Heterodimeric GTPase core of the SRP targeting complex - PubMed (original) (raw)

Heterodimeric GTPase core of the SRP targeting complex

Pamela J Focia et al. Science. 2004.

Abstract

Two structurally homologous guanosine triphosphatase (GTPase) domains interact directly during signal recognition particle (SRP)-mediated cotranslational targeting of proteins to the membrane. The 2.05 angstrom structure of a complex of the NG GTPase domains of Ffh and FtsY reveals a remarkably symmetric heterodimer sequestering a composite active site that contains two bound nucleotides. The structure explains the coordinate activation of the two GTPases. Conformational changes coupled to formation of their extensive interface may function allosterically to signal formation of the targeting complex to the signal-sequence binding site and the translocon. We propose that the complex represents a molecular "latch" and that its disengagement is regulated by completion of assembly of the GTPase active site.

PubMed Disclaimer

Figures

Fig. 1

Overall structure of the heterodimeric Ffh/FtsY NG domain complex. (A) Ribbon representation viewed perpendicular to the dimer axis, which is vertical in the figure. The N domains (blue) and C-terminal helices (golden) of the two proteins are at the top, and their IBD domains (purple) are at the bottom. The two active sites are brought into direct apposition to form an active site chamber at the center of the G domains (gray), where the buried GMPPCP ligands are shown. The motif I P-loops of the two proteins pack adjacent to each other (*). The structure is highly symmetric, with the exception of the smaller N domain of FtsY, and all secondary structure elements adopt the same orientation in both proteins. (B) The structure viewed along the two-fold axis further highlights the symmetry of the complex. The viewpoint is toward the IBD, at the bottom of the diagram in (A).

Fig. 2

An extensive interaction surface. (A) The molecular surfaces of the Ffh monomer (left) and the FtsY monomer (right) are shown, shaded by the change in accessible surface area at each residue between the monomer and in the heterodimer. The blue areas define the protein-protein contact. The GTP binding motifs I to IV are indicated, and the Mg2+ nucleotide ligands are shown in ball and stick representation. A symmetric triangular contact region above the active site cavity is termed the latch. The IBD regions of the two proteins contact one another below the active site cleft. The packing orientation in the complex can be visualized by rotating the monomers to overlay the yellow asterisks. Arrows on the surface of the FtsY monomer highlight the orientation of the Asp/Lys framework (black) and the latch interface (pink) presented in the following panels. (B) The framework formed by Asp229(219) of the DGQ motif (see table S1) and Lys256(246) of motif IV from both monomers is shown superimposed to emphasize the symmetry between Ffh and FtsY in the complex. This symmetric interaction lies approximately along the diagonal ridge located above the active site clefts in(A). The lysine hydrogen bonds to both P-loops, thus bridging the interface. In all figures, residues from FtsY are labeled in gray italics font and from Ffh in black font. (C) The symmetric latch interface between the N and G domains, corresponding to the close loop contacts seen above the adjacent P-loops in Fig. 1A. The conserved hydrophobic residues of the ALLEADV motifs of the N domains (top) and the symmetric glycine pair of the DGQ motifs of the G domains (bottom) are shown along with the pair of bridging aspartate and glutamine residues.

Fig. 3

Conformational changes generate the heterodimer interface. (A) The structure of the Ffh NG domain with GMPPNP bound (1JPJ.pdb) (in lighter colors) is superimposed with its structure in the complex. The N domain moves as a rigid body toward helix α3 of the G domain; this shift, in turn, is coupled to conformational rearrangement in the DGQ motif at the N terminus of α3, enabling formation of the extensive heterodimeric contact there. Helix α4 moves with the N domain, accommodated by an ~2.9 Å translation of the remainder of helix α3. Note the concurrent reorientation of the C-terminal helix. (B) G-domain conformational changes associated with complex formation are limited to the loops of conserved sequence motifs. The magnitude of the shifts are mapped so that the largest shifts (~6.5 Å) are the darkest shaded regions. (C) Reorientation of motifs II and III upon complex formation. The left panel shows the Ffh NG GMPPNP structure, the right panel Ffh NG in the complex. The side chain of motif III residue Leu192 moves to insert into a pocket across the heterodimer interface, between the guanine base and Gly259(249) that follows motif IV. Movement of this leucine and the accompanying rearrangement of the motif III backbone allows the P-loop to open sufficiently to accommodate the nucleotide in an extended conformation (10). Motif II residues Asp135 and Arg138 move into the catalytic chamber. The same configuration is observed in FtsY.

Fig. 4

A composite active site. (A) A stereo image shows the nucleotides interacting directly and symmetrically across the complex interface within the active site chamber. Residues from Ffh are shown in gray, from FtsY in purple. Ffh motif II residue Arg138 extends from the right, and Arg142(138) (FtsY) from the left. A water molecule is located asymmetrically between them (large central red sphere). Each GMPPCP ribose O3′ hydroxyl forms a short hydrogen bond with the γ-phosphate oxygen of the other across the active site chamber. The putative catalytic water molecule at each active center is shown as a large red sphere, hydrogen bonded to Asp135 [and Asp139(135)] and hydrogen bonded to another water molecule that interacts across the dimer interface with invariant Glu284(274). Two other invariant arginines, Arg191 and Arg195(191), point away from the active site chamber but may be reoriented by further conformational rearrangement of motif III. (B) Superimposition of the Ffh NG with Ras/RasGAP in complex with GDP/Mg/AlF3 (1WQ1.pdb). Active site ligands, loops, and side chains from the Ras/RasGAP complex are shown with thicker lines and labeled in gray. Only the nucleotide bound to FtsY is shown for the heterodimer; the other superimposes well with the Ras/RasGAP ligands, as shown by the position of the magnesium ion from Ffh (small purple sphere center) and the putative catalytic water (small red sphere, *). An additional active site water (above right), which hydrogen bonds Wat13 and Glu284, is nearly superimposed with the Ras motif III Gln61 OE2. Gly190 of Ffh motif III is only 2 Å from the AlF3 bound in the Ras complex, suggesting that motif III undergoes a further conformational change to accommodate the transition-state structure of the Ffh/FtsY heterodimer.

References

1. Keenan RJ, Freymann DM, Stroud RM, Walter P. Annu Rev Biochem. 2001;70:755. - PubMed
1. Bernstein HD, et al. Nature. 1989;340:482. - PubMed
1. Römisch K, et al. Nature. 1989;340:478. - PubMed
1. Miller JD, Wilhelm H, Gierasch L, Gilmore R, Walter P. Nature. 1993;366:351. - PubMed
1. Powers T, Walter P. Science. 1995;269:1422. - PubMed

Heterodimeric GTPase core of the SRP targeting complex - PubMed (original) (raw)