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 T. aquaticus. Consistent with previous studies of the
E. 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.
Comment
Here we showed how to assemble a stable complex of the two
GTPases of Ffh and FtsY. We had
previously demonstrated, in unpublished work, that Ffh hydrolyzes GTPgS and
GMPPNP over long timescales (such as would be used in crystallization) and high
temperatures (such as might be used to assemble the complex of the thermophilic
proteins). We settled on the more
stable nucleotide analog GMPPCP.
Key to the assembly of the complex was the decision to carry out the
incubation for days, rather than hours - we found that over that period an
N-terminal peptide was cleaved off, and that this was required for
stable assembly of the targeting complex.
This work provided the biochemical basis for the subsequent
determination of the structure of the complex.