PDBsum entry 1okk

Cell cycle

PDB id: 1okk

Contents

Protein chains

290 a.a. *

265 a.a. *

Ligands

GCP ×2

BZP ×2

SO4 ×11

EDO ×4

Metals

_MG ×2

Waters ×568

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Heterodimeric gtpase core of the srp targeting complex.

Authors

P.J.Focia, I.V.Shepotinovskaya, J.A.Seidler, D.M.Freymann.

Ref.

Science, 2004, 303, 373-377. [DOI no: 10.1126/science.1090827]

PubMed id

14726591

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.

Figure 2.
Fig. 2. An extensive interaction surface. (A) The molecular surfaces of the Ffh monomer (left) and the FtsY monomer (right) are shown, shaded by the change in accessible surface area at each residue between the monomer and in the heterodimer. The blue areas define the protein-protein contact. The GTP binding motifs I to IV are indicated, and the Mg2+ nucleotide ligands are shown in ball and stick representation. A symmetric triangular contact region above the active site cavity is termed the latch. The IBD regions of the two proteins contact one another below the active site cleft. The packing orientation in the complex can be visualized by rotating the monomers to overlay the yellow asterisks. Arrows on the surface of the FtsY monomer highlight the orientation of the Asp/Lys framework (black) and the latch interface (pink) presented in the following panels. (B) The framework formed by Asp229(219) of the DGQ motif (see table S1) and Lys256(246) of motif IV from both monomers is shown superimposed to emphasize the symmetry between Ffh and FtsY in the complex. This symmetric interaction lies approximately along the diagonal ridge located above the active site clefts in (A). The lysine hydrogen bonds to both P-loops, thus bridging the interface. In all figures, residues from FtsY are labeled in gray italics font and from Ffh in black font. (C) The symmetric latch interface between the N and G domains, corresponding to the close loop contacts seen above the adjacent P-loops in Fig. 1A. The conserved hydrophobic residues of the ALLEADV motifs of the N domains (top) and the symmetric glycine pair of the DGQ motifs of the G domains (bottom) are shown along with the pair of bridging aspartate and glutamine residues.

Figure 3.
Fig. 3. Conformational changes generate the heterodimer interface. (A) The structure of the Ffh NG domain with GMPPNP bound (1JPJ [PDB] .pdb) (in lighter colors) is superimposed with its structure in the complex. The N domain moves as a rigid body toward helix 3 of the G domain; this shift, in turn, is coupled to conformational rearrangement in the DGQ motif at the N terminus of 3, enabling formation of the extensive heterodimeric contact there. Helix 4 moves with the N domain, accommodated by an 2.9 Å translation of the remainder of helix 3. Note the concurrent reorientation of the C-terminal helix. (B) G-domain conformational changes associated with complex formation are limited to the loops of conserved sequence motifs. The magnitude of the shifts are mapped so that the largest shifts ( 6.5 Å) are the darkest shaded regions. (C) Reorientation of motifs II and III upon complex formation. The left panel shows the Ffh NG GMPPNP structure, the right panel Ffh NG in the complex. The side chain of motif III residue Leu192 moves to insert into a pocket across the heterodimer interface, between the guanine base and Gly259(249) that follows motif IV. Movement of this leucine and the accompanying rearrangement of the motif III backbone allows the P-loop to open sufficiently to accommodate the nucleotide in an extended conformation (10). Motif II residues Asp135 and Arg138 move into the catalytic chamber. The same configuration is observed in FtsY.

The above figures are reprinted by permission from the AAAs: Science (2004, 303, 373-377) copyright 2004.

Secondary reference #1

Title

Crystallization of the gmppcp complex of the ng domains of thermus aquaticus ffh and ftsy.

Authors

I.V.Shepotinovskaya, P.J.Focia, D.M.Freymann.

Ref.

Acta Crystallogr D Biol Crystallogr, 2003, 59, 1834-1837. [DOI no: 10.1107/S0907444903016573]

PubMed id

14501130

Figure 1.
Figure 1 Crystals of the Ffh/FtsY NG complex. Crystals grew with a number of different morphologies. (a) Sheaves. Crystals were grown from Ffh/FtsY NG complex at 20 mg ml-1 in 50 mM Tris pH 8.0, 200 mM NaCl, 2 mM MgCl[2], 1 mM GMPPCP equilibrated against 1.51 M ammonium sulfate, 100 mM Tris pH 8, 100 mM NaCl, 10 mM MgCl[2] at room temperature. (b) Needles and sheaves. Crystals were grown from the protein used in (a) equilibrated against 1.91 M ammonium sulfate, 100 mM bis-tris pH 6.7, 100 mM NaCl, 20 mM MgCl[2], 4% PEG 400. Sheaves appeared within 1-2 d; however, after 2.5-3 weeks needle clusters appeared, coexisting with the sheaves, which then dissolved such that after a month only needle clusters were present in the drop. (c) A large well formed rod growing among needle clusters. Crystals were grown from protein solution at 9.9 mg ml-1, equilibrated against 1.91 M ammonium sulfate, 100 mM bis-tris pH 6.7, 100 mM NaCl, 4% PEG 400. While the sheaf and needle morphologies were readily reproducible, well formed rods appeared in only a small subset of the crystallization drops.

The above figure is reproduced from the cited reference with permission from the IUCr

Secondary reference #2

Title

Conformational change of the n-Domain on formation of the complex between the gtpase domains of thermus aquaticus ffh and ftsy.

Authors

I.V.Shepotinovskaya, D.M.Freymann.

Ref.

Biochim Biophys Acta, 2002, 1597, 107-114. [DOI no: 10.1016/S0167-4838(02)00287-X]

PubMed id

12009409

Secondary reference #3

Title

The conformation of bound gmppnp suggests a mechanism for gating the active site of the srp gtpase.

Authors

S.Padmanabhan, D.M.Freymann.

Ref.

Structure, 2001, 9, 859-867. [DOI no: 10.1016/S0969-2126(01)00641-4]

PubMed id

11566135

Figure 1.
Figure 1. GMPPNP Binding to the NG DomainOmit difference (F[o] - F[c]) electron density maps contoured at 3 s (light blue) and 6 s (dark blue) for (a) structure N1 and (b) structure N2a. The triplet of electron-dense peaks to the right in each image indicates the positions of the phosphate groups. Two residues, Gln107 and Thr112, define the top and bottom of the P loop jaws

The above figure is reproduced from the cited reference with permission from Cell Press