Mechanism of association and reciprocal activation of two GTPases.
The signal recognition particle (SRP) mediates the cotranslational targeting of nascent proteins to the eukaryotic endoplasmic reticulum membrane or the bacterial plasma membrane. During this process, two GTPases, one in SRP and one in the SRP receptor (named Ffh and FtsY in bacteria, respectively), form a complex in which both proteins reciprocally activate the GTPase reaction of one another. Here, we explore by site-directed mutagenesis the role of 45 conserved surface residues in the Ffh-FtsY interaction. Mutations of a large number of residues at the interface impair complex formation, supporting the importance of an extensive interaction surface. Surprisingly, even after a stable complex is formed, single mutations in FtsY can block the activation of GTP hydrolysis in both active sites. Thus, activation requires conformational changes across the interface that coordinate the positioning of catalytic residues in both GTPase sites. A distinct class of mutants exhibits half-site reactivity and thus allows us to further uncouple the activation of individual GTPases. Our dissection of the activation process suggests discrete conformational stages during formation of the active SRP*SRP receptor complex. Each stage provides a potential control point in the targeting reaction at which regulation by additional components can be exerted, thus ensuring the binding and release of cargo at the appropriate time.
Shan
Shu-ou
SO
Department of Biochemistry and Biophysics, University of California, San Francisco, USA. sshan@itsa.ucsf.edu <sshan@itsa.ucsf.edu>
Stroud
Robert M
RM
Walter
Peter
P
D001426:Bacterial Proteins / Q000737:chemistry / Q000502:physiology*; D001665:Binding Sites; D020134:Catalytic Domain; D004252:DNA Mutational Analysis; D004789:Enzyme Activation; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000502:physiology*; D020558:GTP Phosphohydrolases / Q000737:chemistry* / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000737:chemistry; D006868:Hydrolysis; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008856:Microscopy, Fluorescence; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000502:physiology*; D018271:Signal Recognition Particle / Q000737:chemistry / Q000502:physiology*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C082931:Ffh protein, E coli; C087406:FtsY protein, Bacteria; D018160:Receptors, Cytoplasmic and Nuclear; D018271:Signal Recognition Particle; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D008274:Magnesium