To efficiently insert a protein into an equilibrated and fully hydrated membrane with minimal membrane perturbation we present a computational tool, called g_membed, which is part of the Gromacs suite of programs. The input consists of an equilibrated membrane system, either flat or curved, and a protein structure in the right position and orientation with respect to the lipid bilayer. g_membed first decreases the width of the protein in the xy-plane and removes all molecules (generally lipids and waters) that overlap with the narrowed protein. Then the protein is grown back to its full size in a short molecular dynamics simulation (typically 1000 steps), thereby pushing the lipids away to optimally accommodate the protein in the membrane. After embedding the protein in the membrane, both the lipid properties and the hydration layer are still close to equilibrium. Thus, only a short equilibration run (less then 1 ns in the cases tested) is required to re-equilibrate the membrane. Its simplicity makes g_membed very practical for use in scripting and high-throughput molecular dynamics simulations.

Atomic-resolution X-ray crystallography, functional analyses, and molecular dynamics simulations suggest a novel mechanism for the regulation of water flux through the yeast Aqy1 water channel.

Objective—Inflammatory conditions provoke essential processes in the human vascular system. It leads to the formation of ultralarge von Willebrand factor (VWF) fibers, which are immobilized on the endothelial cell surface and transform to highly adhesive strings under shear conditions. Furthermore, leukocytes release a meshwork of DNA (neutrophil extracellular traps) during the process of the recently discovered cell death program NETosis. In the present study, we characterized the interaction between VWF and DNA and possible binding sites to underline the role of VWF in thrombosis and inflammation besides its function in platelet adhesion. Approach and Results—Both functionalized surfaces and intact cell layers of human umbilical vein endothelial cells were perfused with isolated, protein-free DNA or leukocytes from whole blood at distinct shear rates. DNA–VWF interaction was monitored using fluorescence microscopy, ELISA-based assays, molecular dynamics simulations, and electrostatic potential calculations. Isolated DNA, as well as DNA released by stimulated leukocytes, was able to bind to shear-activated, but not inactivated, VWF. However, DNA–VWF binding does not alter VWF degradation by a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13. Moreover, DNA–VWF interaction can be blocked using unfractionated and low-molecular-weight heparin, and DNA–VWF complexes attenuate platelet binding to VWF. These findings were supported using molecular dynamics simulations and electrostatic calculations of the A1- and A2-domains. Conclusions—Our findings suggest that VWF directly binds and immobilizes extracellular DNA released from leukocytes. Therefore, we hypothesize that VWF might act as a linker for leukocyte adhesion to endothelial cells, supporting leukocyte extravasation and inflammation

Background: Aquaporin-9 is a glycerol-permeable channel expressed in hepatocytes. Results: Newly identified chemical inhibitors, as well as aquaporin-9 gene deletion, eliminate glycerol-dependent murine hepatocyte glucose output. Conclusion: Aquaporin-9 is the primary route of murine hepatocyte glycerol uptake for gluconeogenesis. Significance: Aquaporin-9 may be a drug target in diabetes treatment

Mechanosensing at focal adhesions regulates vital cellular processes. Here, we present results from molecular dynamics (MD) and mechano-biochemical network simulations that suggest a direct role of Focal Adhesion Kinase (FAK) as a mechano-sensor. Tensile forces, propagating from the membrane through the PIP2 binding site of the FERM domain and from the cytoskeleton-anchored FAT domain, activate FAK by unlocking its central phosphorylation site (Tyr576/577) from the autoinhibitory FERM domain. Varying loading rates, pulling directions, and membrane PIP2 concentrations corroborate the specific opening of the FERM-kinase domain interface, due to its remarkably lower mechanical stability compared to the individual alpha-helical domains and the PIP2-FERM link. Analyzing downstream signaling networks provides further evidence for an intrinsic mechano-signaling role of FAK in broadcasting force signals through Ras to the nucleus. This distinguishes FAK from hitherto identified focal adhesion mechano-responsive molecules, allowing a new interpretation of cell stretching experiments.

Aquaporins (AQPs) facilitate the passive flux of water across biological membranes in response to an osmotic pressure. A number of AQPs, for instance in plants and yeast, have been proposed to be regulated by phosphorylation, cation concentration, pH change, or membrane-mediated mechanical stress. Here we report an extensive set of molecular dynamics simulations of AQP1 and AQP4 subject to large membrane potentials in the range of ±1.5 V, suggesting that AQPs may in addition be regulated by an electrostatic potential. As the regulatory mechanism we identified the relative population of two different states of the conserved arginine in the aromatic/arginine constriction region. A positive membrane potential was found to stabilize the arginine in an up-state, which allows rapid water flux, whereas a negative potential favors a down-state, which reduces the single-channel water permeability.

Von Willebrand factor (VWF) plays a central role in hemostasis. Triggered by shear-stress, it adheres to platelets at sites of vascular injury. Inactivation of VWF has been associated to the shielding of its adhesion sites and proteolytic cleavage. However, the molecular nature of this shielding and its coupling to cleavage under shear-forces in flowing blood remain unknown. In this study, we describe, to our knowledge, a new force-sensory mechanism for VWF-platelet binding, which addresses these questions, based on a combination of molecular dynamics (MD) simulations, atomic force microscopy (AFM), and microfluidic experiments. Our MD simulations demonstrate that the VWF A2 domain targets a specific region at the VWF A1 domain, corresponding to the binding site of the platelet glycoprotein Ibα (GPIbα) receptor, thereby causing its blockage. This implies autoinhibition of the VWF for the binding of platelets mediated by the A1-A2 protein-protein interaction. During force-probe MD simulations, a stretching force dissociated the A1A2 complex, thereby unblocking the GPIbα binding site. Dissociation was found to be coupled to the unfolding of the A2 domain, with dissociation predominantly occurring before exposure of the cleavage site in A2, an observation that is supported by our AFM experiments. This suggests that the A2 domain prevents platelet binding in a force-dependent manner, ensuring that VWF initiates hemostasis before inactivation by proteolytic cleavage. Microfluidic experiments with an A2-deletion VWF mutant resulted in increased platelet binding, corroborating the key autoinhibitory role of the A2 domain within VWF multimers. Overall, autoinhibition of VWF mediated by force-dependent interdomain interactions offers the molecular basis for the shear-sensitive growth of VWF-platelet aggregates, and might be similarly involved in shear-induced VWF self-aggregation and other force-sensing functions in hemostasis.

Lipid-protein interactions play pivotal roles in biological membranes. Electron crystallographic studies of the lens-specific water channel aquaporin-0 (AQP0) revealed atomistic views of such interactions, by providing high-resolution structures of annular lipids surrounding AQP0. It remained unclear, however, whether these lipid structures are representative of the positions of unconstrained lipids surrounding an individual protein, and what molecular determinants define the lipid positions around AQP0. We addressed these questions by using molecular dynamics simulations and crystallographic refinement, and calculated time-averaged densities of dimyristoyl-phosphatidylcholine lipids around AQP0. Our simulations demonstrate that, although the experimentally determined crystallographic lipid positions are constrained by the crystal packing, they appropriately describe the behavior of unconstrained lipids around an individual AQP0 tetramer, and thus likely represent physiologically relevant lipid positions.While the acyl chains were well localized, the lipid head groups were not. Furthermore, in silico mutations showed that electrostatic inter actions do not play a major role attracting these phospholipids towards AQP0. Instead, the mobility of the protein crucially modulates the lipid localization and explains the difference in lipid density between extracellular and cytoplasmic leaflets. Moreover, our simulations support a general mechanism in which membrane proteins laterally diffuse accompanied by several layers of localized lipids, with the positions of the annular lipids being influenced the most by the protein surface. We conclude that the acyl chains rather than the head groups define the positions of dimyristoylphosphatidylcholine lipids around AQP0. Lipid localization is largely determined by the mobility of the protein surface, whereas hydrogen bonds play an important but secondary role.electron crystallography | lipd bilayer | atomistic simulations L ipids and membrane proteins form biological membranes that constitute the boundary of cells and their intracellular compartments. Lipids arrange in a bilayer conformation that serve as a 2D fluid for membrane proteins. The lipid bilayer, however, is more than a passive fluid and influences many aspects of membrane proteins, including their insertion into the membrane (1, 2), assembly into complexes (3-5), and activity (6, 7). Conversely, membrane proteins alter the conformational properties of lipid bilayers, mediating for instance pore formation (8), fusogenicity (9), and membrane bending (10, 11). Detailed knowledge of how lipids and membrane proteins interact with each other is therefore crucial to understand the molecular machinery of biological membranes.To date, spectroscopic methods have contributed most to our understanding of lipid-protein interactions, providing insight into the dynamics of such interactions (1, 12). Atomistic views were obtained by structures of membrane proteins either with few specifically bound lipids or surrounded by a compl...