Structure of the GMPPNP-stabilized NG domain complex of the SRP GTPases Ffh and FtsY

Abstract

Ffh and FtsY are GTPase components of the signal recognition particle co-translational targeting complex that assemble during the SRP cycle to form a GTP-dependent and pseudo twofold symmetric heterodimer. Previously the SRP GTPase heterodimer has been stabilized and purified for crystallographic studies using both the non-hydrolysable GTP analog GMPPCP and the pseudo-transition state analog GDP:AlF4, revealing in both cases a buried nucleotide pair that bridges and forms a key element of the heterodimer interface. A complex of Ffh and FtsY from Thermus aquaticus formed in the presence of the analog GMPPNP could not be obtained, however. The origin of this failure was previously unclear, and it was thought to have arisen from either instability of the analog, or, alternatively, from differences in its interactions within the tightly conscribed composite active site chamber of the complex. Using insights gained from the previous structure determinations, we have now determined the structure of the SRP GTPase targeting heterodimer stabilized by the non-hydrolysable GTP analog GMPPNP. The structure demonstrates how the different GTP analogs are accommodated within the active site chamber despite slight differences in the geometry of the phosphate chain. It also reveals a K+ coordination site at the highly conserved DARGG loop at the N/G interdomain interface.

Figure 1. Comparison of the active site structure in Ffh NG:FtsY NG complexes

(A) Overall structure of the GMPPNP-stabilized SRP GTPase heterodimer. The two non-hydrolysable GTP analogs are buried within a composite active site chamber at the interface of the two proteins (arrows). The ‘N’ and ‘G’ domains of Ffh and FtsY are indicated. The extensive ‘latch’ interface is at left of the nucleotide pair, and much of the catalytic machinery is provided by the IBD subdomain, at right. The blue arrowhead indicates the point of view in (C). (B) The nucleotide analogs bound to Ffh within the active site chambers of the GMPPNP- and GMPPCP- stabilized complexes are superimposed (over the ribose and α-phosphate groups, bracketed). There are only small relative shifts of the two analogs, although the orientations of the terminal phosphate moieties change due to the different β–γ bridging group (-CH2- or -NH-). In neither structure do the bridging methylene or amido groups make direct interactions with sidechain or mainchain atoms or with water molecules. (C) Stereo diagram of the sidechain and water interactions of the buried GMPPNP pair. All buried waters and all sidechains that interact with the nucleotides are shown - note that the sidechains are exclusively arranged on the ‘IBD’ face of the nucleotide pair, as all interactions on the opposite face are mediated by mainchain atoms (and not shown in the figure). The nucleophilic (n), auxiliary (a), and shared central (c) waters are indicated. Figures were made with O, Molscript and Raster3D (Jones et al., 1991; Kraulis, 1991; Merritt and Bacon, 1997).

Figure 1. Comparison of the active site structure in Ffh NG:FtsY NG complexes

(A) Overall structure of the GMPPNP-stabilized SRP GTPase heterodimer. The two non-hydrolysable GTP analogs are buried within a composite active site chamber at the interface of the two proteins (arrows). The ‘N’ and ‘G’ domains of Ffh and FtsY are indicated. The extensive ‘latch’ interface is at left of the nucleotide pair, and much of the catalytic machinery is provided by the IBD subdomain, at right. The blue arrowhead indicates the point of view in (C). (B) The nucleotide analogs bound to Ffh within the active site chambers of the GMPPNP- and GMPPCP- stabilized complexes are superimposed (over the ribose and α-phosphate groups, bracketed). There are only small relative shifts of the two analogs, although the orientations of the terminal phosphate moieties change due to the different β–γ bridging group (-CH2- or -NH-). In neither structure do the bridging methylene or amido groups make direct interactions with sidechain or mainchain atoms or with water molecules. (C) Stereo diagram of the sidechain and water interactions of the buried GMPPNP pair. All buried waters and all sidechains that interact with the nucleotides are shown - note that the sidechains are exclusively arranged on the ‘IBD’ face of the nucleotide pair, as all interactions on the opposite face are mediated by mainchain atoms (and not shown in the figure). The nucleophilic (n), auxiliary (a), and shared central (c) waters are indicated. Figures were made with O, Molscript and Raster3D (Jones et al., 1991; Kraulis, 1991; Merritt and Bacon, 1997).

Figure 1. Comparison of the active site structure in Ffh NG:FtsY NG complexes

(A) Overall structure of the GMPPNP-stabilized SRP GTPase heterodimer. The two non-hydrolysable GTP analogs are buried within a composite active site chamber at the interface of the two proteins (arrows). The ‘N’ and ‘G’ domains of Ffh and FtsY are indicated. The extensive ‘latch’ interface is at left of the nucleotide pair, and much of the catalytic machinery is provided by the IBD subdomain, at right. The blue arrowhead indicates the point of view in (C). (B) The nucleotide analogs bound to Ffh within the active site chambers of the GMPPNP- and GMPPCP- stabilized complexes are superimposed (over the ribose and α-phosphate groups, bracketed). There are only small relative shifts of the two analogs, although the orientations of the terminal phosphate moieties change due to the different β–γ bridging group (-CH2- or -NH-). In neither structure do the bridging methylene or amido groups make direct interactions with sidechain or mainchain atoms or with water molecules. (C) Stereo diagram of the sidechain and water interactions of the buried GMPPNP pair. All buried waters and all sidechains that interact with the nucleotides are shown - note that the sidechains are exclusively arranged on the ‘IBD’ face of the nucleotide pair, as all interactions on the opposite face are mediated by mainchain atoms (and not shown in the figure). The nucleophilic (n), auxiliary (a), and shared central (c) waters are indicated. Figures were made with O, Molscript and Raster3D (Jones et al., 1991; Kraulis, 1991; Merritt and Bacon, 1997).

Figure 2. Coordination of the DARGG loop carbonyl crown

(A) In Ffh (left), Arg²⁹⁰ forms an extensive set of hydrogen bonding interactions with the carbonyl oxygens of the Ffh DARGG loop (which has the sequence Asp²⁵⁰AlaArgGlyGly²⁵⁴ in T. aquaticus Ffh). In FtsY (right), a potassium ion (large ball) is coordinated by the carbonyl crown contributed by motif IV/DARGG residues Leu²⁵⁷, Gly²⁵⁹ and Ala²⁶¹, and by three water molecules (small red balls). The 2Fo−Fc electron density map is shown with the backbone atoms of residues 257–262 in ball-and-stick. The ‘DARGG’ loop has the sequence Thr²⁶⁰AlaLysGlyGly²⁶⁴ in T. aquaticus FtsY. (B) The context of the DARGG loop interactions in (A) are shown in two orientations of a surface representation of the heterodimer. The interactions are at the junction of the N and G domains of each protein, and in Ffh, couple to the position of the C-terminal helix (which in the intact protein is linked to the signal sequence recognition subunit, the M-domain). The C-terminus is indicated (C), and the DARGG loop indicated in each case by an arc. The figure was made with PYMOL (DeLano, 2002).

Figure 2. Coordination of the DARGG loop carbonyl crown

(A) In Ffh (left), Arg²⁹⁰ forms an extensive set of hydrogen bonding interactions with the carbonyl oxygens of the Ffh DARGG loop (which has the sequence Asp²⁵⁰AlaArgGlyGly²⁵⁴ in T. aquaticus Ffh). In FtsY (right), a potassium ion (large ball) is coordinated by the carbonyl crown contributed by motif IV/DARGG residues Leu²⁵⁷, Gly²⁵⁹ and Ala²⁶¹, and by three water molecules (small red balls). The 2Fo−Fc electron density map is shown with the backbone atoms of residues 257–262 in ball-and-stick. The ‘DARGG’ loop has the sequence Thr²⁶⁰AlaLysGlyGly²⁶⁴ in T. aquaticus FtsY. (B) The context of the DARGG loop interactions in (A) are shown in two orientations of a surface representation of the heterodimer. The interactions are at the junction of the N and G domains of each protein, and in Ffh, couple to the position of the C-terminal helix (which in the intact protein is linked to the signal sequence recognition subunit, the M-domain). The C-terminus is indicated (C), and the DARGG loop indicated in each case by an arc. The figure was made with PYMOL (DeLano, 2002).