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The osmoregulated betaine transporter BetP is a stable trimer. Structural studies have shown that individual protomers can adopt distinct transport conformations, implying a functional role for the trimeric state in transport, although the role of trimerization in regulation is not yet understood. We designed putative monomeric mutants by molecular-dynamics simulations and in silico alanine-scanning mutagenesis. Several mutants including BetP-W101A/T351A were monomeric in detergent as well as in the membrane, as shown by blue native gel electrophoresis, crosslinking and electron microscopy. This monomeric form retains the ability to accumulate betaine, but is no longer regulated by hyperosmotic shock.

Lactose permease (LacY) is the prototype of the major facilitator superfamily (MFS) of secondary transporters. Available structures of LacY reveal a state in which the substrate is exposed to the cytoplasm but is occluded from the periplasm. However, the alternating-access transport mechanism requires the existence of a periplasm-facing state. We recently showed that inverted-topology structural repeats provide the foundation for the mechanisms of two transporter families with folds distinct from the MFS. Here, we generated a structural model of LacY by swapping the conformations of inverted-topology repeats identified in its two domains. The model exhibits all required properties of an outward-facing conformation, i.e., closure of the binding site to the cytoplasm and exposure to the periplasm. Furthermore, the model agrees with double electron-electron resonance distance changes, accessibility to cysteine-modifying reagents, cysteine cross-linking data, and a recent structure of a distantly related transporter. Analysis of the intradomain differences between the two states suggests a role for conserved sequence motifs in occluding the central pathway through kinking of the pore-lining helices. In addition, predicted re-pairing of critical salt-bridging residues in the binding sites agrees remarkably well with previous proposals, allowing a description of the proton/sugar transport mechanism. More fundamentally, our model demonstrates that inverted-topology repeats provide the foundation for the alternating-access mechanisms of MFS transporters., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

As new atomic structures of membrane proteins are resolved, they reveal increasingly complex transmembrane topologies, and highly irregular surfaces with crevices and pores. In many cases, specific interactions formed with the lipid membrane are functionally crucial, as is the overall lipid composition. Compounded with increasing protein size, these characteristics pose a challenge for the construction of simulation models of membrane proteins in lipid environments; clearly, that these models are sufficiently realistic bears upon the reliability of simulation-based studies of these systems. Here, we introduce GRIFFIN, which uses a versatile framework to automate and improve a widely-used membrane-embedding protocol. Initially, GRIFFIN carves out lipid and water molecules from a volume equivalent to that of the protein, so as to conserve the system density. In the subsequent optimization phase GRIFFIN adds an implicit grid-based protein force-field to a molecular dynamics simulation of the pre-carved membrane. In this force-field, atoms inside the implicit protein volume experience an outward force that will expel them from that volume, whereas those outside are subject to electrostatic and van-der-Waals interactions with the implicit protein. At each step of the simulation, these forces are updated by GRIFFIN and combined with the intermolecular forces of the explicit lipid-water system. This procedure enables the construction of realistic and reproducible starting configurations of the protein-membrane interface within a reasonable timeframe and with minimal intervention. GRIFFIN is a standalone tool designed to work alongside any existing molecular dynamics package, such as NAMD or GROMACS.

The molecular mechanisms of membrane proteins that are activated either by ions or by ATP are just beginning to come into focus, as long-awaited structural data are revealed. This information is being leveraged and supplemented to great effect by molecular modeling and computer simulation studies. Important examples include the homology modeling of eukaryotic protein structures based on distantly related templates, as well as the use of internal structural symmetry for modeling different states in conformational cycles. Molecular simulation studies have elucidated the location and coordination structure of ion binding sites, and explained their selectivity, while also providing tantalizing insights into the mechanisms that couple conformational change to ion translocation or ATP hydrolysis., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Secondary active transporters couple the free energy of the electrochemical potential of one solute to the transmembrane movement of another. As a basic mechanistic explanation for their transport function the model of alternating access was put forward more than 40 years ago, and has been supported by numerous kinetic, biochemical and biophysical studies. According to this model, the transporter exposes its substrate binding site(s) to one side of the membrane or the other during transport catalysis, requiring a substantial conformational change of the carrier protein. In the light of recent structural data for a number of secondary transport proteins, we analyze the model of alternating access in more detail, and correlate it with specific structural and chemical properties of the transporters, such as their assignment to different functional states in the catalytic cycle of the respective transporter, the definition of substrate binding sites, the type of movement of the central part of the carrier harboring the substrate binding site, as well as the impact of symmetry on fold-specific conformational changes. Besides mediating the transmembrane movement of solutes, the mechanism of secondary carriers inherently involves a mechanistic coupling of substrate flux to the electrochemical potential of co-substrate ions or solutes. Mainly because of limitations in resolution of available transporter structures, this important aspect of secondary transport cannot yet be substantiated by structural data to the same extent as the conformational change aspect. We summarize the concepts of coupling in secondary transport and discuss them in the context of the available evidence for ion binding to specific sites and the impact of the ions on the conformational state of the carrier protein, which together lead to mechanistic models for coupling., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Crystal structures of LeuT, a bacterial homologue of mammalian neurotransmitter transporters, show a molecule of bound substrate that is essentially exposed to the extracellular space but occluded from the cytoplasm. Thus, there must exist an alternate conformation for LeuT in which the substrate is accessible to the cytoplasm and a corresponding mechanism that switches accessibility from one side of the membrane to the other. Here, we identify the cytoplasmic accessibility pathway of the alternate conformation in a mammalian serotonin transporter (SERT) (a member of the same transporter family as LeuT). We also propose a model for the cytoplasmic-facing state that exploits the internal pseudosymmetry observed in the crystal structure. LeuT contains two structurally similar repeats (TMs1-5 and TMs 6-10) that are inverted with respect to the plane of the membrane. The conformational differences between them result in the formation of the extracellular pathway. Our model for the cytoplasm-facing state exchanges the conformations of the two repeats and thus exposes the substrate and ion-binding sites to the cytoplasm. The conformational change that connects the two states primarily involves the tilting of a 4-helix bundle composed of transmembrane helices 1, 2, 6, and 7. Switching the tilt angle of this bundle is essentially equivalent to switching the conformation of the two repeats. Extensive mutagenesis of SERT and accessibility measurements, using cysteine reagents, are accommodated by our model. These observations may be of relevance to other transporter families, many of which contain internal inverted repeats.

The recent determination of the crystal structure of the leucine transporter from Aquifex aeolicus (aaLeuT) has provided significant insights into the function of neurotransmitter:sodium symporters. Transport by aaLeuT is Cl(-) independent, whereas many neurotransmitter:sodium symporters from higher organisms depend on Cl(-) ions. However, the only Cl(-) ion identified in the aaLeuT structure interacts with nonconserved residues in extracellular loops, and thus the relevance of this binding site is unclear. Here, we use calculations of pK(A)s and homology modeling to predict the location of a functionally important Cl(-) binding site in serotonin transporter and other Cl(-)-dependent transporters. We validate our model through the site-directed mutagenesis of residues predicted to coordinate the Cl(-) ion and through the observation of sequence conservation patterns in other Cl(-)-dependent transporters. The proposed site is located midway across the membrane and is formed by residues from transmembrane helices 2, 6, and 7. It is close to the Na1 sodium binding site, thus providing an explanation for the coupling of Cl(-) and Na(+) ions during transport. Other implications of the model are also discussed.

We survey computational approaches that tackle membrane protein structure and function prediction. While describing the main ideas that have led to the development of the most relevant and novel methods, we also discuss pitfalls, provide practical hints and highlight the challenges that remain. The methods covered include: sequence alignment, motif search, functional residue identification, transmembrane segment and protein topology predictions, homology and ab initio modeling. In general, predictions of functional and structural features of membrane proteins are improving, although progress is hampered by the limited amount of high-resolution experimental information available. While predictions of transmembrane segments and protein topology rank among the most accurate methods in computational biology, more attention and effort will be required in the future to ameliorate database search, homology and ab initio modeling.