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Structure of the Sec61 Channel Opened by a Signal Sequence

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Structure of the Sec61 Channel Opened by a Signal Sequence RESEARCH | REPORTS We have demonstrated through structure-guided PROTEIN TRANSLOCATION design that neutralization of positive charges in the nt-groove can dramatically decrease off- target indel formation while preserving on-target activity. These data show that eSpCas9(1.1) can Structure of the Sec61 channel be used to increase the specificity of genome- editing applications. Future structure-guided inter- opened by a signal sequence rogation of Cas9 binding and cleavage mechanism will likely enable further optimization of the Rebecca M. Voorhees and Ramanujan S. Hegde* CRISPR-Cas9 genome-editing toolbox. REFERENCES AND NOTES Secreted and integral membrane proteins compose up to one-third of the biological 1. L. Cong et al., Science 339, 819–823 (2013). proteome. These proteins contain hydrophobic signals that direct their translocation 2. P. Mali et al., Science 339, 823–826 (2013). across or insertion into the lipid bilayer by the Sec61 protein–conducting channel. 3. P. D. Hsu et al., Nat. Biotechnol. 31, 827–832 (2013). The molecular basis of how hydrophobic signals within a nascent polypeptide trigger 4. Y. Fu et al., Nat. Biotechnol. 31, 822–826 (2013). – 5. B. Zetsche, S. E. Volz, F. Zhang, Nat. Biotechnol. 33, 139–142 channel opening is not understood. Here, we used cryo electron microscopy to determine (2015). the structure of an active Sec61 channel that has been opened by a signal sequence. 6. K. M. Davis, V. Pattanayak, D. B. Thompson, J. A. Zuris, The signal supplants helix 2 of Sec61a, which triggers a rotation that opens the central D. R. Liu, Nat. Chem. Biol. 11, 316–318 (2015). pore both axially across the membrane and laterally toward the lipid bilayer. Comparisons 7. F. A. Ran et al., Cell 154, 1380–1389 (2013). 8. P. Mali et al., Nat. Biotechnol. 31, 833–838 (2013). with structures of Sec61 in other states suggest a pathway for how hydrophobic signals 9. Y. Fu, J. D. Sander, D. Reyon, V. M. Cascio, J. K. Joung, Nat. engage the channel to gain access to the lipid bilayer. Biotechnol. 32, 279–284 (2014). 10. S. Q. Tsai et al., Nat. Biotechnol. 32, 569–576 (2014). 11. J. P. Guilinger, D. B. Thompson, D. R. Liu, Nat. Biotechnol. 32, he universally conserved Sec complex forms lactin(fig.S1).Translocation,protease-protection, 577–582 (2014). a gated protein translocation channel at the and photo–cross-linking experiments verified that, 12. Y. Fu, J. D. Sander, D. Reyon, V. M. Cascio, J. K. Joung, Nat. Biotechnol. – eukaryotic endoplasmic reticulum (ER) and like the well-characterized native 86-residue inter- 32, 279 284 (2014). 1 10–15 13. S. Q. Tsai et al., Nat. Biotechnol. 33, 187–197 (2015). bacterial plasma membrane ( ). The central mediate ( ), our tagged complex represents et al Cell – T 14. H. Nishimasu ., 156, 935 949 (2014). component of this channel, SecY in bacte- a functional translocation intermediate engaged 15. C. Anders, O. Niewoehner, A. Duerst, M. Jinek, Nature 513, ria and Sec61a in eukaryotes, contains 10 trans- bySec61(figs.S2toS4).Thenascentpolypeptide 569–573 (2014). et al Proc. Natl. Acad. Sci. U.S.A. membrane (TM) helices arranged around a central remains engaged with Sec61 during and after 16. E. Semenova ., 108, 2 10098–10103 (2011). pore ( ). Two single-TM subunits in eukaryotes, purification (fig. S4), which makes it suitable 17. B. Wiedenheft et al., Proc. Natl. Acad. Sci. U.S.A. 108, Sec61b and Sec61g, are peripheral to Sec61a.The for structure determination by single-particle 10092–10097 (2011). central pore in the inactive Sec complex is occluded cryo-EM. Nat. 18. W. Jiang, D. Bikard, D. Cox, F. Zhang, L. A. Marraffini, by a short “plug” helix that must be displaced The structure of this engaged ribosome-Sec61 Biotechnol. 31, 233–239 (2013). 19. S. H. Sternberg, S. Redding, M. Jinek, E. C. Greene, to allow translocation. The interface where TM complex was reconstructed from 101,339 particles J. A. Doudna, Nature 507,62–67 (2014). helices 2 and 3 contact helices 7 and 8 defines a to an overall resolution of 3.6 Å (figs. S5 and S6 20. N. Crosetto et al., Nat. Methods 10,361–365 (2013). “lateral gate” for membrane access of polypep- and table S1). The local resolution of the Sec61 et al Nature – 21. F. A. Ran ., 520, 186 191 (2015). tides (1–3). channel ranged from ~ 3.5 Å near the ribosome 2 4–6 ACKNOWLEDGMENTS Crystal structures of the Sec complex ( , ) to ~7.0 Å at the lumenal loops. Most TM helices We thank J. Dahlman for helpful discussions and a critical lack a translocating polypeptide and likely rep- were at ~4.5 to 5.5 Å resolution (fig. S6), which review of the manuscript; F. A. Ran, R. J. Platt, and J. Joung resent a range of inactive states. Depending on revealed clear helical pitch and many bulky side for experimental assistance; and the entire Zhang laboratory for crystal contacts or translocation partners, the lat- chains in sharpened maps (fig. S7). All 12 TM support and advice. I.S. is supported by the Simons Center for eral gate and plug are in various states of open- helices of the Sec61 complex could be unambig- the Social Brain. W.X.Y. is supported by T32GM007753 from the National Institute of General Medical Sciences and a Paul and ing and displacement. However, the biological uously assigned, leaving a single helix we ascribed Daisy Soros Fellowship. F.Z. is supported by the National Institutes relevance of these channel conformations has to the signal sequence (Fig. 1, A and B, and fig. S8). of Health through NIMH (5DP1-MH100706 and 1R01MH110049) been difficult to interpret without a well-resolved Density visible throughout the ribosomal exit tun- and NIDDK (5R01DK097768-03), a Waterman Award from the and matched active structure. Previous structures nel and in parts of the Sec61 channel (Fig. 1C) sug- National Science Foundation, the Keck, New York Stem Cell, Damon Runyon, Searle Scholars, Merkin, and Vallee Foundations, of translocation or insertion intermediates of the gests a looped configuration for the nascent chain, and B. Metcalfe. F.Z. is a New York Stem Cell Foundation ribosome-Sec complex determined by cryo–electron consistent with earlier cross-linking studies (11). Robertson Investigator. I.S., L.G., B.Z., and F.Z. are inventors on microscopy (cryo-EM) were of moderate resolu- The well-resolved structure of a biochemically provisional patent application 62/181,453 applied for by the tion (7–9), contained heterogeneous substrates validated early translocation intermediate per- Broad Institute and MIT that covers the engineered CRISPR 9 8 proteins described in this manuscript. Plasmid DNA encoding ( ), required artificial stabilization ( ), or were mitted detailed comparisons with other Sec61 eSpCas9(1.0) and eSpCas9(1.1) are available from Addgene at an uncertain stage of insertion (7). Although states to gain insights into the conformational under a Universal Biological Material Transfer Agreement with these earlier structures provided the first views changes accompanying channel opening. A previ- the Broad Institute and MIT. F.Z. is a founder and scientific advisor of substrate-induced structural changes consistent ous cryo-EM structure of the porcine ribosome- for Editas Medicine and a scientific advisor for Horizon 9 Discovery. Further information about the protocols, plasmids, with lateral gate opening, the data could not Sec61 complex lacking a nascent polypeptide ( ) and reagents can be found at the Zhang laboratory website clearly resolve individual Sec61 TM helices or the represents a “primed” state preceding nascent (www.genome-engineering.org). nature of their interactions with the signal. Thus, chain insertion. Relative to this primed structure, a molecular understanding of how substrates the engaged channel is open laterally toward the SUPPLEMENTARY MATERIALS open the channel for translocation or insertion lipid bilayer and axially across the membrane www.sciencemag.org/content/351/6268/84/suppl/DC1 is incomplete. (Fig. 2). The ribosome-Sec61 interaction remains Materials and Methods We devised a strategy to tag and purify the fixed, with only minor movements of the asso- Figs. S1 to S12 canine ribosome-Sec61 complex engaged by the ciated Sec61g and TM helices 6, 7, 8, and 9 of Tables S1 to S3 first 86 residues of the secretory protein prepro- Sec61a. The other seven TM helices of the Sec61 Supplementary DNA Sequences References complex rotate as a rigid body by ~22° (Fig. 2A and movies S1 and S2), which creates space be- 24 September 2015; accepted 18 November 2015 MRC Laboratory of Molecular Biology, Medical Research Published online 1 December 2015 Council, Francis Crick Avenue, Cambridge CB2 0QH, UK. tween helices 2 and 7 for intercalation of the sig- 10.1126/science.aad5227 *Corresponding author. E-mail: [email protected] nal peptide (Fig. 2B). Notably, cryo-tomography 88 1JANUARY2016• VOL 351 ISSUE 6268 sciencemag.org SCIENCE RESEARCH | REPORTS H7 signal Fig. 1. Structure of the signal peptide-engaged signal H8 Sec61 complex. (A) View of the lateral gate (top) or ER lumen from the ER lumen of the Sec61 complex bound to the preprolactin signal peptide (cyan). The mobile regions Sec61α of Sec61a are blue; the comparatively immobile regions Sec61α are red. The b and g subunits are pale green and tan, γ respectively. Helices that compose the lateral gate are Sec61 labeled. (B) Experimental density for the structure cytosol (mesh, filtered to 4.5 Å resolution) superimposed on H2 the backbone trace of the structural model. (C)Density view of observed for the nascent polypeptide through the lateral gate H3 ribosomal tunnel and parts of the Sec61 channel. 90° Signal Sec61γ Sec61 Sec61β H2 H7 H8 H3 view from ER lumen signal tRNA H7 4 7 6 β 3 γ open 7 5 H3 β 4 8 1 10 3 1 H8 rotate β 10 9 down & away 90° 2 4 2 2 8 primed γ 4 3 γ vs.
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