1. X-ray structure of a protein- conducting channel Van Den Berg, Bert, William M. Clemons Jr., Ian Collinson, Yorgo Modis, Enno Harmann, Stephen C. Harrison, and Tom A. Rapoport By Rick Hooy Nature. March 2004 Howard Hughes Medical Institute and Department of Cell Biology Max Planck Institute of Biophysics Howard Hughes Medical Institute University Luebeck, Institute for Biology

2. Introduction Many of the proteins that are synthesized by the cell need to be able to get across membranes – Post-translational modification – Secretion – Insertion into the membrane This paper focuses on the protein complex that allows unfolded proteins to cross membranes in Archaea

4. The protein-conducting channel SecY complex (bacteria and Archaea), Sec61 complex (eukaryotes) The complex consists of an evolutionarily conserved heterotrimer -Alpha (SecY) -Beta (SecG) -Gamma (SecE)

5. The Alpha Subunit (SecY) Forms the channel pore Features: – Two halves: TM1-5 and TM6-10 – The two halves are connected by an external loop between TM5 and TM6 – The two halves convey pseudo symmetry – TM2a consists of a long loop that moves along the external side of the molecule until it leads back into the center of the alpha subunit and ends in a short distorted helix

6. The Beta subunit (SecG) SecG consists of a single TM connected to a disordered cytoplasmic segment SecG is not required for SecY complex function

7. The Gamma Subunit (SecE) Consists of two helices The N-terminal helix is amphipathic and lays in contact with the C-terminal portion of the alpha subunit on the cytoplasmic surface of the membrane The C-terminal helix transverses the membrane at an angle of 35 degrees w.r.t. the plane of the membrane The two helices are oriented in such a way that they clamp the two alpha halves together N C C

8. Obtaining the crystal structure Previous structures were resolved to approximately 8A What this group did to improve resolution: – Selenomethionine-derivatization of the protein yielded a 3.4A structure using SAD Based on the 3.4A model, they identified areas where crystals were in contact with each other, and mutated those areas in hopes of strengthening crystal-crystal interactions – Crystals of the double mutant, Y1, diffracted to 3.2A

9. Left: Overlap of Y1 mutant and Selenomethionine-derivatized models. Mutant is grey. Right: A display of SecYEG mutant dimer. This illustrates where crystal-crystal contacts can be optimized

11. Structure Defines Function The 3.2A X-ray structure allowed them to “see” different parts of the complex with more detail and furthermore allowed them to hypothesize how the SecY complex works Key features of the complex: – The “plug” – The pore ring – The channel – The spacing between helices

12. The Pore The cytosolic interface of SecY is shaped like a funnel The funnel converges on the pore ring near the middle of the channel The inner wall of the pore contains a high number of uncharged, hydrophilic residues 20-25A Closed State Pore ring Surface Representation of Polar Residues

13. The pore ring Consists of 6 hydrophobic residues The ring is about 5-8A in diameter Creates a gasket-like seal around a translocating polypeptide Open State

14. The plug Based on the X-ray structure, it was hypothesized that thehelix portion of TM2a plugs the hole in the pore ring Mutant studies provided evidence to show thatdisplacement of TM2a and subsequent binding to SecEallows for polypeptide translocation – Method: Introduced disulfide bridges between the plug and SecE.Upon activation the channel became constitutively open Glycine Hinge

15. Activating/Opening the Channel Upon entering the SecY funnel, the signal sequence (short stretch of hydrophobic residues) folds into a short helix and intercalates between TM2b and TM7 The two halves of SecY are hinged at loop 5/6. In order to accommodate the signal sequence intercalation, the two halves hinge open 15⁰. Consequently the pore size increases and a peptide sequence can be translocated. It is hypothesized that this cascade of events destabilizes the plugs interactions at the center of the channel, causing it to be displaced and to bind to the gamma subunit.

16. Left: This figure suggests how the complex would reorient after signal sequence binding Right: The signal sequence does get exposed to lipid contact when it is bound in the complex

17. Signal Sequence Suppressor (prl) Mutations Prl mutations mimic the effects of signal- sequence binding such that the channel becomes stabilized in the open state or destabilized in the closed state. Examples: mutate residues in the plug, change hydrophobic residues in the pore ring to hydrophilic residues, induce a new crosslink between the gamma subunit and the plug

18. A model for protein translocation

19. Binding of Cytosolic Channel Partners Ribosomes, Sec A, Sec62/63p C-terminal half of the alpha subunit serves as binding site for channel partners Highly conserved residues on loop 8/9 serve as binding sites for ribosomal subunits and SecA SecA and ribosome cannot bind at same time Binding of channel partners probably induces conformational change in channel which then allows for the signal sequence to intercalate

20. Lateral movement of Membrane Proteins Membrane proteins must exit the channel through the lateral gate Opening the gate requires breaking structural bonds between helices (requires energy) Top: Side view of the lateral gate region and “open face” of the complex Bottom: Cytosolic view highlighting signal sequence intercalation and the lateral gate

21. Does the channel function as a monomer or as an oligomer? The SecY/Sec61 complex is capable of functioning as a monomer. However, functional dimer and tetramer complexes have been observed. – Cysteine cross-linking between a gamma subunit of one monomer to the gamma subunit of another (back-to-back orientation) – Tandem molecules with fused N and C termini; two monomers fused together at the genomic level. – WT dimers, trimers, and tetramers have been observed through EM Introduced disulfide bridge crosslink to create dimer of SecY

22. Structural Homology Sequence homology of the SecY/Sec61 complex across the 3 domains of life suggests structural homology of secretory protein-conducting channels X-ray structure of M. jannaschii SecY complex overlaying 2D electron density map of E. coli SecY complex

23. References Van Den Berg, Bert, William M. Clemons Jr., Ian Collinson, Yorgo Modis, Enno Harmann, Stephen C. Harrison, and Tom A. Rapoport. "X-ray Structure of a Protein-conducting Channel."Nature 427.6969 (2004): 36- 44. PubMed. Web. 1 Mar. 2011. Flower, Ann M. "The SecY Translocation Complex: Convergence of Genetics and Structure."Trends in Microbiology 15.5 (2007): 203-10. Print.