All cells contain mechanisms by which proteins are targeted to different cellular compartments. Co-translational targeting of proteins to cell membranes is mediated by the signal recognition particle (SRP), which recognizes the amino-terminal signal sequence of the nascent polypeptide and targets the translating ribosome to a membrane receptor. Structural and functional elements of the SRP ribonucleoprotein are highly phylogenetically conserved. One protein component of the particle, SRP54 or Ffh (in prokaryotes), is of particular interest because it provides specific recognition of the hydrophobic signal peptide and is also a GTPase which interacts directly with the membrane receptor. GTPases are a ubiquitous family of proteins which use the binding and hydrolysis of GTP to elicit various cellular functions - well studied GTPases are involved in translation, cell regulation, and signal transduction. This proposal is directed towards detailed protein structural understanding of the NG GTPase domain of Ffh. The results will contribute to our understanding of the role of this specific GTPase in the SRP pathway, and, more generally, give insight into how different GTPases build on a common core protein structure to elicit different cellular function. The project has three crystallographic objectives. To obtain highly refined ultra-high (1.0 Angstroms) resolution structural models of the apo- and GDP-bound NG GTPase from crystals now in hand. To improve existing MgGDP-form crystals so that we can describe that structure at a similar level of detail. And, to crystallize the MgGTP-bound NG GTPase using nonhydrolyzable nucleotide analogs or GTP, and to solve its crystal structure as well. The structural data, spanning the ligand states of the GTPase, will provide the basis for detailed and accurate analysis of the interaction between the protein, water, magnesium, and nucleotide. They should also reveal whether structural phenomena not resolved in structures at lower resolution - deviations of the protein stereochemistry, packing imperfections, and conformational substrates - may be relevant to GTPase function. The analyses will address the chemistry of the GTPase and the protein design principles which facilitate mobilization of different structural motifs during the cycle of GTP binding and hydrolysis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM058500-03
Application #
6351259
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Lewis, Catherine D
Project Start
1999-02-01
Project End
2002-07-31
Budget Start
2001-02-01
Budget End
2002-07-31
Support Year
3
Fiscal Year
2001
Total Cost
$242,864
Indirect Cost
Name
Northwestern University at Chicago
Department
Pharmacology
Type
Schools of Medicine
DUNS #
005436803
City
Chicago
State
IL
Country
United States
Zip Code
60611
Ramirez, Ursula D; Focia, Pamela J; Freymann, Douglas M (2008) Nucleotide-binding flexibility in ultrahigh-resolution structures of the SRP GTPase Ffh. Acta Crystallogr D Biol Crystallogr 64:1043-53
Gawronski-Salerno, Joseph; Freymann, Douglas M (2007) Structure of the GMPPNP-stabilized NG domain complex of the SRP GTPases Ffh and FtsY. J Struct Biol 158:122-8
Gawronski-Salerno, Joseph; Coon 5th, John S; Focia, Pamela J et al. (2007) X-ray structure of the T. aquaticus FtsY:GDP complex suggests functional roles for the C-terminal helix of the SRP GTPases. Proteins 66:984-95
Focia, Pamela J; Gawronski-Salerno, Joseph; Coon 5th, John S et al. (2006) Structure of a GDP:AlF4 complex of the SRP GTPases Ffh and FtsY, and identification of a peripheral nucleotide interaction site. J Mol Biol 360:631-43
Ramirez, Ursula D; Freymann, Douglas M (2006) Analysis of protein hydration in ultrahigh-resolution structures of the SRP GTPase Ffh. Acta Crystallogr D Biol Crystallogr 62:1520-34
Focia, Pamela J; Alam, Hena; Lu, Thanh et al. (2004) Novel protein and Mg2+ configurations in the Mg2+GDP complex of the SRP GTPase ffh. Proteins 54:222-30
Focia, Pamela J; Shepotinovskaya, Irina V; Seidler, James A et al. (2004) Heterodimeric GTPase core of the SRP targeting complex. Science 303:373-7
Shepotinovskaya, Irina V; Focia, Pamela J; Freymann, Douglas M (2003) Crystallization of the GMPPCP complex of the NG domains of Thermus aquaticus Ffh and FtsY. Acta Crystallogr D Biol Crystallogr 59:1834-7
Shepotinovskaya, Irina V; Freymann, Douglas M (2002) Conformational change of the N-domain on formation of the complex between the GTPase domains of Thermus aquaticus Ffh and FtsY. Biochim Biophys Acta 1597:107-14
Ramirez, Ursula D; Minasov, George; Focia, Pamela J et al. (2002) Structural basis for mobility in the 1.1 A crystal structure of the NG domain of Thermus aquaticus Ffh. J Mol Biol 320:783-99

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