Heterodimeric GTPase Core of the SRP Targeting Complex

Abstract

Two structurally homologous guanosine triphosphatase (GTPase) domains interact directly during signal recognition particle (SRP)-mediated cotranslational targeting of proteins to the membrane. The 2.05 angstrom structure of a complex of the NG GTPase domains of Ffh and FtsY reveals a remarkably symmetric heterodimer sequestering a composite active site that contains two bound nucleotides. The structure explains the coordinate activation of the two GTPases. Conformational changes coupled to formation of their extensive interface may function allosterically to signal formation of the targeting complex to the signal-sequence binding site and the translocon. We propose that the complex represents a molecular "latch" and that its disengagement is regulated by completion of assembly of the GTPase active site.

Structure of a GDP:AlF4 Complex of the SRP GTPases Ffh and FtsY, and Identification of a Peripheral Nucleotide Interaction Site

“…The GMPPCP-stabilized structure represents a "ground state" of the targeting complex, before interaction with other components of the SRP and the conformational rearrangements that deliver the translating ribosome to the membrane translocon. Subsequent structural and biochemical studies support the notion that multiple structural states occur along the path from assembly to disengagement of the targeting complex, 13,15 and that these as yet uncharacterized structural changes may provide points of regulation. Indeed, mutagenesis of E. coli FtsY 15 has identified residues at the heterodimer interface that have little effect on assembly but that severely affect the subsequent hydrolysis of GTP by the complex.…”

“…13 The overall structure of the heterodimer is essentially unchanged, with an RMSD over the N and G domains of both Ffh and FtsY of only 0.56 Å over 549 C α atoms. Remarkably, the configurations of the ligands in the two structures are nearly identical as well.…”

“…The X-ray structure of the heterodimeric complex of the GTPase domains of Thermus aquaticus Ffh and FtsY stabilized using the GTP analog GMPPCP 11 in the absence of the SRP RNA [12][13][14] reveals that they adopt an approximately 2-fold symmetric arrangement that brings their two active sites together to form a shared catalytic chamber at the interface between them. The GMPPCP-stabilized structure represents a "ground state" of the targeting complex, before interaction with other components of the SRP and the conformational rearrangements that deliver the translating ribosome to the membrane translocon.…”

Electrostatics and Intrinsic Disorder Drive Translocon Binding of the SRP Receptor FtsY

“…The flexibility of the A domain allows the NG domain to scan a large area for binding to the NG domain of RNC‐bound Ffh/SRP, such that it can recruit an RNC to the translocon by forming the heterodimeric complex of the homologous NG domains17, 18 of translocon‐bound FtsY and RNC‐bound SRP. Thus the A domain of FtsY provides an example for an intrinsically disordered protein region that has a tethering function in a multicomponent protein assembly and illustrates how simple physical principles, such as electrostatic interactions, can drive complex cellular processes, such as the assembly of the quaternary transfer complex at the membrane.…”

“…Electrostatic attraction between the negatively charged central A domain (red) and the positively charged and exposed C4/C5 loop region (blue) of the translocon SecYEG directs FtsY to the translocon. The NG domain of translocon‐bound FtsY searches for the NG domain of RNC‐bound SRP/Ffh (green) to initiate quaternary transfer complex formation by establishing interactions between the NG domains of FtsY and Ffh 17, 18…”