Microsecond-Level Simulations Reveal Membrane Protein Insertion Mechanism of Insertase YidC

Abstract

YidC, a member of the YidC/Alb3/Oxa1 insertase family, mediates membrane protein assembly and insertion both with and without the involvement of Sec machinery. The mechanistic details of the insertion process, however, remain elusive at the molecular level partly due to experimental limitations associated with structural studies. Here microsecond-level all-atom molecular dynamics (MD) simulations are employed to investigate the structural dynamics of YidC both in its apo form and bound to a Pf3 coat protein in order to characterize the Sec-independent protein insertion mechanism of YidC. Structural studies suggest that the cooperative interaction between the cytoplasmic loops (C1 and C2), the conserved hydrophilic groove of YidC, and the incoming peptide are the framework for the binding/insertion mechanism of YidC. Our simulations provide a dynamic picture of protein structure at atomic resolution that complements the limited structural data. We have modeled YidC both without the C2 loop, which is missing in the crystal structure, and with a modeled C2 loop. Both systems were modeled in the natural environment of the protein involving lipids, water, and ions, followed by microsecond-level simulations. We have also modeled the same systems with a docked Pf3 coat protein in different poses and performed microsecond-level simulations. The data provided by our extensive set of simulations suggest a key role not only for the C1 loop, which has already been suggested, but also for the missing C2 loop that stabilizes YidC according to our model. Importantly, the simulations illustrate the role of water dynamics in the insertion process of the Pf3 coat protein, which involves several stages of entering and exiting the waters to the core region of the protein while the substrate is being inserted in the membrane.