Structure and function of YidC and homologous membrane proteins
Final Report Abstract
Most integral membrane proteins use the co-translational targeting and insertion machinery of the signal recognition particle (SRP) in conjunction with the SecYEG translocation channel. However, in bacteria, mitochondria and chloroplasts a number of membrane proteins are inserted and assembled by the YidC/Oxa1/Alb3 family of membrane insertases. Despite the low sequence conservation, all members share a hydrophobic core with five transmembrane helices (TMs), which harbor the central protein insertase function. The N- and C-terminal regions are more diverse and are thought have specialised functions. The mechanism of membrane protein insertion by YidC was not known and structural information was not available at the beginning of this project. We therefore focused on the structural and functional characterization of YidC and its homologs. Starting with E. coli YidC, we found that the periplasmic domain (P1D) has a stabilizing effect and determined the P1D structure at 1.8Å resolution. It shares the conserved super-β-sandwich fold of carbohydrate binding proteins connected to a C-terminal α-helical linker region that might be involved in lipid interaction. Although the function of the P1D is not clear, it has been implicated in folding of periplasmic domains in substrate proteins. As we obtained crystals of different YidC constructs rather quickly, we embarked on an elaborate screen of YidC (Oxa1 and Alb3) variants from a number of organisms. Motivated by the success of carrier-driven crystallization in the GPCR field, we made a huge effort using this technology for YidC. Although we could not improve our crystals significantly, we transfered the strategies and protocols successfully e.g. to the interaction of the chloroplast homolog Alb3 with the targeting machinery (cpSRP43). With respect to eukaryotic ribosome associated chaperones and enzymes, we increased our efforts in the structure/function analysis in the second funding period. We focused on the ribosome associated complex (RAC) that plays a central role in co-translational folding (with the Beckmann lab). RAC consists of the Hsp70 protein Ssz and the Hsp40 protein Zuotin, and forms a chaperone triade at the ribosome together with the Hsp70 protein Ssb. We characterized RAC by small-angle X- ray scattering (SAXS), X-ray crystallography and ribosome-binding assays. We determined the crystal structures of the ribosome-binding domain of Zuotin and the catalytically inactive ATPase domain of Ssz. Cryo-EM analysis shows that RAC crouches over the ribosomal tunnel exit and seems to be stabilized by interaction with expansion segment 27 (ES27) of ribosomal RNA. As RAC acts as a cochaperone that stimulates the ATPase activity of Ssb via the J-domain of Zuotin, we determined a 3.6Å structure of Ssb and a 3.2Å structure of a first Ssz/Zuotin complex. These structures together with functional assays provide the basis to dissect the molecular mechanisms of this unique chaperone system. Among enzymatic modifications of nascent chains, N-terminal acetylation is the most common one in eukaryotes. In yeast all three Nα-terminal acetyltransferase (Nat) complexes seem to bind close to the exit tunnel. We obtained a 3.4Å structure of a trimeric NatA complex. As the third subunit has a regulatory role, we currently perform a detailed kinetic characterization. Ribosome binding assays have been started with the Beckmann lab and are continued.
Publications
- (2008) Purification, crystallization and preliminary structural characterization of the periplasmic domain P1 of the Escherichia coli membrane-protein insertase YidC Acta Crystallogr Sect F Struct Biol Cryst Commun 64 Pt 2: 144-148
Ravaud, S., Wild, K. and Sinning, I.
(See online at https://doi.org/10.1107/S1744309108002364) - (2008) The crystal structure of the periplasmic domain of the Escherichia coli membrane protein insertase YidC contains a substrate binding cleft, J. Biol. Chem. 283: 9350-8
Ravaud, S., Stjepanovic, G., Wild, K., Sinning, I.
(See online at https://doi.org/10.1074/jbc.M710493200) - (2009) Delivering proteins for export from the cytosol, Nat. Rev. Mol. Cell. Biol. 10: 255-64
Cross, B.C.S., Sinning, I., Luirink, J., High, S.
(See online at https://doi.org/10.1038/nrm2657) - (2009) Signal sequences get active. Nat Chem Biol. 5: 146-7
Sinning, I., Wild, K., Bange, G.
(See online at https://doi.org/10.1038/nchembio0309-146) - (2010) cpSRP43 is a novel chaperone specific for light-harvesting chlorophyll a,b binding proteins, J. Biol. Chem. 285: 21655-61
Falk, S. & Sinning I.
(See online at https://doi.org/10.1074/jbc.C110.132746) - (2010) The C terminus of Alb3 interacts with the chromodomains 2 and 3 of cpSRP43. J. Biol. Chem. 285: le25-6
Falk, S., Sinning, I.
(See online at https://dx.doi.org/10.1074%2Fjbc.L110.160093) - (2010) The C-terminus of the Alb3 membrane insertase recruits cpSRP43 to the thylakoid membrane, J. Biol. Chem. 285: 5954-62
Falk, S., Ravaud, S., Koch, J., Sinning, I.
(See online at https://doi.org/10.1074/jbc.M109.084996) - (2011) Mdm38, is a 14-3-3-like receptor that associates with the protein synthesis machinery at the inner mitochondrial membrane. Traffic 12: 1457-66
Lupo, D., Vollmer, C., Deckers, M., Mick, D.U., Tews, I., Sinning, I., Rehling, P.
(See online at https://doi.org/10.1111/j.1600-0854.2011.01239.x) - (2013) Consistent mutational paths predict eukaryotic thermostability, BMC Evol Biol 13: 7
van Noort, V., Bradatsch, B., Arumugam, M., Amlacher, S., Bange, G., Creevey, C., Falk, S., Mende, D.R., Sinning, I., Hurt, E., Bork, P.
(See online at https://doi.org/10.1186/1471-2148-13-7) - (2013) Structural characterization of a eukaryotic co-translational chaperone, the ribosome-associated complex (RAC), Nat. Struct. Mol. Biol. 20: 23-8
Leidig, C., Bange, G., Kopp, J., Amlacher, S., Aravind, A., Wickles, S., Witte, G., Hurt, E., Beckmann, R., Sinning, I.
(See online at https://doi.org/10.1038/nsmb.2447) - (2014) Construction of the central protuberance requires roatation of 5S RNP during 60S ribosome biogenesis, Nat. Commun. 5: 3491
Leidig, C., Thoms, M., Holdermann, I., Bradatsch, B., Berninghausen, O., Bange, G., Sinning, I., Hurt, E., Beckmann, R.
(See online at https://doi.org/10.1038/ncomms4491)
