Analyzing lipid distributions and curvature in molecular dynamics simulations of complex membranes.

We describe methods to analyze lipid distributions and curvature in membranes with complex lipid mixtures and embedded membrane proteins. We discuss issues involved in these analyses, available tools to calculate curvature preferences of lipids and proteins, and focus on tools developed in our group for visual analysis of lipid-protein interactions and the analysis of membrane curvature.

Molecular dynamics simulations of lipid-protein interactions in SLC4 proteins.

The SLC4 family of secondary bicarbonate transporters is responsible for the transport of HCO3-, CO32-, Cl-, Na+, K+, NH3, and H+, which are necessary for regulation of pH and ion homeostasis. They are widely expressed in numerous tissues throughout the body and function in different cell types with different membrane properties. Potential lipid roles in SLC4 function have been reported in experimental studies, focusing mostly on two members of the family: AE1 (Cl-/HCO3- exchanger) and NBCe1 (Na+-CO32-cotransporter). Previous computational studies of the outward-facing state of AE1 with model lipid membranes revealed enhanced protein-lipid interactions between cholesterol (CHOL) and phosphatidylinositol bisphosphate (PIP2). However, the protein-lipid interactions in other members of the family and other conformation states are still poorly understood and this precludes the detailed studies of a potential regulatory role for lipids in the SLC4 family. In this work, we performed coarse-grained and atomistic molecular dynamics simulations on three members of the SLC4 family with different transport modes: AE1, NBCe1, and NDCBE (an Na+-CO32-/Cl- exchanger), in model HEK293 membranes consisting of CHOL, PIP2, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin. The recently resolved inward-facing state of AE1 was also included in the simulations. Lipid-protein contact analysis of the simulated trajectories was performed with the ProLint server, which provides a multitude of visualization tools for illustration of areas of enhanced lipid-protein contact and identification of putative lipid binding sites within the protein matrix. We observed enrichment of CHOL and PIP2 around all proteins with subtle differences in their distribution depending on the protein type and conformation state. Putative binding sites were identified for CHOL, PIP2, phosphatidylcholine, and sphingomyelin in the three studied proteins, and their potential roles in the SLC4 transport function, conformational transition, and protein dimerization are discussed.

Martini 3 Coarse-Grained Force Field for Cholesterol.

Cholesterol plays a crucial role in biomembranes by regulating various properties, such as fluidity, rigidity, permeability, and organization of lipid bilayers. The latest version of the Martini model, Martini 3, offers significant improvements in interaction balance, molecular packing, and inclusion of new bead types and sizes. However, the release of the new model resulted in the need to reparameterize many core molecules, including cholesterol. Here, we describe the development and validation of a Martini 3 cholesterol model, addressing issues related to its bonded setup, shape, volume, and hydrophobicity. The proposed model mitigates some limitations of its Martini 2 predecessor while maintaining or improving the overall behavior.

Coarse-grained molecular dynamics simulations of lipid-protein interactions in SLC4 proteins.

The SLC4 family of secondary bicarbonate transporters is responsible for the transport of HCO 3 -, CO 3 2- , Cl - , Na + , K + , NH 3 and H + necessary for regulation of pH and ion homeostasis. They are widely expressed in numerous tissues throughout the body and function in different cell types with different membrane properties. Potential lipid roles in SLC4 function have been reported in experimental studies, focusing mostly on two members of the family: AE1 (Cl - /HCO 3 - exchanger) and NBCe1 (Na + -CO 3 2- cotransporter). Previous computational studies of the outward facing (OF) state of AE1 with model lipid membranes revealed enhanced protein-lipid interactions between cholesterol (CHOL) and phosphatidylinositol bisphosphate (PIP2). However, the protein-lipid interactions in other members of the family and other conformation states are still poorly understood and this precludes the detailed studies of a potential regulatory role for lipids in the SLC4 family. In this work, we performed multiple 50 µs coarse-grained molecular dynamics simulations on three members of the SLC4 family with different transport modes: AE1, NBCe1 and NDCBE (a Na + -CO 3 2- /Cl - exchanger), in model HEK293 membranes consisting of CHOL, PIP2, phosphatidylcholine (POPC), phosphatidylethanolamine (POPE), phosphatidylserine (POPS), and sphingomyelin (POSM). The recently resolved inward-facing (IF) state of AE1 was also included in the simulations. Lipid-protein contact analysis of the simulated trajectories was performed with the ProLint server, which provides a multitude of visualization tools for illustration of areas of enhanced lipid-protein contact and identification of putative lipid binding sites within the protein matrix. We observed enrichment of CHOL and PIP2 around all proteins with subtle differences in their distribution depending on the protein type and conformation state. Putative binding sites were identified for CHOL, PIP2, POPC, and POSM in the three studied proteins and their potential roles in the SLC4 transport function, conformational transition and protein dimerization were discussed. Statement of significance: The SLC4 protein family is involved in critical physiological processes like pH and blood pressure regulation and maintenance of ion homeostasis. Its members can be found in various tissues. A number of studies suggest possible lipid regulation of the SLC4 function. However, the protein-lipid interactions in the SLC4 family are still poorly understood. Here we make use of long coarse-grained molecular dynamics simulations to assess the protein-lipid interactions in three SLC4 proteins with different transport modes, AE1, NBCe1, and NDCBE. We identify putative lipid binding sites for several lipid types of potential mechanistic importance, discuss them in the framework of the known experimental data and provide a necessary basis for further studies on lipid regulation of SLC4 function.

Predicting a Stable Dimeric Form of the PD1-PDL1 Complex: Implications for Understanding the PD1 Activation Mechanism.

The activation of T cells is typically accompanied by inhibitory mechanisms within which the programmed cell death (PD1) receptor stands out. Upon binding the ligands PDL1 and PDL2, PD1 drives T cells to an unresponsive state called exhaustion, characterized by a markedly decreased capacity to exert effector functions. For this reason, PD1 has become one of the most important targets in cancer immunotherapy. Despite the numerous studies about PD1 signaling modulation, how the PD1 signaling is activated upon the ligands' binding remains an open question. Several experimental facts suggest that the activation of the PD1-PLD1 pathway depends on the interaction with an unknown partner at the cellular membrane. In this work, we investigate the possibility that the target of PD1-PDL1 is the same PD1-PDL1 complex. We combined molecular docking with molecular dynamics and umbrella sampling simulations to explore different binding modes and assess the complexes' stability. We predicted a stable dimeric form of the extracellular domains of the PD1-PDL1 complex. This dimeric complex has an affinity comparable to the PD1-PDL1 interaction and resembles the form of a linear lattice. We proposed a new model for PD1 activation where the PD1-PDL1 dimeric form could facilitate the interaction of the intracellular domains of PD1 and the further binding and activation of the SHP2 phosphatase. This model might explain the inhibitory effect of anti-PD1/PDL1 antibodies through the prevention of the formation of the PD1-PDL1 dimers and, subsequently, the abrogation of the SHP2 phosphatase activation.

In silico identification of new potentially active brassinosteroid analogues.

Brassinosteroids (BRs) are hormones found in a wide range of plant species, they are synthesized at low concentrations and are essential for normal growth and development. These phytohormones are recognized by the Leucine-rich-repeat ectodomain of the receptor-like-kinase BRI1 which leads to subsequent downstream signaling mediating plant growth/development. In spite of the advantages that BRs offer, their extraction from natural sources is highly expensive, which constitutes one of its major limitations. Thus, the design and synthesis of structural and/or functional analogues of BRs with higher activity and lower cost has a great practical significance in world agriculture. In this matter, in silico methods, such as molecular docking, are valuable tools for the prediction and design of new compounds with improved activity. In this work we performed molecular docking simulations of 20 synthetic steroids in order to identify active compounds. Contact based analysis, level of exposure of polar groups to the solvent and binding affinity were the parameters used to consider if a synthetic steroid was active. Our results suggested that 17 out of a total of 20 of the analyzed steroids can potentially activate BRI1 receptor.