Periodic Reporting for period 2 - MCS-MD (The Molecular Dynamics of Membrane Contact Sites) Reporting period: 2020-12-01 to 2022-05-31 Summary of the context and overall objectives of the project The goal of this project is to obtain an atomistic structural and dynamical characterization of the inner workings of membrane contact sites (MCS) between intracellular organelles, in order to understand how molecular processes such as non-vesicular lipid transport at MCS might modulate lipid homeostatic processes at the whole-cell scale.Investigation of the mechanisms taking place at MCS has emerged as a central topic in cellular biology in the last few years, and it has led to a large amount of novel cellular, biochemical and structural data that has drastically revolutionized our general understanding of lipid homeostasis in the cell. Yet, due to limitations of experimental methods, a high-resolution understanding of how MCS proteins work is still limited, and the specific molecular details of these mechanisms are still under intense debate, and especially concerning the specificity of lipid transport or the discrimination between lipid sensing and lipid transport.To understand these processes with unprecedented molecular detail, we have been developing high-throughput protocols based on atomistic and coarse-grain molecular dynamics simulations that leverage and take advantage of all the available, yet often scattered, experimental data. With these approaches, that have not been used so far to investigate MCS because of the extreme complexity of these cellular machineries, we are obtaining a detailed understanding of key molecular processes taking place at MCS, including the specificity of membrane binding, the mechanism of lipid uptake and release, the influence of confinement on protein activity, and the role of membrane lipid composition in the regulation of lipid transport. This approach will drive forward our perception of the limits of structure-based in-silico methods, and it will contribute to our mechanistic understanding of key cellular biology processes by providing new quantitative results that are beyond the current possibilities of experimental approaches. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far During the first half of the project, in line with the proposed work, we have focused on four main aspects. First, we have extensively investigated and elucidated the mechanism of membrane targeting by MCS proteins. As a first step, we have thoroughly assessed the capability of current all-atom (AA) and coarse-grain (CG) models to investigate peripheral protein-membrane interactions. Next, we have applied the best-identified methods to investigate the association of several MCS protein domains to different model membranes. This detailed molecular understanding of the membrane interface of MCS proteins is a significant progress with respect to current knowledge, as current structural biology approaches often do not provide this piece of information.Second, we have started investigating the internal dynamics of individual domains of MCS proteins as well as their assemblies. This has helped us characterize the relationship between the dynamics of MCS proteins and their functionThird, we have characterized the mechanism of lipid uptake and release for one specific family of MCS proteins. Our preliminary results recapitulate experimental measurements on transfer rates, and they provide a mechanistic explanation of why lipid transport proteins have a wide range of transport efficiencies. Fourth, we have worked on improving force field parameters for molecular dynamics simulations in order to correctly reproduce experimental observations. So far, this has led to improved parameters for neutral lipids diacylglycerol and triacylglycerol, but further developments are under way. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) So far, we have focused on the investigation of the ability of current computational methodologies to reliably describe processes that are important in lipid transport at MCS, such as membrane association or lipid desorption. By doing so, we identified strengths and weaknesses of available methods and, whenever needed, we provided alternative solutions to solve issues that have emerged. Taking advantage of the acquired knowledge, we have extensively applied these approaches to study mechanistic aspects of MCS protein’s function.In the remaining half of the project, (i) we will continue our development efforts to provide the community with improved technological means to investigate lipid transport at MCS using computer simulations, and (ii) we will establish correlations between MCS protein’s molecular mechanism and their physiological function, in order to elucidate molecular details of cellular lipid homeostasis.