Date Published: October 21, 2004
Publisher: Public Library of Science
Author(s): Shu-ou Shan, Robert M Stroud, Peter Walter
Abstract: 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.
Partial Text: GTPases comprise a superfamily of proteins that provide molecular switches to regulate many cellular processes, including translation, signal transduction, cytoskeletal organization, vesicle transport, nuclear transport, and spindle assembly (Gilman 1987; Bourne et al. 1991). In many cases, the GTPases exert their regulatory function through a “GTPase switch” mechanism (Bourne et al. 1991) in which the GTPase assumes two alternative conformational states: an active, GTP-bound state and an inactive, GDP-bound state. Each state is kinetically stable, and interconversion between these states is facilitated by external regulatory factors, such as GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs).
In light of the recently published structures of the Ffh•FtsY complex, kinetic analyses become increasingly valuable in unraveling the dynamic nature of the Ffh-FtsY interaction. To this end, we generated 45 site-directed mutants that were made in surface residues of FtsY. As previously described (Egea et al. 2004), all but one mutation that functionally compromise the Ffh-FtsY interaction map to the extensive interaction surface between the two proteins (Figure 1). As we show below, dissection of the mutational effect on individual steps allows us to divide the deleterious mutants into distinct classes: Class I mutants primarily affect complex formation, Class II mutants primarily affect the reciprocal GTPase activation, Class III mutants are defective in both steps, and Class IV or half-site mutants block the activation of only one GTPase site in the complex (Table 1).
The mutational analyses described here define four distinct classes of mutants that map to the Ffh-FtsY interface. Each mutant class blocks the reaction in a different way and at a distinct stage, demonstrating that (i) multiple conformational rearrangements are required to form an activated Ffh•FtsY complex and (ii) some rearrangements can be blocked without preventing other rearrangements from taking place. The different classes of mutant interrupt the reaction in different ways, as represented by the states depicted in Figure 6A, in the pathway of Ffh•FtsY complex formation and reciprocal GTPase activation. The most plausible interpretation of our analysis and the crystallographic analysis of the Ffh•FtsY complex suggest that each of the states blocked by the mutants represents a step on the pathway for the wild-type protein. However, we cannot rule out that some of the rearrangements could occur independently of one another, in which case their depicted order represents only one of the possibilities. Our analysis leads to the conclusion that perturbations, such as those introduced here by site-specific mutations, can modulate specific conformational changes during the Ffh-FtsY interaction. Each of these states provides a potential regulatory point during the protein-targeting reaction, at which analogous effects could be exerted by the cargoes of SRP and SR—the ribosome, signal sequence, and translocon.