We acknowledge the following funding:
“Physical modeling of non-equilibrium processes in biological systems”
“How allosteric effectors influence protein translocation mediated by the Sec61 complex”
In eukaryotes, the protein biosynthesis is carried out either by cytosolic ribosomes or by ribosomes that are attached to the membrane of the endoplasmatic reticulum (ER). In the latter case, the newly synthesized “nascent peptide chain” (NC) is typically translocated into the ER or laterally inserted into the ER membrane via an integral membrane protein complex. In eukaryotes, its central component is termed the Sec61 complex. Proteins that should be translocated across the ER membrane carry a “signal Peptide” (SP) at their N-terminus. In the ER, this sequence is then cleaved off by the enzyme signal peptidase. Upon exiting from the ribosome, the signal recognition particle (SRP) binds SP of the newly synthesized NC at the ribosome. Subsequently, upon interaction of SRP and SRP receptor at the ER membrane, SRP dissociates from SP which then is free to insert into the Sec61 channel. This pathway is termed the co-translational, SRP-dependent pathway, which is the focus of the proposed work. Not all NCs that should pass Sec61 may do this on their own. Certain NCs require the presence of additional accessory membrane proteins such as TRAP and Sec62-Sec63.
Until recently, it was unclear which features of SPs guide them through different translocation pathways. In prior published work, we have contributed to unraveling physico chemical features of SPs that distinguish TRAP clients or Sec62-63 clients from those proteins that do not require aid by such accessory proteins. Still, the mechanistic role of these accessory proteins and how they mechanistically affect cotranslational translocation is unknown. Furthermore, several small molecule effectors have been described in the literature (e.g. eeyarestatins, mycolactone, and Ipomoeassin F) that modulate the protein translocation efficiency of the Sec61 complex. In prior published work, we combined ligand docking with data from electrophysiology to suggest how eeyarestatins derivatives bind differentially in the Sec61 pore. Based on our published findings and on recent advances in structure determination of the Sec61 complex, we now aim at unravelling the mechanistic details how allosteric effectors influence protein translocation mediated by the Sec61 complex. We will implement an integrated computational and experimental approach.
Specific aims of the proposed project are:
- We want to characterize how SPs bind in the Sec61 pore,
- we want to characterize how accessory proteins alter the conformational sampling of SP in the Sec61 pore,
- we want to characterize the molecular consequences upon binding of small-molecule effectors to Sec61.
“How posttranslational modifications and alternative splicing affect protein-peptide interactions”
Large-scale proteomics and transcriptomics studies have unravelled that about half of all proteins in biological cells are targets of post-translational modifications and 95% of all multi-exon genes in higher eukaryotes are alternatively spliced. Since both processes may have crucial consequences on the protein interactions involving the respective proteins, this severely complicates our understanding of the cellular protein interactome. Only few model systems have sofar been characterized in structural and thermodynamic terms.
For this project, we selected two such model systems, 14-3-3 domains and PDZ domains, that both bind to hundreds of other proteins in human cells. Based on X-ray crystallographic data for the bound complexes, we will study how well molecular dynamics computer simulations can capture the influence of peptide phosphorylation on their binding to 14-3-3 adaptor domains and the influence of alternative splicing on the binding of peptides to PDZ domains. We will characterize the dynamic conformations of bound complexes, association-, dissociation-pathways and binding free energies of the peptide ligands, and the competitive binding between target peptides and small-molecule protein-protein inhibitors.
This project will explore the potential and limitations of molecular dynamics simulations to contribute to the systematic proteomic mapping of the consequences of post-translational modifications and alternative splicing on the cellular protein interactome.