The long term objective of the proposed research is to obtain a precise functional understanding of the molecular mechanisms of the two GTPases that play a central role in signal recognition particle (SRP) mediated targeting of secreted and membrane proteins. The two proteins, Ffh, the signal sequence recognition subunit of the SRP, and FtsY, its membrane-associated receptor, undergo a molecular 'handshake' during transfer of the translating ribosome nascent chain complex from the cytosolic SRP to the membrane translocon. Remarkably, the GTPase domains of the two proteins are structural homologs and interact directly. The mechanism for formation of this transient protein:protein complex is central to understanding the fundamentally important SRP targeting mechanism. Here, three lines of investigation to address its structural basis are proposed: First, the structures of the apo- and nucleotide-bound Fth GTPase 'NG' domain are being determined at -~ 1.0 A resolution. The completed structures should allow detailed and accurate analysis of functionally important structural elements that are not revealed in structures determined at lower resolution. Second, the stable complex of the NG domains of Ffh and FtsY has been trapped in the presence of a non-hydrolyzable GTP analog and will be characterized biochemically with the aim of crystallizing the complex and determining its X-ray structure. Small 'lead' crystals have been obtained. The structure of the FfhIFtsY NG complex will be fundamental for understanding the molecular details of the interaction of SRP with its receptor. Finally, biochemical and site-directed mutagenesis studies will be carried out to test specific hypotheses that address the structural basis of the interaction between the two proteins. This work will build on the results of the two previous aims. Future studies will be directed towards understanding why two homologous 'NG' GTPases occur at subsequent steps in the SRP targeting pathway. Because the SRP GTPases are members of a poorly understood group of GTPases that exhibit a functional logic different from the classic 'GTPase switch', an understanding of the structural basis for formation of the targeting complex will be of importance not only with respect to the SRP, but also to understanding how 'assembly-activated' GTPases build on the common GTPase fold to harness GTP binding and hydrolysis to organize cellular components for function.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM058500-05
Application #
6611055
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Flicker, Paula F
Project Start
1999-02-01
Project End
2006-07-31
Budget Start
2003-08-01
Budget End
2004-07-31
Support Year
5
Fiscal Year
2003
Total Cost
$259,948
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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