The functional core proteins of the signal recognition particle (SRP) and the SRP receptor (SR) (called Ffh and FtsY in bacteria) contain GTPase domains and form a distinct subfamily of GTPases. These GTPases mediate cotranslational targeting of secretory and membrane proteins to the endoplasmic reticulum (ER) membrane in eukaryotes or the plasma membrane in prokaryotes (for a review, see 
). The classical GTPase motifs I-IV (also referred to as G1-G4) 
are present in both SRP GTPases and show marked conservation with p21Ras. Present in and unique to SRP and SR are four additional elements, the insertion box domain (IBD), the closing loop, the ‘DARGG’ motif and the ‘ALLEADV’ motif. These elements contain essential structural functionality for SRP GTPases (for a review, see 
). In contrast to the ‘classical’ model of GTPase regulation by external factors, SRP GTPases interact directly to reciprocally stimulate GTP hydrolysis and neither requires an exchange factor for nucleotide release 
. The SRP family of GTPases thus provides a unique variation to the ‘classical’ GTPase model and the elucidation of the underlying mechanisms involved in regulating the targeting reaction is at the core of current structural and biochemical studies.
A family of crystal structures of prokaryotic SRP GTPases illustrates this unique mechanism of activation. In particular, apo structures of Ffh-NG, a truncated version of the prokaryotic SRP core protein containing the amino- and GTPase domains, and FtsY, the SR protein, show the stabilization of an “open” state through interactions of the GTPase and SRP conserved sequence motif residues 
. “Open” state conformations in the presence of either bound nonhydrolyzable GTP substrate analog guanine 5′-imidotriphosphate (GMPPNP) or product GDP have been shown for Ffh-NG 
. Similarly a structure of FtsY with the product, GDP has been obtained 
. In this structure, the GDP is coordinated with canonical binding interactions and reveals the importance of the C-terminal helix in the NG packing interface. In addition, three additional apo structures of FtsY have been solved and show distinct properties from FtsY in complex with Ffh 
. Low measured intrinsic GTPase activities of Ffh and of FtsY (0.09 min−1
, 0.01 min−1
imply that proteins bind GTP and remain in “open” conformational states. Low specificity for nucleotide in monomeric FtsY has also been shown and further suggests a novel structural regulation of GTPase activity for FtsY 
The structures of the FtsY·Ffh-NG complex in presence of the non-hydrolyzable substrate analog GMPPCP 
, GMPPNP 
, or GDP:AlF4 
show the formation of a composite, active site sequestered from solvent, through the catalytic interactions between the classical GTPase and SRP specific conserved elements from both GTPases and the bound nucleotides. Nucleotide hydrolysis in each active site drives dissociation of the SRP·SRP receptor complex, allowing the SRP and SRP receptor components to be recycled 
. Interestingly, the FtsY·Ffh-NG complex structures also suggest that (i)
the structures observed represent a ground state of the GTP hydrolysis reaction, and (ii)
that additional conformational changes in the active site are necessary to progress to the transition state. Recently, mutational studies of FtsY have revealed that site-specific mutations can modulate discrete conformational changes during Ffh·FtsY complex formation 
. These specific conformational states involve the sequential activation of the Ffh and FtsY active sites after binding of GTP and during the formation of the targeting complex.
Here we report the structures of two conformations of FtsY in complex with the substrate analogue GMPPNP. These structures reveal two novel active site architectures for SR GTPase-nucleotide complexes. These structures, along with the structure of apo FtsY and FtsY in the Ffh-NG·FtsY complex, can be interpreted as a series of discrete conformational states along the pathway of step-wise activation of the SRP GTPases during the formation of the SRP·SR complex and provide structural explanations for the biochemical differences observed for FtsY along the targeting cycle.