Docking and Homology Modeling Explain Inhibition of the Human Vesicular Glutamate Transporters
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Downloaded from www.proteinscience.org on September 10, 2007 Docking and homology modeling explain inhibition of the human vesicular glutamate transporters Jonas Almqvist, Yafei Huang, Aatto Laaksonen, Da-Neng Wang and Sven Hovmöller Protein Sci. 2007 16: 1819-1829; originally published online Jul 27, 2007; Access the most recent version at doi:10.1110/ps.072944707 References This article cites 54 articles, 17 of which can be accessed free at: http://www.proteinscience.org/cgi/content/full/16/9/1819#References Email alerting Receive free email alerts when new articles cite this article - sign up in the box at the service top right corner of the article or click here Notes To subscribe to Protein Science go to: http://www.proteinscience.org/subscriptions/ © 2007 Cold Spring Harbor Laboratory Press Downloaded from www.proteinscience.org on September 10, 2007 Docking and homology modeling explain inhibition of the human vesicular glutamate transporters 1 1 2 3 JONAS ALMQVIST, YAFEI HUANG, AATTO LAAKSONEN, DA-NENG WANG, AND SVEN HOVMO¨ LLER1 1Division of Structural Chemistry, Arrhenius Laboratory, Stockholm University, S-10691 Stockholm, Sweden 2Division of Physical Chemistry, Arrhenius Laboratory, Stockholm University, S-10691 Stockholm, Sweden 3Kimmel Center for Biology and Medicine of the Skirball Institute, and Department of Cell Biology, New York University School of Medicine, New York, New York 10016, USA (RECEIVED April 12, 2007; FINAL REVISION May 23, 2007; ACCEPTED May 28, 2007) Abstract As membrane transporter proteins, VGLUT1–3 mediate the uptake of glutamate into synaptic vesicles at presynaptic nerve terminals of excitatory neural cells. This function is crucial for exocytosis and the role of glutamate as the major excitatory neurotransmitter in the central nervous system. The three transporters, sharing 76% amino acid sequence identity in humans, are highly homologous but differ in regional expression in the brain. Although little is known regarding their three-dimensional structures, hydropathy analysis on these proteins predicts 12 transmembrane segments connected by loops, a topology similar to other members in the major facilitator superfamily, where VGLUT1–3 have been phylogenetically classified. In this work, we present a three-dimensional model for the human VGLUT1 protein based on its distant bacterial homolog in the same superfamily, the glycerol-3- phosphate transporter from Escherichia coli. This structural model, stable during molecular dynamics simulations in phospholipid bilayers solvated by water, reveals amino acid residues that face its pore and are likely to affect substrate translocation. Docking of VGLUT1 substrates to this pore localizes two different binding sites, to which inhibitors also bind with an overall trend in binding affinity that is in agreement with previously published experimental data. Keywords: vesicular glutamate transporter; homology modeling; membrane protein structure; inhibitor; docking; molecular dynamics The amino acid glutamate is used by the mammalian as its central component (Takamori 2006). Unlike plasma central nervous system as the major excitatory neuro- membrane glutamate transporters, which work with high transmitter. Following its exocytotic release into the affinity to terminate glutamate signaling by clearing the synaptic cleft, glutamate binds post-synaptic receptors, neurotransmitter molecules present in the synaptic cleft, ligand-gated ion channels that depolarize the post-syn- the vesicular transporters transport glutamate with an aptic neuron. However, for this to happen, a presynaptic affinity that is 100–1000-fold lower (Shigeri et al. transport system is needed to upload the neurotransmitter 2004), but against much higher concentration gradients into small vesicles at the axon terminals. This uploading (Otis 2001). A total of three VGLUT subtypes have been system uses vesicular glutamate transporters (VGLUTs) identified in mammals: VGLUT1–3 (Aihara et al. 2000; Fremeau et al. 2002; Gras et al. 2002). These proteins are highly homologous in the amino acid sequence, but Reprint requests to: Jonas Almqvist, Structural Chemistry, Arrhe- display markedly different and complementary regional nius Laboratory, Stockholm University, S-10691 Stockholm, Sweden; expression patterns (Fremeau et al. 2004). e-mail: [email protected]; fax: 46-8-163118. Article published online ahead of print. Article and publication date The human VGLUT1 protein comprises 560 amino are at http://www.proteinscience.org/cgi/doi/10.1110/ps.072944707. acids. Due to its significant sequence homology (up to Protein Science (2007), 16:1819–1829. Published by Cold Spring Harbor Laboratory Press. Copyright Ó 2007 The Protein Society 1819 Downloaded from www.proteinscience.org on September 10, 2007 Almqvist et al. 30% identity) to mammalian Na+-dependent inorganic play significant lower homology. In the first 60 residues, phosphate cotransporters, together with experiments although the degree of homology is low, there is some + demonstrating elevated levels of Na -dependent Pi uptake sequence conservation between all three proteins. The afterVGLUT1mRNAinjectionintofrogoocytes, amino acid composition is indicative of water-soluble VGLUT1 was originally identified as a brain-specific parts rather than membrane spanning, with roughly 50% inorganic phosphate cotransporter (Ni et al. 1994). How- of the residues being charged or polar. The overall charge, ever, more recent observations have established vesicular however, is close to neutral. In contrast, the last 60 C- glutamate transport as its predominant role in vivo terminal residues are the least conserved in the entire (Bellocchio et al. 2000; Takamori et al. 2000). The sequences, and heavily enriched in aspartate and gluta- functional difference between plasma membrane and mate, resulting in a net charge of À7to–9forthisregion. vesicular glutamate transporters is also reflected in the Given the high number of negative charges, this part of phylogenetic separation between the two groups of thesequenceisalsounlikelytospanthevesiclemem- proteins. The vesicular glutamate transporters have been brane. Furthermore, for VGLUT1, this C-terminal region classified as members of the anion-cation subfamily contains a very high number of proline residues (13 out (ACS) of the major facilitator superfamily (MFS) (Pao of 43, or 30%). Outside a phospholipid membrane, the et al. 1998), the largest family of secondary transporter presence of tri- and tetra-proline peptides precludes proteins known today. With over 5000 members, the MFS secondary structure elements such as a-helices or b- proteins are represented in all kingdoms of life. They sheets (Lise and Jones 2005), and suggests a flexible facilitate the transport across the membrane of a variety structure in this region. This is also confirmed in two of hydrophilic solutes, such as ions, sugars, nucleosides, recent studies (De Gois et al. 2006; Vinatier et al. 2006), amino acids, small peptides, drugs, and neurotransmitters. which identifies protein–protein interactions between Most MFS transporters are believed to feature 12 trans- two isoforms of endophilins to a proline-rich motif membrane helices (Abramson et al. 2003b; Lemieux et al. (PPRPPPP) in the C-terminal region of VGLUT1. Finally, 2004), and their sequences display a weak internal besides these two peripheral N- and C-terminal regions, homology between the N- and C-terminal halves of the another predominantly hydrophilic section is present in protein, which are structurally related through a twofold the center of the sequences. Situated in the middle of an psuedo-symmetry in the MFS structures (Abramson et al. overall hydrophobic milieu, a region of 40 amino acids 2003a; Huang et al. 2003; Yin et al. 2006a). (starting from S256 in VGLUT1) probably constitutes a While structure determination through overexpression, loop connecting two transmembrane domains. Thus, three purification, and crystallization of mammalian membrane large regions of the sequences appear as extramembrane. proteins is notoriously challenging, inferring structural With the alignment parsed in this way, the transmembrane information from bacterial homologs of mammalian MFS segments and loops connecting them should be confined transporters has been proven to be very successful to the remaining two parts of the VGLUT sequences, (Almqvist et al. 2004; Vardy et al. 2004). Recently, a which together consist of 468 residues with 79% mutagenesis study on human VGLUT2 identified residues sequence identity between the three VGLUT isoforms, essential for full transport activity of L-glutamate (Juge in sharp contrast to the 17% for the N-terminal section, et al. 2006), and the position of these residues in the the 2% for the C-terminal section, and the 53% for the backbone structure of a homology model was shown. middle section. The alignment between VGLUT1 and ThismodelwasbasedontheX-raycrystalstructureof GlpT (Fig. 1) was then used to build our three-dimen- a distant bacterial homolog—the glycerol-3-phosphate sional model of the transmembrane region of VGLUT1. transporter GlpT from Escherichia coli. To gain further For clarity we have chosen to limit the modeling to insight into the structure and mechanism of the VGLUTs, VGLUT1. However, because of the high sequence iden- we present a three-dimensional model of VGLUT1 tity between the three vesicular glutamate transporters in validated through molecular dynamics (MD) simulations, the modeled region, results should be representative