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UCSD BIBC 100 - X-ray Structure of a Protein-conducting Channel

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X-ray structure of a protein-conductingchannelBert van den Berg1*, William M. Clemons Jr1*, Ian Collinson2, Yorgo Modis3, Enno Hartmann4, Stephen C. Harrison3& Tom A. Rapoport11Howard Hughes Medical Institute and Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA2Max Planck Institute of Biophysics, Marie-Curie-Strasse 13-15, D-60439 Frankfurt am Main, Germany3Howard Hughes Medical Institute, Children’s Hospital and Harvard Medical School, 320 Longwood Avenue, Boston, Massachusetts 02115, USA4University Luebeck, Institute for Biology, Ratzeburger Allee 160, Luebeck, D-23538, Germany* These authors contributed equally to this work...........................................................................................................................................................................................................................A conserved heterotrimeric membrane protein complex, the Sec61 or SecY complex, forms a protein-conducting channel, allowingpolypeptides to be transferred across or integrated into membranes. We report the crystal structure of the complex fromMethanococcus jannaschii at a resolution of 3.2 A˚. The structure suggests that one copy of the heterotrimer serves as a functionaltranslocation channel. Thea-subunit has two linked halves, transmembrane segments 1–5 and 6–10, clamped together by theg-subunit. A cytoplasmic funnel leading into the channel is plugged by a short helix. Plug displacement can open the channel intoan ‘hourglass’ with a ring of hydrophobic residues at its constriction. This ring may form a seal around the translocatingpolypeptide, hindering the permeation of other molecules. The structure also suggests mechanisms for signal-sequencerecognition and for the lateral exit of transmembrane segments of nascent membrane proteins into lipid, and indicates bindingsites for partners that provide the driving force for translocation.A decisive step in the biosynthesis of many secretory and plasma-membrane proteins is their transport across the endoplasmicreticulum (ER) membrane in eukaryotes or across the cytoplasmicmembrane in prokaryotes (for a review, see ref. 1). These polypep-tides are first targeted to the membrane by hydrophobic amino-acidsequences, which are either cleavable signal sequences or transmem-brane segments (TM) of membrane proteins. Soluble proteins, suchas those destined for secretion, are subsequently transported acrossthe membrane through a protein-conducting channel with a hydro-philic interior2,3. In the case of membrane proteins, when ahydrophobic TM arrives in the channel, it is released through anopening in the channel wall into the surrounding lipid phase. Thecapacity of the channel to open laterally towards the lipid and thewide variety of substrates that it must transport distinguish it frommany other channels.An evolutionarily conserved heterotrimeric complex of mem-brane proteins, called the Sec61 complex in eukaryotes and the SecYcomplex in eubacteria and archaea, forms the channel (for a review,see ref. 4). Thea-subunits (Sec61ain mammals, Sec61p inSaccharomyces cerevisiae, SecY in bacteria and archaea) andg-subunits (Sec61gin mammals, Sss1p in S. cerevisiae,SecEinbacteria and archaea) show significant sequence conservation (seeSupplementary Fig. S1). Both subunits are required for cell viabilityin S. cerevisiae and Escherichia coli. Theb-subunits (Sec61binmammals, Sbh in S. cerevisiae, Secbin archaea) are not essential forcell viability in these organisms; they are similar in eukaryotes andarchaea, but show no obvious homology to the corresponding SecGsubunits in bacteria. Thea-subunit forms the channel pore, and it isthe crosslinking partner of polypeptide chains passing through themembrane5. Reconstitution experiments have shown that theSec61/SecY complex is the essential membrane component forprotein translocation6,7.The channel itself is a passive conduit for polypeptides and musttherefore associate with other components that provide a drivingforce1. In co-translational translocation, the major partner is theribosome. The elongating polypeptide chain moves directly fromthe ribosome into the associated membrane channel. The energy fortranslocation comes from GTP hydrolysis during translation. Many(or perhaps all) cells also have post-translational translocation, inwhich polypeptides are completed in the cytosol and then trans-ported across the membrane. In yeast (and probably in all eukary-otes), the post-translational translocation partners are anothermembrane protein complex (the tetrameric Sec62/63p complex)and the lumenal protein BiP, a member of the Hsp70 family ofATPases8,9. BiP promotes translocation by acting as a molecularratchet, preventing the polypeptide chain from sliding back into thecytosol10. In the eubacterial post-translational pathway, the cytoso-lic ATPase SecA pushes polypeptides through the channel11.Inaddition, an electrochemical gradient across the membrane stimu-lates translocation in vitro and is essential in vivo12. Archaea lackSecA and the Sec62/63p complex, and it is unclear how they performpost-translational translocation13. Despite the differences betweenthe pathways, most mechanistic aspects of translocation that relateto the channel itself are probably similar. Specifically, in all cases thechannel partner—either the ribosome, the Sec62/63p complex orSecA—binds first, and the signal sequence or TM of a translocationsubstrate associates with the channel subsequently, priming it forpolypeptide translocation.An understanding of the mechanisms that underlie proteintranslocation requires detailed structural information. Low-resol-ution structures have been obtained by single-particle electronmicroscopy (EM) of either the isolated Sec61/SecY complex orthe Sec61 complex bound to the ribosome14–18. A recent structure ofthe E. coli SecY complex, derived from electron cryo-microscopy oftwo-dimensional (2D) crystals in a phospholipid bilayer, indicatedthe expected number of TM helices, but the resolution (about 8 A˚in-plane) was insufficient for their identification19. We now reportthe structure of the SecY complex from the archae M. jannaschii,determined by X-ray diffraction at 3.2 A˚resolution.Structure determination of the SecY complexWe purified and crystallized the complex from M. jannaschii in thedetergent diheptanoylphosphatidyl choline. Crystals of seleno-methionine-derivatized protein diffract


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UCSD BIBC 100 - X-ray Structure of a Protein-conducting Channel

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