Two Dimensional Window Exchange Umbrella Sampling for Transmembrane Helix Assembly

The method of window exchange umbrella sampling molecular dynamics (WEUSMD) with a pre-optimized parameter set was recently used to obtain the most probable conformations and the energetics of transmembrane (TM) helix assembly of a generic TM sequence. When applied to glycophorin A TM domain (GpA-TM) using the restraint potentials along the helix-helix distance, however, tight interfacial packing of GpA-TM resulted in insufficient conformational sampling at short helix-helix separation. This sampling issue is addressed by extending the WEUSMD into two dimensions with the restraint potentials along the helix-helix distance and crossing angle. The two-dimensional WEUSMD results demonstrate that the incomplete sampling in the one-dimensional WEUSMD arises from high barriers along the crossing angle between the GpA-TM helices. Together with the faster convergence in both the assembled conformations and the potential of mean force, the 2D-WEUSMD can be a general and efficient approach in computational studies of TM helix assembly

PIP2-Binding Site in Kir Channels: Definition by Multiscale Biomolecular Simulations†

Phosphatidylinositol bisphosphate (PIP(2)) is an activator of mammalian inwardly rectifying potassium (Kir) channels. Multiscale simulations, via a sequential combination of coarse-grained and atomistic molecular dynamics, enabled exploration of the interactions of PIP(2) molecules within the inner leaflet of a lipid bilayer membrane with possible binding sites on Kir channels. Three Kir channel structures were investigated: X-ray structures of KirBac1.1 and of a Kir3.1-KirBac1.3 chimera and a homology model of Kir6.2. Coarse-grained simulations of the Kir channels in PIP(2)-containing lipid bilayers identified the PIP(2)-binding site on each channel. These models of the PIP(2)-channel complexes were refined by conversion to an atomistic representation followed by molecular dynamics simulation in a lipid bilayer. All three channels were revealed to contain a conserved binding site at the N-terminal end of the slide (M0) helix, at the interface between adjacent subunits of the channel. This binding site agrees with mutagenesis data and is in the proximity of the site occupied by a detergent molecule in the Kir chimera channel crystal. Polar contacts in the coarse-grained simulations corresponded to long-lived electrostatic and H-bonding interactions between the channel and PIP(2) in the atomistic simulations, enabling identification of key side chains

Identification and structural characterization of FYVE domain-containing proteins of Arabidopsis thaliana

<p>Abstract</p> <p>Background</p> <p>FYVE domains have emerged as membrane-targeting domains highly specific for phosphatidylinositol 3-phosphate (PtdIns(3)<it>P</it>). They are predominantly found in proteins involved in various trafficking pathways. Although FYVE domains may function as individual modules, dimers or in partnership with other proteins, structurally, all FYVE domains share a fold comprising two small characteristic double-stranded β-sheets, and a C-terminal α-helix, which houses eight conserved Zn²⁺ion-binding cysteines. To date, the structural, biochemical, and biophysical mechanisms for subcellular targeting of FYVE domains for proteins from various model organisms have been worked out but plant FYVE domains remain noticeably under-investigated.</p> <p>Results</p> <p>We carried out an extensive examination of all <it>Arabidopsis </it>FYVE domains, including their identification, classification, molecular modeling and biophysical characterization using computational approaches. Our classification of fifteen <it>Arabidopsis </it>FYVE proteins at the outset reveals unique domain architectures for FYVE containing proteins, which are not paralleled in other organisms. Detailed sequence analysis and biophysical characterization of the structural models are used to predict membrane interaction mechanisms previously described for other FYVE domains and their subtle variations as well as novel mechanisms that seem to be specific to plants.</p> <p>Conclusions</p> <p>Our study contributes to the understanding of the molecular basis of FYVE-based membrane targeting in plants on a genomic scale. The results show that FYVE domain containing proteins in plants have evolved to incorporate significant differences from those in other organisms implying that they play a unique role in plant signaling pathways and/or play similar/parallel roles in signaling to other organisms but use different protein players/signaling mechanisms.</p

Interactions of the pleckstrin homology domain with phosphatidylinositol phosphate and membranes: characterization via molecular dynamics simulations.

The mechanism of interaction of pleckstrin homology (PH) domains with phosphatidylinositol 4,5-bisphosphate (PIP 2)-containing lipid bilayers remains uncertain. While crystallographic studies have emphasized PH-inositol 1,4,5-trisphosphate (IP 3) interactions, biophysical studies indicate a degree of less specific protein-bilayer interactions. We have used molecular dynamics simulations to characterize the interactions of the PH domain from phospholipase C-delta1 with IP 3 and with PIP 2, the latter in lipid bilayers and in detergent micelles. Simulations of the PH domain in water reveal a reduction in protein flexibility when IP 3 is bound. Simulations of the PH domain bound to PIP 2 in lipid bilayers indicate a tightening of ligand-protein interactions relative to the PH-IP 3 complex, alongside formation of H-bonds between PH side chains and lipid (PC) headgroups, and a degree of penetration of hydrophobic side chains into the core of the bilayer. Comparison with simulations of the PH-bound domain to a PC bilayer in the absence of PIP 2 suggests that the presence of PIP 2 increases the extent of PH-membrane interactions. Thus, comparative molecular dynamics simulations reveal how a PI-binding domain undergoes changes in conformational dynamics on binding to a PIP 2-containing membrane and how interactions additional to those with the PI headgroup are formed

Interactions of the pleckstrin homology domain with phosphatidylinositol phosphate and membranes: characterization via molecular dynamics simulations.

The mechanism of interaction of pleckstrin homology (PH) domains with phosphatidylinositol 4,5-bisphosphate (PIP 2)-containing lipid bilayers remains uncertain. While crystallographic studies have emphasized PH-inositol 1,4,5-trisphosphate (IP 3) interactions, biophysical studies indicate a degree of less specific protein-bilayer interactions. We have used molecular dynamics simulations to characterize the interactions of the PH domain from phospholipase C-delta1 with IP 3 and with PIP 2, the latter in lipid bilayers and in detergent micelles. Simulations of the PH domain in water reveal a reduction in protein flexibility when IP 3 is bound. Simulations of the PH domain bound to PIP 2 in lipid bilayers indicate a tightening of ligand-protein interactions relative to the PH-IP 3 complex, alongside formation of H-bonds between PH side chains and lipid (PC) headgroups, and a degree of penetration of hydrophobic side chains into the core of the bilayer. Comparison with simulations of the PH-bound domain to a PC bilayer in the absence of PIP 2 suggests that the presence of PIP 2 increases the extent of PH-membrane interactions. Thus, comparative molecular dynamics simulations reveal how a PI-binding domain undergoes changes in conformational dynamics on binding to a PIP 2-containing membrane and how interactions additional to those with the PI headgroup are formed

PX- and FYVE-mediated interactions with membranes: simulation studies.

Molecular dynamics simulations have been used to explore the interactions of two PI(3)P-binding domains with their PI ligands and with a phospholipid bilayer. Three simulations each of the EEA1-FYVE domain and the p40(phox)-PX domain have been compared: with the protein in an apo state, with a bound Ins(1,3)P(2) molecule, and bound to a PI(3)P molecule embedded in a lipid bilayer. Two main questions were addressed in analysis of the simulations: (i) the location of these domains relative to the lipid bilayer and (ii) their interactions with the lipids, both specific interactions via bound PI(3)P and nonspecific interactions with bilayer phospholipids. Both domains underwent a decrease in dynamic flexibility on binding to the ligand and to the membrane, this being more pronounced for the FYVE domain. Compared to their starting locations [docked to a membrane-inserted PI(3)P molecule], each of the domains penetrated more deeply into the lipid bilayer. For FYVE, nonspecific protein-lipid interactions were formed mainly by the N-terminal hydrophobic region of the protein. For PX, both the alpha1-alpha2 and the beta1-beta2 regions penetrated the bilayer. There appeared to be more marked dynamic fluctuations in hydrogen bonds between basic side chains and PI(3)P for FYVE than for PX, but for both domains, such interactions were maintained throughout the simulations. The simulations agree well with available biophysical data, suggesting this computational method may be used to predict protein-bilayer interactions for other PI-binding proteins

Molecular dynamics simulations of the dimerization of transmembrane alpha-helices.

Membrane proteins account for nearly a quarter of all genes, but their structure and function remain incompletely understood. Most membrane proteins have transmembrane (TM) domains made up of bundles of hydrophobic alpha-helices. The lateral association of TM helices within the lipid bilayer is a key stage in the folding of membrane proteins. It may also play a role in signaling across cell membranes. Dimerization of TM helices is a simple example of such lateral association. Molecular dynamics (MD) simulations have been used for over a decade to study membrane proteins in a lipid bilayer environment. However, direct atomistic (AT) MD simulation of self-assembly of a TM helix bundle remains challenging. AT-MD may be complemented by coarse-grained (CG) simulations, in which small numbers of atoms are grouped together into particles. In this Account, we demonstrate how CG-MD may be used to simulate formation of dimers of TM helices. We also show how a serial combination of CG and AT simulation provides a multiscale approach for generating and refining models of TM helix dimers. The glycophorin A (GpA) TM helix dimer represents a paradigm for helix-helix packing, mediated by a GxxxG sequence motif. It is well characterized experimentally and so is a good test case for evaluating computational methods. CG-MD simulations in which two separate TM helices are inserted in a lipid bilayer result in spontaneous formation of a right-handed GpA dimer, in agreement with NMR structures. CG-MD models were evaluated via comparison with data on destabilizing mutants of GpA. Such mutants increased the conformational flexibility and the dissociation constants of helix dimers. GpA dimers have been used to evaluate a multiscale approach: A CG model is converted to an AT model, which is used as the basis of an AT-MD simulation. Comparison of three AT-MD simulations of GpA, one starting from a CG model and two starting from NMR structures, leads to convergence to a common refined structure for the dimer. CG-MD self-assembly has also been used to model dimerization of the TM domain of the syndecan-2 receptor protein. This TM helix contains a GxxxG motif, which mediates right-handed helix packing comparable to that of the GxxxG motif in GpA. The multiscale approach has been applied to a more complex system, the heterodimeric alphaIIb/beta3 integrin TM helix dimer. In CG-MD, both right-handed and left-handed structures were formed. Subsequent AT-MD simulations showed that the right-handed structure was more stable, yielding a dimer in which the GxxxG motif of the alphaIIb TM helix packed against a hydrophobic surface of the beta3 helix in a manner comparable to that observed in two recent NMR studies. This work demonstrates that the multiscale simulation approach can be used to model simple membrane proteins. The method may be applied to more complex proteins, such as the influenza M2 channel protein. Future refinements, such as extending the multiscale approach to a wider range of scales (from CG through QM/MM simulations, for example), will expand the range of applications and the accuracy of the resultant models

MD simulations of Mistic: conformational stability in detergent micelles and water.

Mistic is an unusual membrane protein from Bacillus subtilis. It appears to fold and insert autonomously into a lipid bilayer and has been suggested as a tool that aids the targeting of eukaryotic membrane proteins to bacterial membranes. The NMR structure of Mistic in detergent (LDAO) micelles has revealed it to be a four alpha-helix bundle. From a structural perspective, Mistic does not resemble other membrane proteins. Its external surface is not very hydrophobic, and standard methods do not predict any of its helices to be in the transmembrane orientation. Molecular dynamics simulations (simulation times approximately 30 ns) in water and in detergent micelles have been used to explore the conformational stability of Mistic as a function of its environment. In water, the protein is stable, exhibiting no significant change in fold on a 30 ns time scale. In contrast, in three simulations in detergent micelles, the partial unfolding of Mistic occurred, whereby the H4 helix drifted away from the H1-H3 core. This was due to the penetration of detergent molecules between H4 and the remainder of the protein. This is unlike the behavior of several other membrane proteins, both alpha-helix bundles and beta-barrels, in comparable detergent micelle simulations. The unfolding of H4 from the H1-H3 core of Mistic could be partially reversed by a simulation in which the detergent molecules were removed, and the unfolded protein was simulated in water. These results suggest that Mistic may not be a stable integrated membrane protein but rather that it may undergo a conformational change upon interaction with a membrane or membrane-like environment