The MemProtMD database : a resource for membrane-embedded protein structures and their lipid interactions

Integral membrane proteins fulfil important roles in many crucial biological processes, including cell signalling, molecular transport and bioenergetic processes. Advancements in experimental techniques are revealing high resolution structures for an increasing number of membrane proteins. Yet, these structures are rarely resolved in complex with membrane lipids. In 2015, the MemProtMD pipeline was developed to allow the automated lipid bilayer assembly around new membrane protein structures, released from the Protein Data Bank (PDB). To make these data available to the scientific community, a web database (http://memprotmd.bioch.ox.ac.uk) has been developed. Simulations and the results of subsequent analysis can be viewed using a web browser, including interactive 3D visualizations of the assembled bilayer and 2D visualizations of lipid contact data and membrane protein topology. In addition, ensemble analyses are performed to detail conserved lipid interaction information across proteins, families and for the entire database of 3506 PDB entries. Proteins may be searched using keywords, PDB or Uniprot identifier, or browsed using classification systems, such as Pfam, Gene Ontology annotation, mpstruc or the Transporter Classification Database. All files required to run further molecular simulations of proteins in the database are provided

The energetics of protein-lipid interactions as viewed by molecular simulations

Membranes are formed from a bilayer containing diverse lipid species with which membrane proteins interact. Thus, integral membrane proteins are embedded in a bilayer, where they interact with lipids from their surroundings, whilst peripheral membrane proteins bind to lipids at the surface of membranes. Lipid interactions can influence the function of membrane proteins, either directly or allosterically. Both experimental (structural) and computational approaches can reveal lipid binding sites on membrane proteins. It is therefore important to understand the free energies of these interactions. This affords a more complete view of the engagement of a particular protein with the biological membrane surrounding it. Here, we describe a number of computational approaches currently in use for this purpose, including recent advances using both free energy and unbiased simulation methods. In particular we focus on interactions of integral membrane proteins with cholesterol, and with anionic lipids such as phosphatidylinositol 4,5-bisphosphate and cardiolipin. Peripheral membrane proteins are exemplified via interactions of PH domains with phosphoinositide-containing membranes. We summarise the current state of the field and provide an outlook on likely future directions of investigation

CG2AT2 : an enhanced fragment-based approach for serial multi-scale molecular dynamics simulations

Coarse-grained molecular dynamics provides a means for simulating the assembly and interactions of macromolecular complexes at a reduced level of representation, thereby allowing both longer timescale and larger sized simulations. Here, we describe an enhanced fragment-based protocol for converting macromolecular complexes from coarse-grained to atomistic resolution, for further refinement and analysis. While the focus is upon systems that comprise an integral membrane protein embedded in a phospholipid bilayer, the technique is also suitable for membrane-anchored and soluble protein/nucleotide complexes. Overall, this provides a method for generating an accurate and well-equilibrated atomic-level description of a macromolecular complex. The approach is evaluated using a diverse test set of 11 system configurations of varying size and complexity. Simulations are assessed in terms of protein stereochemistry, conformational drift, lipid/protein interactions, and lipid dynamics

Structural basis for substrate specificity and regulation of nucleotide sugar transporters in the lipid bilayer

Nucleotide sugars are the activated form of monosaccharides used by glycosyltransferases during glycosylation. In eukaryotes the SLC35 family of solute carriers are responsible for their selective uptake into the Endoplasmic Reticulum or Golgi apparatus. The structure of the yeast GDP-mannose transporter, Vrg4, revealed a requirement for short chain lipids and a marked difference in transport rate between the nucleotide sugar and nucleoside monophosphate, suggesting a complex network of regulatory elements control transport into these organelles. Here we report the crystal structure of the GMP bound complex of Vrg4, revealing the molecular basis for GMP recognition and transport. Molecular dynamics, combined with biochemical analysis, reveal a lipid mediated dimer interface and mechanism for coordinating structural rearrangements during transport. Together these results provide further insight into how SLC35 family transporters function within the secretory pathway and sheds light onto the role that membrane lipids play in regulating transport across the membrane

From bench to biomolecular simulation : phospholipid modulation of potassium channels

Potassium (K+) ion channels are crucial in numerous cellular processes as they hyperpolarise a cell through K+ conductance, returning a cell to its resting potential. K+ channel mutations result in multiple clinical complications such as arrhythmia, neonatal diabetes and migraines. Since 1995, the regulation of K+ channels by phospholipids has been heavily studied using a range of interdisciplinary methods such as cellular electrophysiology, structural biology and computational modelling. As a result, K+ channels are model proteins for the analysis of protein-lipid interactions. In this review, we will focus on the roles of lipids in the regulation of K+ channels, and how atomic-level structures, along with experimental techniques and molecular simulations, have helped guide our understanding of the importance of phospholipid interactions

Atomistic mechanism of transmembrane helix association

Transmembrane helix association is a fundamental step in the folding of helical membrane proteins. The prototypical example of this association is formation of the glycophorin dimer. While its structure and stability have been well-characterized experimentally, the detailed assembly mechanism is harder to obtain. Here, we use all-atom simulations within phospholipid membrane to study glycophorin association. We find that initial association results in the formation of a non-native intermediate, separated by a significant free energy barrier from the dimer with a native binding interface. We have used transition-path sampling to determine the association mechanism. We find that the mechanism of the initial bimolecular association to form the intermediate state can be mediated by many possible contacts, but seems to be particularly favoured by formation of non-native contacts between the C-termini of the two helices. On the other hand, the contacts which are key to determining progression from the intermediate to the native state are those which define the native binding interface, reminiscent of the role played by native contacts in determining folding of globular proteins. As a check on the simulations, we have computed association and dissociation rates from the transition-path sampling. We obtain results in reasonable accord with available experimental data, after correcting for differences in native state stability. Our results yield an atomistic description of the mechanism for a simple prototype of helical membrane protein folding

Substrate-triggered position switching of TatA and TatB during Tat transport in <i>Escherichia coli</i>

The twin-arginine protein transport (Tat) machinery mediates the translocation of folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. The Escherichia coli Tat system comprises TatC and two additional sequence-related proteins, TatA and TatB. The active translocase is assembled on demand, with substrate-binding at a TatABC receptor complex triggering recruitment and assembly of multiple additional copies of TatA; however, the molecular interactions mediating translocase assembly are poorly understood. A 'polar cluster' site on TatC transmembrane (TM) helix 5 was previously identified as binding to TatB. Here, we use disulfide cross-linking and molecular modelling to identify a new binding site on TatC TM helix 6, adjacent to the polar cluster site. We demonstrate that TatA and TatB each have the capacity to bind at both TatC sites, however in vivo this is regulated according to the activation state of the complex. In the resting-state system, TatB binds the polar cluster site, with TatA occupying the TM helix 6 site. However when the system is activated by overproduction of a substrate, TatA and TatB switch binding sites. We propose that this substrate-triggered positional exchange is a key step in the assembly of an active Tat translocase

Insights into membrane protein–lipid interactions from free energy calculations

Integral membrane proteins are regulated by specific interactions with lipids from the surrounding bilayer. The structures of protein–lipid complexes can be determined through a combination of experimental and computational approaches, but the energetic basis of these interactions is difficult to resolve. Molecular dynamics simulations provide the primary computational technique to estimate the free energies of these interactions. We demonstrate that the energetics of protein–lipid interactions may be reliably and reproducibly calculated using three simulation-based approaches: potential of mean force calculations, alchemical free energy perturbation, and well-tempered metadynamics. We employ these techniques within the framework of a coarse-grained force field and apply them to both bacterial and mammalian membrane protein–lipid systems. We demonstrate good agreement between the different techniques, providing a robust framework for their automated implementation within a pipeline for annotation of newly determined membrane protein structures

Evaluating inositol phospholipid interactions with inward rectifier potassium channels and characterising their role in disease

Membrane proteins are frequently modulated by specific protein-lipid interactions. The activation of human inward rectifying potassium (hKir) channels by phosphoinositides (PI) has been well characterised. Here, we apply a coarse-grained molecular dynamics free energy perturbation (CG-FEP) protocol to capture the energetics of binding of PI lipids to hKir channels. By using either a single- or multi-step approach, we establish a consistent value for the binding of PIP2 to hKir channels, relative to the binding of the bulk phosphatidylcholine phospholipid. Furthermore, by perturbing amino acid side chains on hKir6.2, we show that the neonatal diabetes mutation E179K increases PIP2 affinity, while the congenital hyperinsulinism mutation K67N results in a reduced affinity. We show good agreement with electrophysiological data where E179K exhibits a reduction in neomycin sensitivity, implying that PIP2 binds more tightly E179K channels. This illustrates the application of CG-FEP to compare affinities between lipid species, and for annotating amino acid residues