The aim is to understand signal recognition particle (SRP) dependent targeting of membrane and secretory proteins to the translocation machinery of the cell, at the level of molecular structure and chemical mechanism. We have prepared, and solved structures for, the 'N' and GTPase domains of the prokaryotic homologue of the 54 kD protein component (Ffh) of the SRP from T. aquaticus and the entire Ffh including the signal sequence and SRP RNA binding M domain. We seek to understand binding of signal sequences at the level of affinity, specificity and three-dimensional structure. Mutations of hydrophobic residues that line the signal-binding site will be correlated with signal sequence binding and Ffh structure. Crystal structures will elucidate the mechanism by which binding of the SRP to its membrane bound receptor (SR) is coupled to GTPase activation. Mutations made in the conserved interface between 'N' and GTPase domains will elucidate the mechanism of allosteric coupling of nucleotide binding to receptor binding in unidirectional targeting to the membrane. The role of RNA in SRP structure and function and in mediating translation arrest of ribosomal synthesis will be defined using crystallography and electron microscopy. Components of the membrane pore are to be prepared and characterized for structural approaches to translocation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM060641-03
Application #
6490234
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Lewis, Catherine D
Project Start
2000-01-01
Project End
2003-12-31
Budget Start
2002-01-01
Budget End
2002-12-31
Support Year
3
Fiscal Year
2002
Total Cost
$281,788
Indirect Cost
Name
University of California San Francisco
Department
Biochemistry
Type
Schools of Medicine
DUNS #
073133571
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Egea, Pascal F; Muller-Steffner, Hélène; Kuhn, Isabelle et al. (2012) Insights into the mechanism of bovine CD38/NAD+glycohydrolase from the X-ray structures of its Michaelis complex and covalently-trapped intermediates. PLoS One 7:e34918
Korennykh, Alexei V; Egea, Pascal F; Korostelev, Andrei A et al. (2011) Cofactor-mediated conformational control in the bifunctional kinase/RNase Ire1. BMC Biol 9:48
Egea, Pascal F; Stroud, Robert M (2010) Lateral opening of a translocon upon entry of protein suggests the mechanism of insertion into membranes. Proc Natl Acad Sci U S A 107:17182-7
Korennykh, Alexei V; Egea, Pascal F; Korostelev, Andrei A et al. (2009) The unfolded protein response signals through high-order assembly of Ire1. Nature 457:687-93
Egea, Pascal F; Napetschnig, Johanna; Walter, Peter et al. (2008) Structures of SRP54 and SRP19, the two proteins that organize the ribonucleic core of the signal recognition particle from Pyrococcus furiosus. PLoS One 3:e3528
Egea, Pascal F; Tsuruta, Hiro; de Leon, Gladys P et al. (2008) Structures of the signal recognition particle receptor from the archaeon Pyrococcus furiosus: implications for the targeting step at the membrane. PLoS One 3:e3619
Reyes, Christopher L; Rutenber, Earl; Walter, Peter et al. (2007) X-ray structures of the signal recognition particle receptor reveal targeting cycle intermediates. PLoS One 2:e607
Credle, Joel J; Finer-Moore, Janet S; Papa, Feroz R et al. (2005) On the mechanism of sensing unfolded protein in the endoplasmic reticulum. Proc Natl Acad Sci U S A 102:18773-84
Egea, Pascal F; Stroud, Robert M; Walter, Peter (2005) Targeting proteins to membranes: structure of the signal recognition particle. Curr Opin Struct Biol 15:213-20
Chu, Feixia; Shan, Shu-ou; Moustakas, Demetri T et al. (2004) Unraveling the interface of signal recognition particle and its receptor by using chemical cross-linking and tandem mass spectrometry. Proc Natl Acad Sci U S A 101:16454-9