Mechanism of association and reciprocal activation of two GTPases

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.

Figure 1. The Mutational Effects in E. coli FtsY Mapped onto the Crystal Structure of the Ffh•FtsY Complex

The bound nucleotides are shown as black sticks, and the dotted white lines in the interface view outline the contact surface of Ffh with FtsY. The colors denote different classes of mutational effects: blue, Class I mutants defective in complex formation; red, Class II mutants defective in the reciprocal GTPase activation; magenta, Class III mutants defective in both steps; green, Class IV mutants exhibiting half-site reactivity; yellow, Class V or neutral mutants.

Figure 2. The Effect of FtsY Mutations on the Reciprocally Stimulated GTPase Reaction between Ffh and FtsY

The stimulated GTPase reactions of (A) mutant FtsYE475(274)K (•), (B) FtsY T307(112)A (•), (C) FtsYA335(140)W (•), (D) FtsYR333(138)A (•), and wild-type FtsY (○) were determined as described in Materials and Methods. The insets show the reaction curve of the mutant FtsYs on an expanded scale.

Figure 3. Determination of Complex Formation between Ffh and FtsY Mutants

(A) Inhibition assay for determining the affinity of mutant FtsY proteins for Ffh, as described in the text and in Materials and Methods. (B and C) Representative inhibition curves are shown for FtsY mutants (B) T307(112)A and (C) A335(140)W. The data were fit to equation 3 in Materials and Methods.

Figure 4. Fluorescence Characterization of Complex Formation between Ffh and Mutant FtsYA335(140)W

(A) The tryptophan fluorescence of mutant FtsYA335(140)W changes upon complex formation with Ffh. Complex formation was initiated by the addition of Mg²⁺, as described previously (Shan and Walter 2003). Other Class II mutants do not exhibit as significant a fluorescence change (unpublished data). Thus, the conformational change that alters the environment surrounding the fluorescent W343(148) does not occur even though these mutants can form stable complexes with Ffh. (B) Association rate constants for complex formation with mutant FtsYA335(140)W (○) and wild-type FtsY(•). Linear fits to the data gave association rate constants of 6.36 × 10⁴ and 6.34 × 104 M⁻¹ s⁻¹ for wild-type and mutant FtsY, respectively. (C) Dissociation rate constants of the Ffh•FtsY complexes formed by mutant FtsYA335(140)W (upper curve) and wild-type FtsY (lower curve). First-order fits to the data gave dissociation rate constants of 3.6 × 10⁻³ and 4.2 × 10⁻³ s⁻¹ for wild-type and mutant FtsY, respectively.

Figure 5. Half-Site Mutants Are Compromised in the Hydrolysis Reaction from the FtsY but Not Ffh Active Site

(A) The reciprocally stimulated GTPase reaction with wild-type Ffh for wild-type FtsY (○) and mutant FtsYG455(254)W (•). (B) The FfhD251N-stimulated GTPase reaction from wild-type FtsY (○) and mutant FtsYG455(254)W (•), determined as described in Materials and Methods. (C) The XTP hydrolysis reaction from FfhD251N stimulated by wild-type FtsY (○) and mutant FtsYG455(254)W (•), determined as described in Materials and Methods.

Figure 6. Model for Conformational Changes during Ffh-FtsY Reciprocal GTPase Activation and Implications for the Protein-Targeting Reaction

(A) Model for conformational changes during formation of an activated Ffh-FtsY complex. Step a is the rearrangement of both proteins from the open to the closed state during complex formation. Step b is the coordinate docking of the IBD loops into the active sites, and step c is the docking of the Arg191s. Step d is the additional rearrangement of residues that completes one or the other GTPase site. Step e is the rearrangement that completes the other active site. GTP can be hydrolyzed from either the hemiactivated complexes (step f) or the activated complex (step g) to drive complex dissociation. (B–D) Catalytic interactions made by residues exhibiting the Class II phenotype. FtsY is in surface representation, the catalytic residues from FtsY are depicted as red sticks, the nucleotides bound to FtsY and Ffh are in dark green and dark blue, respectively, and the dotted lines depict hydrogen bonds or van der Waals contacts. (B) Interaction of IBD loop with GTP in the FtsY active site. The blue ball represents the attacking water molecule (A.W.); the violet red ball represents the active site Mg²⁺. (C) Interactions of Asn111(107) at the Ffh-FtsY interface. The residue homologous to Asn111, Gln107 in Ffh, is in violet red. (D) Arg195(191) is in a “pending” position. The residue homologous to Arg191 in Ras, Gln61, is in violet red. (E) Conformational changes in the GTPase domains of SRP and SR provide potential regulatory points during the protein-targeting reaction. Step 1, SR undergoes an open → closed conformational change upon association with the membrane translocon. Step 2, SRP undergoes an open → closed conformational change upon association with the ribosome and nascent polypeptide. Step 3, complex formation between SRP and SR delivers the cargo to the membrane. Step 4, cargo release from SRP allows the SRP•SR complex to undergo additional conformational changes to activate GTP hydrolysis. Step 5, SRP dissociates from SR after GTP is hydrolyzed. Note that steps 1–3 correspond to Ffh-FtsY binding (step a) in the model shown in (A), step 4 corresponds to Ffh•FtsY activation (steps b–e) in the model shown in (A), and step 5 corresponds to Ffh•FtsY complex dissociation (step g) in the model shown in (A).