Alan Chetwynd

Coarse-grained MD simulations of membrane protein-bilayer self-assembly

Complete determination of a membrane protein structure requires knowledge of the protein position within the lipid bilayer. As the number of determined structures of membrane proteins increases so does the need for computational methods which predict their position in the lipid bilayer. Here we present a coarse-grained molecular dynamics approach to lipid bilayer self-assembly around membrane proteins. We demonstrate that this method can be used to predict accurately the protein position in the bilayer for membrane proteins with a range of different sizes and architectures.

Exploring Peptide-Membrane Interactions with Coarse-Grained MD Simulations

The interaction of α-helical peptides with lipid bilayers is central to our understanding of the physicochemical principles of biological membrane organization and stability. Mutations that alter the position or orientation of an α-helix within a membrane, or that change the probability that the α-helix will insert into the membrane, can alter a range of membrane protein functions. We describe a comparative coarse-grained molecular dynamics simulation methodology, based on self-assembly of a lipid bilayer in the presence of an α-helical peptide, which allows us to model membrane transmembrane helix insertion. We validate this methodology against available experimental data for synthetic model peptides (WALP23 and LS3). Simulation-based estimates of apparent free energies of insertion into a bilayer of cystic fibrosis transmembrane regulator-derived helices correlate well with published data for translocon-mediated insertion. Comparison of values of the apparent free energy of insertion from self-assembly simulations with those from coarse-grained molecular dynamics potentials of mean force for model peptides, and with translocon-mediated insertion of cystic fibrosis transmembrane regulator-derived peptides suggests a nonequilibrium model of helix insertion into bilayers.

CGDB: A database of membrane protein/lipid interactions by coarse-grained molecular dynamics simulations

Membrane protein function and stability has been shown to be dependent on the lipid environment. Recently, we developed a high-throughput computational approach for the prediction of membrane protein/lipid interactions. In the current study, we enhanced this approach with the addition of a new measure of the distortion caused by membrane proteins on a lipid bilayer. This is illustrated by considering the effect of lipid tail length and headgroup charge on the distortion caused by the integral membrane proteins MscS and FLAP, and by the voltage sensing domain from the channel KvAP. Changing the chain length of lipids alters the extent but not the pattern of distortion caused by MscS and FLAP; lipid headgroups distort in order to interact with very similar but not identical regions in these proteins for all bilayer widths investigated. Introducing anionic lipids into a DPPC bilayer containing the KvAP voltage sensor does not affect the extent of bilayer distortion.

Dimerization of the EphA1 Receptor Tyrosine Kinase Transmembrane Domain: Insights into the Mechanism of Receptor Activation

EphA1 is a receptor tyrosine kinase (RTK) that plays a key role in developmental processes, including guidance of the migration of axons and cells in the nervous system. EphA1, in common with other RTKs, contains an N-terminal extracellular domain, a single transmembrane (TM) α-helix, and a C-terminal intracellular kinase domain. The TM helix forms a dimer, as seen in recent NMR studies. We have modeled the EphA1 TM dimer using a multiscale approach combining coarse-grain (CG) and atomistic molecular dynamics (MD) simulations. The one-dimensional potential of mean force (PMF) for this system, based on interhelix separation, has been calculated using CG MD simulations. This provides a view of the free energy landscape for helix–helix interactions of the TM dimer in a lipid bilayer. The resulting PMF profiles suggest two states, consistent with a rotation-coupled activation mechanism. The more stable state corresponds to a right-handed helix dimer interacting via an N-terminal glycine zipper motif, consistent with a recent NMR structure (2K1K). A second metastable state corresponds to a structure in which the glycine zipper motif is not involved. Analysis of unrestrained CG MD simulations based on representative models from the PMF calculations or on the NMR structure reveals possible pathways of interconversion between these two states, involving helix rotations about their long axes. This suggests that the interaction of TM helices in EphA1 dimers may be intrinsically dynamic. This provides a potential mechanism for signaling whereby extracellular events drive a shift in the repopulation of the underlying TM helix dimer energy landscape.

The Energetics of Transmembrane Helix Insertion into a Lipid Bilayer

Free energy profiles for insertion of a hydrophobic transmembrane protein α-helix (M2 from CFTR) into a lipid bilayer have been calculated using coarse-grained molecular dynamics simulations and umbrella sampling to yield potentials of mean force along a reaction path corresponding to translation of a helix across a lipid bilayer. The calculated free energy of insertion is smaller when a bilayer with a thinner hydrophobic region is used. The free energies of insertion from the potentials of mean force are compared with those derived from a number of hydrophobicity scales and with those derived from translocon-mediated insertion. This comparison supports recent models of translocon-mediated insertion and in particular suggests that: 1), helices in an about-to-be-inserted state may be located in a hydrophobic region somewhat thinner than the core of a lipid bilayer; and/or 2), helices in a not-to-be-inserted state may experience an environment more akin (e.g., in polarity/hydrophobicity) to the bilayer/water interface than to bulk water.

Membrane Insertion of a Voltage Sensor Helix

Most membrane proteins contain a transmembrane (TM) domain made up of a bundle of lipid-bilayer-spanning α-helices. TM α-helices are generally composed of a core of largely hydrophobic amino acids, with basic and aromatic amino acids at each end of the helix forming interactions with the lipid headgroups and water. In contrast, the S4 helix of ion channel voltage sensor (VS) domains contains four or five basic (largely arginine) side chains along its length and yet adopts a TM orientation as part of an independently stable VS domain. Multiscale molecular dynamics simulations are used to explore how a charged TM S4 α-helix may be stabilized in a lipid bilayer, which is of relevance in the context of mechanisms of translocon-mediated insertion of S4. Free-energy profiles for insertion of the S4 helix into a phospholipid bilayer suggest that it is thermodynamically favorable for S4 to insert from water to the center of the membrane, where the helix adopts a TM orientation. This is consistent with crystal structures of Kv channels, biophysical studies of isolated VS domains in lipid bilayers, and studies of translocon-mediated S4 helix insertion. Decomposition of the free-energy profiles reveals the underlying physical basis for TM stability, whereby the preference of the hydrophobic residues of S4 to enter the bilayer dominates over the free-energy penalty for inserting charged residues, accompanied by local distortion of the bilayer and penetration of waters. We show that the unique combination of charged and hydrophobic residues in S4 allows it to insert stably into the membrane.

Primary and Secondary Dimer Interfaces of the Fibroblast Growth Factor Receptor 3 Transmembrane Domain: Characterization via Multiscale Molecular Dynamics Simulations

Receptor tyrosine kinases are single-pass membrane proteins that form dimers within the membrane. The interactions of their transmembrane domains (TMDs) play a key role in dimerization and signaling. Fibroblast growth factor receptor 3 (FGFR3) is of interest as a G380R mutation in its TMD is the underlying cause of ~99% of the cases of achondroplasia, the most common form of human dwarfism. The structural consequences of this mutation remain uncertain: the mutation shifts the position of the TMD relative to the lipid bilayer but does not alter the association free energy. We have combined coarse-grained and all-atom molecular dynamics simulations to study the dimerization of wild-type, heterodimer, and mutant FGFR3 TMDs. The simulations reveal that the helices pack together in the dimer to form a flexible interface. The primary packing mode is mediated by a Gx3G motif. There is also a secondary dimer interface that is more highly populated in heterodimer and mutant configurations that may feature in the molecular mechanism of pathology. Both coarse-grained and atomistic simulations reveal a significant shift of the G380R mutant dimer TMD relative to the bilayer to allow interactions of the arginine side chain with lipid headgroup phosphates.

Insertion Properties of Cftr Explored with High Throughput, Coarse Grain Molecular Dynamics

Transmembrane helix insertion into the membrane occurs through a complex process, involving dedicated cellular machinery. Recent experimental work has been able to show that the insertion of peptides by the translocon shows high correlation with hydrophobic scales based on water/octanol partitioning, but that the absolute energies of insertion of different amino acids are consistently different by an order of magnitude. Similarly, energies of transmembrane insertion from explicit energy calculations on detailed molecular model also appear to differ, by up to 2 orders of magnitude. Coarse grain (CG) techniques are an increasingly popular approach for the molecular modelling of biomolecules, which increase the effective timescale or system size which can be modelled compared to more common atomistic techniques. We adopt a high throughput, CG approach to understanding helix insertion into the membrane. Using self assembly of systems of peptides derived from the cystic fibrosis protein, we are able to predict transmembrane insertion energies with a correlation coefficient of up to 0.86, and energies within a factor of 2 of the experimentally determined energies. Additionally, we show that the insertion behaviour observed is sensitive to membrane thickness, and in agreement with explicit energy calculations and experimental evidence, find that thinner membrane bilayers favour a transmembrane conformation. Alongside results from PMF calculations, the results here appear to suggest that the energy differences measured in the translocon experiments represent the differences in energy between the interfacial and the transmembrane conformations for a helix.

Molecular Dynamics Simulations of the Transmembrane Helix of the FGFR3 Receptor in POPC and DPPC

Fibroblast growth factor receptor 3 (FGFR3) is a receptor tyrosine kinase that negatively regulates bone growth. Elevated FGFR3 activity results in achondroplasia, the most common form of human dwarfism. In the majority (∼98%) of cases the underlying mutation is G380R in the FGFR3 transmembrane domain. We have used coarse-grained molecular dynamics simulations to study the dimerization behaviour of wild-type, heterodimer, and mutant homodimer 33-residue transmembrane FGFR3 constructs in both POPC and DPPC bilayers. FGFR3 dimers are stable once formed in POPC, but dissociations are observed in DPPC. All three FGFR3 constructs exhibit bimodal helix crossing angle distributions, in contrast to the strong preference for right-handed crossing in glycophorin A (GpA) control simulations. We present evidence for a primary FGFR3 dimer interface and a less stable secondary interface. The latter is more pronounced for mutant than wild-type constructs in POPC, but not in DPPC. The helix crossing angle is right-handed at the secondary dimer interface for both heterodimer and mutant homodimer FGFR3 constructs in POPC. G370, A374, and R397 are prevalent FGFR3 dimer contacts, while the same analysis procedure on GpA control simulations selects the most important interfacial residues established by experiment. We suggest subtle differences, relative to wild-type, in the dimerization properties of G380R FGFR3 transmembrane domains.