PDBsum entry 1k5d

Signaling protein/signaling activator

PDB id: 1k5d

Contents

Protein chains

206 a.a. *

146 a.a. *

344 a.a. *

Ligands

GNP ×4

Metals

_MG ×4

Waters ×342

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Rangap mediates gtp hydrolysis without an arginine finger.

Authors

M.J.Seewald, C.Körner, A.Wittinghofer, I.R.Vetter.

Ref.

Nature, 2002, 415, 662-666. [DOI no: 10.1038/415662a]

PubMed id

11832950

Abstract

GTPase-activating proteins (GAPs) increase the rate of GTP hydrolysis on guanine nucleotide-binding proteins by many orders of magnitude. Studies with Ras and Rho have elucidated the mechanism of GAP action by showing that their catalytic machinery is both stabilized by GAP binding and complemented by the insertion of a so-called 'arginine finger' into the phosphate-binding pocket. This has been proposed as a universal mechanism for GAP-mediated GTP hydrolysis. Ran is a nuclear Ras-related protein that regulates both transport between the nucleus and cytoplasm during interphase, and formation of the mitotic spindle and/or nuclear envelope in dividing cells. Ran-GTP is hydrolysed by the combined action of Ran-binding proteins (RanBPs) and RanGAP. Here we present the three-dimensional structure of a Ran-RanBP1-RanGAP ternary complex in the ground state and in a transition-state mimic. The structure and biochemical experiments show that RanGAP does not act through an arginine finger, that the basic machinery for fast GTP hydrolysis is provided exclusively by Ran and that correct positioning of the catalytic glutamine is essential for catalysis.

Figure 2.
Figure 2: Details of the Ran–RanGAP interface. a, Worm plot of Ran and RanGAP, with colours as in Fig. 1 and important residues shown in ball and stick representation. The uncomplexed RanGAP (cyan) is superimposed; arrows indicate the movement of Lys 76 and Arg 74, and asterisk symbolizes the potential clash of Leu 43 and Lys 76 on complex formation. b, Switch II of Ran (green) superimposed on the Ran–ranBD1 structure (blue), illustrating the reorientation of Gln 69 into a catalytically competent conformation by the residues Tyr 39 from Ran and Asn 131 fron RanGAP.

Figure 3.
Figure 3: The active site. a, The nucleotide and relevant residues of Ran and RanGAP; interactions are indicated by dashed lines. The catalytic water ('W') has very weak density (probably owing to the limited resolution) and was not included in the final model, but is shown here to illustrate its potential interaction partners. It sits in a similar position as in the Ras–RasGAP complex. Asterisk denotes the hydroxyl group of Thr 42 of Ran. The rest of the side chain is omitted for clarity. b, The 2F[o] - F[c] electron density map contoured at 1.6 for the structure with GDP and aluminium fluoride, the latter modelled as AlF[3].

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nature (2002, 415, 662-666) copyright 2002.

Secondary reference #1

Title

The ras-Rasgap complex: structural basis for gtpase activation and its loss in oncogenic ras mutants.

Authors

K.Scheffzek, M.R.Ahmadian, W.Kabsch, L.Wiesmüller, A.Lautwein, F.Schmitz, A.Wittinghofer.

Ref.

Science, 1997, 277, 333-338. [DOI no: 10.1126/science.277.5324.333]

PubMed id

9219684

Figure 1.
Fig. 1. Stereo view of a segment of the 2F[o] F[c] electron density map (contoured at 1.2 ) covering the active site region in the^ complex, with Ras in blue, GAP-334 in red, and waters in light blue.

Figure 2.
Fig. 2. The complex between GAP-334 and Ras. (A) Ribbon representation of the complex model drawn with Molscript (52) and^ Raster3D (53) according to the assignment of secondary structure^ elements obtained with the program DSSP (54). The extra and^ catalytic domains of GAP-334 are shown in green and red (respectively), regions of GAP contacting Ras in light brown, Ras in yellow, and^ GDP and AlF[3] as ball-and-stick models. Regions involved in the^ interface are labeled, Sw I and Sw II indicating the switch regions, C the COOH-terminal, and N the NH[2]-terminal. (B) Schematic^ drawing with selected interactions. Polar interactions between individual residues of GAP-334 and Ras are shown as red lines for interactions of side chains, and as red arrows for contacts from side chain to main chain atoms, where the arrowhead marks the residue contributing the main chain group. Yellow lines indicate^ van der Waals or hydrophobic interactions. Some water molecules (marked W) from the interface region are included. Residues belonging to the interacting regions of Ras indicated in (A) are denoted^ with specified boxes, as indicated. Interaction between Lys88 and Thr791 is shown by a dashed arrow, because the electron density in this region is presently not of sufficient quality to unambiguously define the contact. Amino acid abbreviations are in (55).

The above figures are reproduced from the cited reference with permission from the AAAs

Secondary reference #2

Title

Structure of a ran-Binding domain complexed with ran bound to a gtp analogue: implications for nuclear transport.

Authors

I.R.Vetter, C.Nowak, T.Nishimoto, J.Kuhlmann, A.Wittinghofer.

Ref.

Nature, 1999, 398, 39-46. [DOI no: 10.1038/17969]

PubMed id

10078529

Figure 1.
Figure 1: Representative electron density around switch I in Ran dot-GppNHp and the conserved WKER motif of RanBD1 (residues 57–60). Residues from RanBD1 are represented by white carbon traces, residues from Ran by yellow carbon traces. The omit map is contoured at 1.1 . The figure was prepared with BOBSCRIPT^46.

Figure 5.
Figure 5: Molecular embrace and the DEDDDL motif. Surface representation of RanBD1, showing the basic region where the DEDDDL motif of Ran is expected to bind after the C terminus wraps itself around RanBD1. Ran is shown as a backbone (green), and GppNHp and the magnesium ion as ball and stick. The figure was produced using GRASP^50.

The above figures are reproduced from the cited reference with permission from Macmillan Publishers Ltd

Secondary reference #3

Title

The crystal structure of RNA1p: a new fold for a gtpase-Activating protein.

Authors

R.C.Hillig, L.Renault, I.R.Vetter, T.Drell, A.Wittinghofer, J.Becker.

Ref.

Mol Cell, 1999, 3, 781-791. [DOI no: 10.1016/S1097-2765(01)80010-1]

PubMed id

10394366

Figure 2.
Figure 2. NCS-Averaged Experimental Electron Density MapPart of the map showing the region of LRR8 (residues 217–243). The refined model (ball-and-stick) is superimposed on the experimental electron density map after 2-fold NCS averaging, solvent flattening, and histogram matching, contoured at 1.0 σ. Figure prepared using Bobscript ([28]).

Figure 4.
Figure 4. Structural Overlay of the 11 LRRs of rna1pLRRs that deviate markedly from the ideal LRR structure are presented in blue (LRR1), red (LRR3), and green (LRR5). The position of Arg-74 is marked by a red ball. The LRR consensus residues of LRR8 are depicted as ball-and-stick models (yellow) illustrating the formation of the hydrophobic core. Figure produced with Bobscript ([28]).

The above figures are reproduced from the cited reference with permission from Cell Press

Secondary reference #4

Title

Rna1 encodes a gtpase-Activating protein specific for gsp1p, The ran/tc4 homologue of saccharomyces cerevisiae.

Authors

J.Becker, F.Melchior, V.Gerke, F.R.Bischoff, H.Ponstingl, A.Wittinghofer.

Ref.

J Biol Chem, 1995, 270, 11860-11865.

PubMed id

7744835

Secondary reference #5

Title

The acidic c-Terminal domain of RNA1p is required for the binding of ran.Gtp and for rangap activity.

Authors

J.Haberland, J.Becker, V.Gerke.

Ref.

J Biol Chem, 1997, 272, 24717-24726. [DOI no: 10.1074/jbc.272.39.24717]

PubMed id

9305944

Figure 5.
Fig. 5. GAP activity of S. pombe rna1p in the presence of increasing concentrations of the C-terminally truncated mutant protein rna1 p341. 1 µM Ran loaded with [ -32P]GTP was incubated at 30 °C with buffer ( circle ), 0.025 nM rna1p ( ), 0.05 nM rna1p ( ), or 0.1 nM rna1p ( ). In the latter case (1^ µM Ran, 0.1 nM rna1p) the reaction was carried out in the absence^ ( ) or presence of 0.025 nM rna1p 341 ( ), 0.05 nM rna1p 341 ( ), 0.1 nM rna1p 341 (×), 0.2 nM rna1p 341 ( ), 0.5 nM rna1p 341 ( ), 2 nM rna1p 341 ( ), or 20 nM rna1p 341 ( ). GTPase activity was determined as decrease of the nitrocellulose-bound [ -32P]GTP, and results are given as the percentage of radioactivity bound to Ran 30 s after addition of the respective GAP. The individual experiments were carried out at least three times, and curves of a representative experiment are shown. Note that the addition of increasing amounts ot the rna1p 341 does not affect the GAP activity of 0.1 nM wild-type rna1p. All curves characterizing the reactions containing 1 µM Ran and 0.1 nM rna1p are nearly identical and partially mask one another.

Figure 10.
Fig. 10. GAP activities of the rna1p 361/QQDQ and GST-rna1pCT mutant proteins. A, 1 µM Ran complexed with [ -32P]GTP was incubated at 30 °C with buffer alone ( circle ), with 0.1 nM rna1p ( ), with 0.1 nM rna1p 361 ( ), or with 0.1 nM rna1p 361/QQDQ ( ). B, mixtures consisted of 1 µM [ -32P]GTP-loaded Ran and buffer alone ( circle ), 0.1 nM rna1p ( ), 0.1 nM GST ( ), 0.1 nM GST-rna1pCT ( ), or 50 nM GST-rna1pCT ( ). GTPase^ activities were determined as the decrease of nitrocellulose bound^ [ -32P]GTP, and results are given as percentage of the radioactivity bound to Ran after the addition of buffer, rna1p, rna1p 361, rna1p 361/QQDQ, GST, or GST-rna1pCT, respectively. All curves characterizing reactions containing GST or GST fusion proteins are indistinguishable from the control with buffer alone.

The above figures are reproduced from the cited reference with permission from the ASBMB