Membrane protein folding in the crowded environment
Biochemistry
Cell Biology
Final Report Abstract
Cellular membranes are intrinsically complex environments composed of a broad variety of lipids and characterized by a high protein density, known as macromolecular crowding. Next to specific protein:protein and protein:lipid interactions, macromolecular crowding is an important factor that determines the membrane organization, as it affects diffusion, assembly of macromolecular complexes, and protein conformational dynamics. Insertion and folding of nascent membrane proteins mediated by SecYEG/Sec61 translocon and YidC-type insertases must occur within this heterogeneous and crowded environment. However, the complexity is rarely addressed experimentally, and most structural insights on membrane protein folding have been acquired from lipid-free systems. Within the project, we developed several approaches for preparing lipid membranes, where crowding at the interface and within the lipid bilayer can be tailored and controlled. We also designed a range of fluorescence-based sensors for the membrane-associated crowding that are applicable for both model membranes and living cells. Using synthetic crowded membranes, we demonstrated that interfacial crowding inhibits interactions of SecYEG with both ribosomes and the secretion-dedicated ATPase SecA, and so modulates protein insertion and transport. Nanodisc-reconstituted SecYEG was further employed in cryo-electron microscopy (cryo-EM) analysis to determine the structure of the translocon upon the insertion of the nascent membrane protein in the lipid bilayer. The study revealed the most complete up to date structure of SecYEG:ribosome complex and documented a novel translocon conformation with a partially folded substrate egressing into the lipid bilayer. Moreover, the cryo-EM analysis also provided the structure of the nanodisc-embedded YidC:ribosome complex. The accompanying biophysical and biochemical analysis suggested that both the lipid charge and the fatty acid composition affect the insertion of the nascent protein. Notably, we discovered that unsaturated fatty acids within the physiologically fluid membrane have an immense stimulatory effect on SecYEG-mediated protein transport. A comprehensive molecular analysis allowed us to relate this stimulation to the enhanced binding of SecA ATPase to the lipid bilayer, followed by SecA:SecYEG association and the substrate translocation. The effect of the membrane lipid composition on SecA:SecYEG activity may be an important factor for the bacterial protein secretion under low temperatures, where the synthesis of unsaturated fatty acids is promoted. In summary, our biochemical and biophysical studies show that the complex membrane environment and the macromolecular crowding affect both co-translational and posttranslational protein transport via the universal Sec translocon, and the structural analysis provide first views on protein insertion and folding in the native-like lipid membrane environment.
Publications
- (2016) Structural dynamics of the YidC insertase upon membrane protein insertion. Cell Rep 17, 2943-54
Kedrov A, Wickles S, Crevenna AH, van der Sluis E, Buschauer R, Berninghausen O, Lamb DC & Beckmann R
(See online at https://doi.org/10.1016/j.celrep.2016.11.059) - (2018) Biophysical analysis of Sec-mediated protein translocation in nanodiscs. In: Advances in Biomembranes and Lipid Self- Assembly 28, 41-85 (edited by Aleš Iglič, Michael Rappolt, and Ana J. García-Sáez)
Koch S, Driessen AJ & Kedrov A
(See online at https://doi.org/10.1016/bs.abl.2018.05.003) - (2019) Partially inserted nascent chain unzips the lateral gate of the Sec translocon. EMBO Rep 20, e48191
Kater L, Frieg B, Berninghausen O, Gohlke H, Beckmann R & Kedrov A
(See online at https://doi.org/10.15252/embr.201948191) - (2019) Shaping the lipid composition of bacterial membranes for membrane protein production. Microb Cell Factories 18. 131
Kanonenberg K, Royes J, Kedrov A, Poschmann G, Angius F, Solgadi A, Spitz O, Kleinschrodt D, Stühler K, Miroux B and Schmitt L
(See online at https://doi.org/10.1186/s12934-019-1182-1) - (2020) The more the merrier: Multifaceted effects of macromolecular crowding on dynamics and structure of biological membranes. FEBS J
Löwe M, Kalacheva M, Boersma AJ & Kedrov A
(See online at https://doi.org/10.1111/febs.15429)
