High Resolution Structural Biology of the SRP GTPase

Project: Research project

Description

DESCRIPTION (provided by applicant): 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.
StatusFinished
Effective start/end date8/1/027/31/07

Funding

  • National Institute of General Medical Sciences (5 R01 GM058500-07)

Fingerprint

Signal Recognition Particle
GTP Phosphohydrolases
Guanosine Triphosphate
Proteins
Membranes
Mutagenesis
Protein Sorting Signals
Hydrolysis
Membrane Proteins
Nucleotides
Switches