Periodic Reporting for period 4 - MCS-MD (The Molecular Dynamics of Membrane Contact Sites)
Reporting period: 2023-12-01 to 2024-12-31
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, directionality and driving forces of lipid transport.
To understand these processes with unprecedented molecular detail, we have been developing 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 have obtained 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.
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, directionality and driving forces of lipid transport.
To understand these processes with unprecedented molecular detail, we have been developing 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 have obtained 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.
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 membrane contact site (MCS) proteins. We have thoroughly assessed the capability of current all-atom (AA) and coarse-grain (CG) models to investigate peripheral protein-membrane interactions and, based on our results, 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 binding mode 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 characterising the relationship between the dynamics of MCS proteins and their function, identifying new potential players in lipid transport machineries (e.g. protein insertases) as well as characterising a novel assembly mechanism for the lipid transporter ERMES.
Third, we have elucidated the mechanism of lipid uptake and release for several MCS proteins. Our data provide a mechanistic explanation of why lipid transport proteins have a wide range of transport efficiencies and even allowed us to identify new lipid transport proteins.
Fourth, we have worked on improving force field parameters for molecular dynamics simulations in order to correctly reproduce experimental observations. This has led to significant improved parameters for neutral lipids diacylglycerol and triacylglycerol, ions, and intrinsically disordered domains.
First, we have extensively investigated and elucidated the mechanism of membrane targeting by membrane contact site (MCS) proteins. We have thoroughly assessed the capability of current all-atom (AA) and coarse-grain (CG) models to investigate peripheral protein-membrane interactions and, based on our results, 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 binding mode 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 characterising the relationship between the dynamics of MCS proteins and their function, identifying new potential players in lipid transport machineries (e.g. protein insertases) as well as characterising a novel assembly mechanism for the lipid transporter ERMES.
Third, we have elucidated the mechanism of lipid uptake and release for several MCS proteins. Our data provide a mechanistic explanation of why lipid transport proteins have a wide range of transport efficiencies and even allowed us to identify new lipid transport proteins.
Fourth, we have worked on improving force field parameters for molecular dynamics simulations in order to correctly reproduce experimental observations. This has led to significant improved parameters for neutral lipids diacylglycerol and triacylglycerol, ions, and intrinsically disordered domains.
Our investigations have pushed forward the ability of current computational methodologies to reliably describe processes that are important in lipid transport at MCS, such as membrane association or lipid uptake. 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. Thanks to these innovative approaches, we have identified new players in lipid transport processes and elucidated the molecular details of important lipid transport processes.