Conformational change of the N-domain on formation of the complex between the GTPase domains of Thermus aquaticus Ffh and FtsY

Abstract

The structural basis for the GTP-dependent co-translational targeting complex between the signal recognition particle (SRP) and its receptor is unknown. The complex has been shown to have unusual kinetics of formation, and association in vivo is likely to be dependent on catalysis by the SRP RNA. We have determined conditions for RNA-independent association of the 'NG' GTPase domains of the prokaryotic homologs of the SRP components, Ffh and FtsY, from Thermus aquaticus. Consistent with previous studies of the Escherichia coli proteins, the kinetics of association and dissociation are slow. The T. aquaticus FtsY is sensitive to an endogenous proteolytic activity that cleaves at two sites--the first in a lengthy linker peptide that spans the interface between the N and G domains, and the second near the N-terminus of the N domain of FtsY. Remarkably, this second cleavage occurs only on formation of the Ffh/FtsY complex. The change in protease sensitivity of this region, which is relatively unstructured in the FtsY but not in the Ffh NG domain, implies that it undergoes conformational change on formation of the complex between the two proteins. The N domain, therefore, participates in the interactions that mediate the GTP-dependent formation of the targeting complex.

Fig. 1

Ffh/FtsY NG complex formation demonstrated by gel filtration chromatography. The 100-µl reactions were prepared with 10 µM FtsY, 15 µM Ffh NG and 1 mM GMPPCP in 50 mM HEPES (pH 7.5), 2 mM MgCl₂, 50 mM NaCl and were incubated for the indicated time interval and temperature. The full reaction mixture was injected onto a Superdex 200 HR 10/30 column equilibrated with 50 mM HEPES (pH 7.5), 2 mM MgCl₂, 50 mM NaCl. HPLC traces (280 nm) correspond to: (a) 42 °C overnight, (b) 3 days 37 °C, and (c) 4 days at 37 °C. No complex was observed after short incubations (1 h to overnight) over temperatures ranging from RT to 70 °C. Results from control reactions (all carried out at 37 °C, 4 days) are shown: (d) FtsY alone with GMPPCP, (e) Ffh/FtsY complex reaction with GDP substituted for GMPPCP; and (f) the reaction mixture plus 2 mM EDTA. The position of the complex peak is indicated (asterisk), and the elution volumes of 29 and 150 kDa standards are shown. The expected ratio of the total absorbance at 280 nm for the complex peak relative to the monomer peak assuming 100% complex yield under these conditions is ~ 8.3–1.

Fig. 2

A slow dissociation rate for the complex. After a preincubation of a reaction mixture at 37 °C (100 µl, containing 10 µM FtsY, 15 µM Ffh NG, 50 mM HEPES, pH 7.5, 2 mM MgCl₂, 50 mM NaCl, 1 mM GMPPCP), GDP was added to 5 mM final concentration and the incubation continued at RT. Samples were taken at the indicated times (0–48 h). Dissociation of the complex in the presence of excess GDP was monitored using anion exchange chromatography on a Poros HQ column equilibrated with 50 mM Tris (pH 8.0), 2 mM MgCl₂ and eluted with linear gradient of 1 M NaCl. After 18 h, ~ 50% of the initial complex was still intact.

Fig. 3

Different patterns of cleavage of FtsY free in solution and in the Ffh/FtsY complex. Protein samples (10 µM FtsY and 15 µM NG) were incubated in ‘low-salt’ conditions (50 mM HEPES (pH 7.5), 2 mM MgCl₂, 50 mM NaCl) and ‘high-salt’ conditions (50 mM HEPES (pH 7.5), 2 mM MgCl₂, 300 mM NaCl) at 37 °C. GMPPCP or GDP were used at a final concentration of 1 mM. (a) ‘Low-salt’ experiments. Lane 1—Mark 12 MW standard; lanes 2 and 3—Ffh and FtsY NG stored at −20 °C; lane 4—FtsY, nucleotide-free, 4 days at 37 °C; lane 5—Ffh/FtsY complex purified after 4 days at 37 °C. (b) ‘High-salt’ experiments. Lane 1—Standard; lane 2—Ffh and FtsY NG nucleotide-free control, co-purified by gel filtration; lane 3—Ffh and FtsY NG incubated with GDP at 37 °C for 4 days; lane 4—Ffh/FtsY NG complex formation reaction mix, incubated with GMPPCP 37 °C for 4 days; lane 5—Ffh/FtsY complex purified by gel filtration. Note the appearance of the FtsY cleavage product during the GMPPCP incubation (lane 4) but not the GDP incubation (lane 3). After blotting to PVDF membranes, the fragments marked by arrowheads were cut out and sequenced. The bands are labeled (N-terminus and sites 1 and 2) corresponding to the N-terminal sequences obtained (see Fig. 4). Note that on this gel system FtsY and its larger cleavage products migrate slow relative to the markers and its smaller cleavage products migrate anomalously fast.

Fig. 4

Mapping the sites of proteolysis of FtsY. The sequences of the cleavage products of FtsY (indicated in Fig. 3) are shown on a linear map of the N domain of FtsY NG. Sequences identified the N-terminus (GFFDR), overlapping sites at position 1 (KLGFN and GFNPQ), and overlapping sites at position 2 (LKAIPWG and AIPxG). The secondary structure of the domain is diagrammed based on the published structure of the E. coli FtsY NG domain [19], patterns of sequence identity, and the position of the conserved ‘ALLEADV’ motif between helices αN2 and αN3 (indicated by asterisks). The N domain comprises the first ~ 82 residues of the protein, the ‘linker’ peptide, residues ~ 83–101, and the G domain, residues ~ 102–304. The three helices of the N domain that can be identified with confidence and are indicated (αN2, αN3, αN4). The N-terminal helix αN1 does not appear as such in the E. coli FtsY structure (see Fig. 5); the approximate position of the helix in Ffh NG is indicated (‘~ αN1 ~’). The diagram is shaded by the percentage of sequence conservation at each position in the sequence (white, < 30%; light grey, 30–60%; grey 60–100%).

Fig. 5

A structural model for the locations of the cleavage sites. The positions of the two cleavage sites 1 and 2 are indicated relative to the structure of the E. coli FtsY NG domain (the G domain is truncated at right). Cleavage position 1 can be mapped to the linker peptide that extends between the N and G domains. This region varies widely in sequence and structure between different members of the SRP GTPase family, and may well be disordered and accessible to proteolysis in T. aquaticus FtsY. Cleavage position 2 maps to an N-terminal region that has somewhat irregular conformation and packs against the hydrophobic core of the N domain. The polypeptide backbone at position 2 is presumably inaccessible to proteolysis in the E. coli FtsY conformation, but would become accessible if the region underwent conformational change or if it became displaced in the complex. The position of the conserved ‘ALLEADV’ motif is indicated (asterisks). This figure was generated using Molscript and Raster3d [43,44].