Significance
C&S4

For most membrane proteins, the process of insertion and integration into lipid membranes takes place concurrent with their translation and synthesis by the ribosome. Given their therapeutic relevance, the insertion pathways of nascent trans-membrane proteins into lipid membranes deserve more attention than they have hitherto received. The ribosomal insertion machinery consists mainly of YidC, SecY and the holo-translocon supercomplex; the holo-translocon supercomplex itself is composed of YidC, SecY and SecDF. Depending on size and nature of the inserting nascent protein, the membrane insertion process is performed by either YidC or SecY alone, or by both of them working in concert within the holo-translocon supercomplex. Here, we plan to (1) model an all-atom structure of a membrane-embedded ribosome-bound holo-translocon supercomplex, and then (2) investigate how conformational changes of the holo-translocon supercomplex regulates membrane proteins insertion pathways.

Methods

Most experimental probes fail to catch non-stationary states of non-equilibrium processes such as nascent protein insertion from the ribosome into the membrane. Computational techniques have proved instrumental in investigating such processes. A detailed investigation of protein insertion pathways via the holo-translocon, which necessitates the application of MD simulations, requires several difficult steps. The first involves building an all-atom structure, based on a low-resolution (8-10 Å) cryo-EM density map, for a membrane-embedded ribosome-bound holo-translocon. Center-developed hybrid modeling tools (TRD3) combining de novo structure prediction with MDFF will be employed to address the structure analysis step for a system of ~4 M atoms. Once the all-atom structure is available and simulated, the second difficulty is the millisecond timescale characteristic for protein insertion by the holo-translocon. The enhanced sampling features of NAMD (TRD1) permit one to overcome the timescale problem.

State of the work

In collaboration with Schaffitzel, a detailed all-atom model of an isolated holo-translocon (without the ribosome) has already been determined employing MDFF (TRD3), a hybrid modeling tool developed at the Center. The stability of the model in a realistic membrane environment was assessed over the course of a μs MD trajectory. The MD simulation of the holo-translocon in the membrane further revealed the key residues that stabilize the holo-translocon supercomplex and revealed also how the inherent flexibility of the supercomplex allows it to adapt its conformation to the requirements of its protein substrates. The computationally identified key residues are currently being tested with mutational experiments (Schaffitzel) to confirm their individual and cumulative effects on the stability of the holo-translocon.

Research plan

Having the isolated holo-translocon model available, in the upcoming research a ribosome-bound model of the holo-translocon in a membrane will be constructed. Representing the holo-translocon in its natural environment will provide a viable starting point for studying the holo-translocon mediated protein insertion pathway. In particular, the insertion pathway of a FoC helix will be probed; the FoC helix makes up the rotary motor of ATP synthase. In analogy to prior work on SecY, steered MD simulations will be employed to derive a preliminary FoC insertion pathway. Further refinements of this pathway will be accomplished utilizing enhanced sampling features of NAMD (TRD1). Similar to an earlier successful experimental-computational collaboration with the Beckmann group on YidC, the experimental collaborator (Schaffitzel) will perform cross-linking studies to confirm the computationally predicted relative position of FoC with respect to the constituent proteins of the holo-translocon, and particularly with respect to YidC which is expected to provide an exit way to FoC into the membrane. Our prior work on isolated YidC and SecY as well as colaborating with a world-leading experimental group offers the unique opportunity to probe how YidC and SecY work together within the holo-translocon supercomplex, and, thus, compare the key features of the three most ubiquitous pathways of membrane protein insertion.

Publications
Publications Database
  • A structural model of the active ribosome-bound membrane protein insertase YidC. Stephan Wickles, Abhishek Singharoy, Jessica Andreani, Stefan Seemayer, Lukas Bischoff, Otto Berninghausen, Johannes Soeding, Klaus Schulten, Eli van der Sluis, and Roland Beckmann. eLife, 3:e03035, 2014. (17 pages).
  • Reconciling the roles of kinetic and thermodynamic factors in membrane-protein insertion. James C. Gumbart, Ivan Teo, Benoit Roux, and Klaus Schulten. Journal of the American Chemical Society, 135:2291-2297, 2013.
  • Free energy of nascent-chain folding in the translocon. James Gumbart, Christophe Chipot, and Klaus Schulten. Journal of the American Chemical Society, 133:7602-7607, 2011.
  • Free-energy cost for translocon-assisted insertion of membrane proteins. James Gumbart, Christophe Chipot, and Klaus Schulten. Proceedings of the National Academy of Sciences, USA, 108:3596-3601, 2011.
  • Cryo-EM structure of the ribosome-SecYE complex in the membrane environment. Jens Frauenfeld, James Gumbart, Eli O. van der Sluis, Soledad Funes, Marco Gartmann, Birgitta Beatrix, Thorsten Mielke, Otto Berninghausen, Thomas Becker, Klaus Schulten, and Roland Beckmann. Nature Structural & Molecular Biology, 18:614-621, 2011.
  • Structure of monomeric yeast and mammalian Sec61 complexes interacting with the translating ribosome. Thomas Becker, Shashi Bhushan, Alexander Jarasch, Jean-Paul Armache, Soledad Funes, Fabrice Jossinet, James Gumbart, Thorsten Mielke, Otto Berninghausen, Klaus Schulten, Eric Westhof, Reid Gilmore, Elisabet C. Mandon, and Roland Beckmann. Science, 326:1369-1373, 2009.
  • Regulation of the protein-conducting channel by a bound ribosome. James Gumbart, Leonardo G. Trabuco, Eduard Schreiner, Elizabeth Villa, and Klaus Schulten. Structure, 17:1453-1464, 2009.
  • The roles of pore ring and plug in the SecY protein-conducting channel. James Gumbart and Klaus Schulten. Journal of General Physiology, 132:709-719, 2008.
  • Structural determinants of lateral gate opening in the protein translocon. James Gumbart and Klaus Schulten. Biochemistry, 46:11147-11157, 2007.
  • Mechanics of force propagation in TonB-dependent outer membrane transport. James Gumbart, Michael C. Wiener, and Emad Tajkhorshid. Biophysical Journal, 93:496-504, 2007.
  • Molecular dynamics studies of the archaeal translocon. James Gumbart and Klaus Schulten. Biophysical Journal, 90:2356-2367, 2006.