Docking studies showed that the different conformational states experienced by FLD, such as phosphorylation and oligomerization, play an essential role in the ability to make homo and hetero‐complexes. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 393–413, 2016.     

Modeling of glycerol-3-phosphate transporter suggests a potential 'tilt' mechanism involved in its function

Many major facilitator superfamily (MFS) transporters have similar 12-transmembrane alpha-helical topologies with two six-helix halves connected by a long loop. In humans, these transporters participate in key physiological processes and are also, as in the case of members of the organic anion transporter (OAT) family, of pharmaceutical interest. Recently, crystal structures of two bacterial representatives of the MFS family--the glycerol-3-phosphate transporter (GlpT) and lac-permease (LacY)--have been solved and, because of assumptions regarding the high structural conservation of this family, there is hope that the results can be applied to mammalian transporters as well. Based on crystallography, it has been suggested that a major conformational "switching" mechanism accounts for ligand transport by MFS proteins. This conformational switch would then allow periodic changes in the overall transporter configuration, resulting in its cyclic opening to the periplasm or cytoplasm. Following this lead, we have modeled a possible "switch" mechanism in GlpT, using the concept of rotation of protein domains as in the DynDom program17 and membranephilic constraints predicted by the MAPAS program.(23) We found that the minima of energies of intersubunit interactions support two alternate positions consistent with their transport properties. Thus, for GlpT, a "tilt" of 9 degrees -10 degrees rotation had the most favorable energetics of electrostatic interaction between the two halves of the transporter; moreover, this confirmation was sufficient to suggest transport of the ligand across the membrane. We conducted steered molecular dynamics simulations of the GlpT-ligand system to explore how glycerol-3-phosphate would be handled by the "tilted" structure, and obtained results generally consistent with experimental mutagenesis data. While biochemical data remain most consistent with a single-site alternating access model, our results raise the possibility that, while the "rocker switch" may apply to certain MFS transporters, intermediate "tilted" states may exist under certain circumstances or as transitional structures. Although wet lab experimental confirmation is required, our results suggest that transport mechanisms in this transporter family should probably not be assumed to be conserved simply based on standard structural homology considerations. Furthermore, steered molecular dynamics elucidating energetic interactions of ligands with amino acid residues in an appropriately modeled transporter may have predictive value in understanding the impact of mutations and/or polymorphisms on transporter function.     

Markov state modeling of membrane transport proteins

The flux of ions and molecules in and out of the cell is vital for maintaining the basis of various biological processes. The permeation of substrates across the cellular membrane is mediated through the function of specialized integral membrane proteins commonly known as membrane transporters. These proteins undergo a series of structural rearrangements that allow a primary substrate binding site to be accessed from either side of the membrane at a given time. Structural insights provided by experimentally resolved structures of membrane transporters have aided in the biophysical characterization of these important molecular drug targets. However, characterizing the transitions between conformational states remains challenging to achieve both experimentally and computationally. Though molecular dynamics simulations are a powerful approach to provide atomistic resolution of protein dynamics, a recurring challenge is its ability to efficiently obtain relevant timescales of large conformational transitions as exhibited in transporters. One approach to overcome this difficulty is to adaptively guide the simulation to favor exploration of the conformational landscape, otherwise known as adaptive sampling. Furthermore, such sampling is greatly benefited by the statistical analysis of Markov state models. Historically, the use of Markov state models has been effective in quantifying slow dynamics or long timescale behaviors such as protein folding. Here, we review recent implementations of adaptive sampling and Markov state models to not only address current limitations of molecular dynamics simulations, but to also highlight how Markov state modeling can be applied to investigate the structure–function mechanisms of large, complex membrane transporters.     

构建和评估猪氨基酰化酶I的三维结构

运用同源模建的方法构建了pACY1三维结构模型,并在能量最小化后对模型进行分子动力学模拟和结构合理性评估。同源模建生成了50个原始模型,经过PROCHECK评测后,筛选出模型A、B进行能量最小化,并得到模型A1、B1。分子动力学模拟结果表明模型B1二聚体结构较稳定。PROCHECK、ProSa以及WHATIF检测结果验证了模型B1属于合理性结构。得到的猪氨基酰化酶Ⅰ(pACY1)的三维结构,为研究其结构与功能关系打下基础。     

Eukaryotic major facilitator superfamily transporter modeling based on the prokaryotic GlpT crystal structure

The major facilitator superfamily (MFS) of transporters represents the largest family of secondary active transporters and has a diverse range of substrates. With structural information for four MFS transporters, we can see a strong structural commonality suggesting, as predicted, a common architecture for MFS transporters. The rate for crystal structure determination of MFS transporters is slow, making modeling of both prokaryotic and eukaryotic transporters more enticing. In this review, models of eukaryotic transporters Glut1, G6PT, OCT1, OCT2 and Pho84, based on the crystal structures of the prokaryotic GlpT, based on the crystal structure of LacY are discussed. The techniques used to generate the different models are compared. In addition, the validity of these models and the strategy of using prokaryotic crystal structures to model eukaryotic proteins are discussed. For comparison, E. coli GlpT was modeled based on the E. coli LacY structure and compared to the crystal structure of GlpT demonstrating that experimental evidence is essential for accurate modeling of membrane proteins.     

Study of human Orexin-1 and -2 G-protein-coupled receptors with novel and published antagonists by modeling, molecular dynamics simulations, and site-directed mutagenesis

The class A G-protein-coupled receptors (GPCRs) Orexin-1 (OX1) and Orexin-2 (OX2) are located predominantly in the brain and are linked to a range of different physiological functions, including the control of feeding, energy metabolism, modulation of neuro-endocrine function, and regulation of the sleep-wake cycle. Site-directed mutagenesis (SDM) and domain exchange (chimera) studies have provided important insight into key features of the OX1 and OX2 binding sites. However, the precise determinants of antagonist binding and selectivity are still not fully known. In this work, we used homology modeling of OX receptors to direct further SDM studies. These SDM studies were followed by molecular dynamics (MD) simulations to rationalize the full scope of the SDM data and to explain the role of each mutated residue in the binding and selectivity of a set of OX antagonists: Almorexant (dual OX1 and OX2 antagonist), SB-674042 (OX1 selective antagonist), EMPA (OX2 selective antagonist), and others. Our primary interest was focused on transmembrane helix 3 (TM3), which is identified as being of great importance for the selectivity of OX antagonists. These studies revealed conformational differences between the TM3 helices of OX1 and OX2, resulting from differences in amino acid sequences of the OX receptors that affect key interhelical interactions formed between TM3 and neighboring TM domains. The MD simulation protocol used here, which was followed by flexible docking studies, went beyond the use of static models and allowed for a more detailed exploration of the OX structures. In this work, we have demonstrated how even small differences in the amino acid sequences of GPCRs can lead to significant differences in structure, antagonist binding affinity, and selectivity of these receptors. The MD simulations allowed refinement of the OX receptor models to a degree that was not possible with static homology modeling alone and provided a deeper rationalization of the SDM data obtained. To validate these findings and to demonstrate that they can be usefully applied to the design of novel, very selective OX antagonists, we show here two examples of antagonists designed in house: EP-109-0092 (OX1 selective) and EP-009-0513 (OX2 selective).     

Eukaryotic major facilitator superfamily transporter modeling based on the prokaryotic GlpT crystal structure (Review)

The major facilitator superfamily (MFS) of transporters represents the largest family of secondary active transporters and has a diverse range of substrates. With structural information for four MFS transporters, we can see a strong structural commonality suggesting, as predicted, a common architecture for MFS transporters. The rate for crystal structure determination of MFS transporters is slow, making modeling of both prokaryotic and eukaryotic transporters more enticing. In this review, models of eukaryotic transporters Glut1, G6PT, OCT1, OCT2 and Pho84, based on the crystal structures of the prokaryotic GlpT, based on the crystal structure of LacY are discussed. The techniques used to generate the different models are compared. In addition, the validity of these models and the strategy of using prokaryotic crystal structures to model eukaryotic proteins are discussed. For comparison, E. coli GlpT was modeled based on the E. coli LacY structure and compared to the crystal structure of GlpT demonstrating that experimental evidence is essential for accurate modeling of membrane proteins.     

Kinetic and mutational analysis of the Trypanosoma brucei NBT1 nucleobase transporter expressed in Saccharomyces cerevisiae reveals structural similarities between ENT and MFS transporters

Parasitic protozoa are unable to synthesise purines de novo and thus depend on the uptake of nucleosides and nucleobases across their plasma membrane through specific transporters. A number of nucleoside and nucleobase transporters from Trypanosoma brucei brucei and Leishmania major have recently been characterised and shown to belong to the equilibrative nucleoside transporter (ENT) family. A number of studies have demonstrated the functional importance of particular transmembrane segments (TMS) in nucleoside-specific ENT proteins. TbNBT1, one of only three bona fide nucleobase-selective members of the ENT family, has previously been shown to be a high-affinity transporter for purine nucleobases and guanosine. In this study, we use the Saccharomyces cerevisiae expression system to build a biochemical model of how TbNBT1 recognises nucleobases. We next performed random in vitro and site-directed mutagenesis to identify residues critical for TbNBT1 function. The identification of residues likely to contribute to permeant binding, when combined with a structural model of TbNBT1 obtained by homology threading, yield a tentative three-dimensional model of the transporter binding site that is consistent with the binding model emerging from the biochemical data. The model strongly suggests the involvement of TMS5, TMS7 and TMS8 in TbNBT1 function. This situation is very similar to that concerning transporters of the major facilitator superfamily (MFS), one of which was used as a template for the threading. This point raises the possibility that ENT and MFS carriers, despite being considered evolutionarily distinct, might in fact share similar topologies and substrate translocations pathways.     

Molecular modelling of human 5-hydroxytryptamine receptor (5-HT2A) and virtual screening studies towards the identification of agonist and antagonist molecules

The serotonin receptors, also known as 5-hydroxytryptamine (5-HT) receptors, are a group of G protein-coupled receptors (GPCRs) and ligand-gated ion channels found in the central and peripheral nervous systems. GPCRs have a characteristic feature of activating different signalling pathways upon ligand binding and these ligands display several efficacy levels to differentially activate the receptor. GPCRs are primary drug targets due to their central role in several signal transduction pathways. Drug design for GPCRs is also most challenging due to their inherent promiscuity in ligand recognition, which gives rise to several side effects of existing drugs. Here, we have performed the ligand interaction study using the two prominent states of GPCR, namely the active and inactive state of the 5-HT_2A receptor. Active state of 5-HT_2A receptor model enhances the understanding of conformational difference which influences the ligand-binding site. A 5-HT_2A receptor active state model was constructed by homology modelling using active state β₂-adrenergic receptor (β₂-AR). In addition, virtual screening and docking studies with both active and inactive state models reveal potential small molecule hits which could be considered as agonist-like and antagonist-like molecules. The results from the all-atom molecular dynamics simulations further confirmed that agonists and antagonists interact in different modes with the receptor.     

The role of local hydration and hydrogen-bonding dynamics in ion and solute release from ion-coupled secondary transporters

Recent progress in crystallographic studies of sodium-coupled secondary transporters has revealed striking similarities in the structural organization of ion and solute binding. Previous reports suggested that the Na2 sodium binding site in the neurotransmitter sodium symporter (NSS) leucine transporter (LeuT) is conserved across sodium/proton coupled secondary transporters of many distantly related families. This site is implicated in the conformational dynamics controlled by the binding and release of both translocated solute and ion(s) through a mechanism that largely remains unknown. In this study, we used extensive equilibrium molecular dynamics simulations, potential of mean force (PMF) computations, and quasi-harmonic analysis of the LeuT transporter with and without sodium ion bound at the Na2 site to delineate the role of this site in the conformational dynamics of the protein. PMF computations show that in presence of the sodium ion in Na2 the conserved T354 residue is locked into a single rotameric state in contrast to two degenerate states available in the absence of ion in Na2. Molecular dynamics (MD) simulations suggest the formation of a stable water wire from the cytoplasm to the Na2 site in the occluded state. It is plausible that local hydration plays an important role in transport cycle facilitating release of the ion from Na2. An unbinding of the ion from the Na2 site leads to a tightening of the extracellular thin gates and a destabilization of the intracellular thin gate and thus may promote an unbinding of the cotransported substrate. The study lends additional support to the hypothesis that one of the main drivers in the transport cycle of Na-coupled secondary transporters is the binding of the Na2 ion that controls dynamical equilibrium between an inward-facing to an outward-facing conformation.