Archaeal SRP RNA and SRP19 facilitate the assembly of SRP54-FtsY targeting complex

N-terminal helices and A domain of archaeal FtsY facilitate SRP54 binding and the association with cell membrane

“…Deletion of those helices consequently affected the association between the two proteins. The Association of the wildtype TC in S. acidocaldarius was recorded previously by a similar FRET experiment which was found to be 40.5 ± 10.8 nM (Gupta et al, 2021). Deletion of 1-72 residues (αN1) augmented the equilibrium dissociation constant for SRP54-binding by almost two folds (K d = 75 ± 5.4 nM).…”

“…The construction of pAG3 and pAG351, expressing wild type SRP54 and FtsY respectively, was described earlier (Gupta et al, 2021). The deletion mutants were generated using the pAG351 backbone following site-directed mutagenesis protocol.…”

“…The basal GTPase activity and the kinetics of dual GTP hydrolysis were measured following the protocol discussed earlier (Gupta et al, 2021). Briefly, for the kinetic analyses, 1-10 μM of protein was assayed for the GTPase activity over a range of 5-30 mins.…”

“…Individual mutagenesis of the residues along the interaction surface of FtsY in E. coli severely depleted the reciprocally stimulated GTP hydrolysis by Ffh-FtsY but the basal GTPase activity of FtsY was unaffected (Egea et al, 2004). The association of the targeting complex and its dual GTPase activity depended extensively on the full-length SRP RNA both in bacteria and in archaea (Zhang et al, 2008; Gupta et al, 2021). Crystal structure of the bacterial TC showed profound domain rearrangement of Ffh upon binding the 4.5S RNA at the tetraloop region via the M domain leading to the movement of the NG domain to the distal region of helix 5 (Ataide et al, 2011).…”

“…This was further substantiated by the binding analyses between NG and M domains of Ffh and FtsY in the presence or absence of SRP RNA (Buskiewicz et al, 2005). The binding of FtsY-NG with the SRP54/Ffh-NG is facilitated due to this positional reorientation in the latter and enhanced greatly by the presence of GTP (Jagath et al, 2000; Gupta et al, 2021). Together, the two reoriented NG domains form a composite active center of the TC that binds and hydrolyses GTP, and finally, the activated TC complexed with the RNC interacts with the membrane-bound translocon SecYEG via FtsY (Draycheva et al, 2016).…”