PDBsum entry 1ksh

Signaling protein/hydrolase

PDB id: 1ksh

Contents

Protein chains

165 a.a. *

142 a.a. *

Ligands

PO4

GDP

Metals

_MG

Waters ×124

* Residue conservation analysis

References listed in PDB file

Key reference

Title

The complex of arl2-Gtp and pde delta: from structure to function.

Authors

M.Hanzal-Bayer, L.Renault, P.Roversi, A.Wittinghofer, R.C.Hillig.

Ref.

EMBO J, 2002, 21, 2095-2106. [DOI no: 10.1093/emboj/21.9.2095]

PubMed id

11980706

Abstract

Arf-like (Arl) proteins are close relatives of the Arf regulators of vesicular transport, but their function is unknown. Here, we present the crystal structure of full-length Arl2-GTP in complex with its effector PDE delta solved in two crystal forms (Protein Data Bank codes 1KSG, 1KSH and 1KSJ). Arl2 shows a dramatic conformational change from the GDP-bound form, which suggests that it is reversibly membrane associated. PDE delta is structurally closely related to RhoGDI and contains a deep empty hydrophobic pocket. Further experiments show that H-Ras, Rheb, Rho6 and G alpha(i1) interact with PDE delta and that, at least for H-Ras, the intact C-terminus is required. We suggest PDE delta to be a specific soluble transport factor for certain prenylated proteins and Arl2-GTP a regulator of PDE delta-mediated transport.

Figure 3.
Figure 3 Overall view of Arl2-GTP:PDE and comparison with Rap1A-GppNHp:RafRBD and Cdc42-GDP:RhoGDI. (A) Ribbon diagram of the complex of Arl2-GTP and PDE , with color coding of Arl2 as in Figure 1B. (B) Complex of Rap1A-GppNHp and the Ras binding domain of Raf (Nassar et al., 1995). Color coding as in (A). (C) Complex of Cdc42-GDP and RhoGDI1 (Hoffman et al., 2000). Color coding as in (A). C-terminal geranylgeranyl modification is shown in light blue.

Figure 4.
Figure 4 The interface of Arl2 and PDE . (A) Stereo representation of a view along the − interface (Arl2- 2 and PDE - 7). (B) Schematic representation. Color coding as in Figure 3. Interactions as dotted lines. Residues are boxed, water molecules are represented by circles.

The above figures are reprinted from an Open Access publication published by Macmillan Publishers Ltd: EMBO J (2002, 21, 2095-2106) copyright 2002.

Secondary reference #1

Title

Coexpression, Copurification, Crystallization and preliminary x-Ray analysis of a complex of arl2-Gtp and pde delta.

Authors

L.Renault, M.Hanzal-Bayer, R.C.Hillig.

Ref.

Acta Crystallogr D Biol Crystallogr, 2001, 57, 1167-1170. [DOI no: 10.1107/S0907444901009556]

PubMed id

11468408

Figure 1.
Figure 1 Crystals of ARL2-GTP:PDE . (a) Clusters of micro-needles of crystal form 1 as found with the initial condition. (b) Rod-shaped crystals optimized by streak seeding as used for data collection. The largest rod has dimensions of [102]~ 20 × 40 × 800 µm. (c) Selenomethionine crystals of crystal form 2 (maximum size about 30 × 80 × 100 µm) as used for data set `SeMet'. (d) Large native crystals of form 2 (edges [103]~ 400 µm) as used for `Native-2', found 13 months after setting up of the drop. In the background aged needle bundles of crystal form 1 are still visible.

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

Secondary reference #2

Title

Structural and biochemical properties show arl3-Gdp as a distinct gtp binding protein.

Authors

R.C.Hillig, M.Hanzal-Bayer, M.Linari, J.Becker, A.Wittinghofer, L.Renault.

Ref.

Structure, 2000, 8, 1239-1245. [DOI no: 10.1016/S0969-2126(00)00531-1]

PubMed id

11188688

Figure 2.
Figure 2. The N-Terminal Region in ARL3-GDP(a) Stereoview (C[a] trace) of the N-terminal and interswitch regions of ARL3-GDP (yellow and green, respectively), human Arf1-GDP (blue), and Arf1D17-GppNHp (orange). Loop L5 from the interswitch region of ARL3-GDP protrudes farther upward compared to Arf1-GDP, and this results in the N-terminal region of ARL3 bulging farther out.(b) Stereoview showing the hydrophobic anchoring of the N-terminal region of ARL3 (yellow) to a shallow groove formed by loop L5 of the interswitch region (green), loop L3 (cyan), and the C-terminal helix a5 (magenta).(c) Schematic representation of the hydrophobic interactions between residues of the N-terminal region and the core of the protein in ARL3-GDP and Arf1-GDP. Structurally corresponding residues are horizontally aligned, and the color code is as in Figure 1 and Figure 2

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

Secondary reference #3

Title

The delta subunit of rod specific cyclic gmp phosphodiesterase, Pde delta, Interacts with the arf-Like protein arl3 in a gtp specific manner.

Authors

M.Linari, M.Hanzal-Bayer, J.Becker.

Ref.

FEBS Lett, 1999, 458, 55-59. [DOI no: 10.1016/S0014-5793(99)01117-5]

PubMed id

10518933

Figure 1.
Fig. 1. Interaction of Arl3 with PDE δ in the two-hybrid system. a: The plasmid combinations used for yeast transformation are shown on the left panel, the growth of the appropriate yeast clones on medium selecting either for the presence of both plasmids (UTL) or for protein-protein interaction (THULLy) is shown on the right panel. Arl3 interacts with human and with the 98% identical murine PDE δ in both plasmid combinations. As negative controls, lamin and the small GTPase Ran were used. b: Interaction of WT-Arl3 and its mutants T31N and Q71L with murine PDE δ in the two-hybrid system. Arl3 and Arl3Q71L grew on THULLy medium as indicator for protein-protein interaction whereas Arl3T31N did not interact with PDE δ.

Figure 2.
Fig. 2. a: Expression of WT-Arl3 and its mutants Arl3T31N and Q71L in transfected 293 cells. Human kidney 293 cells were transfected with Arl3 in pcDNA3 or its mutants, respectively. 48 h after transfection, the cell lysates were prepared for Western blotting. Proteins were detected by rabbit anti-ARL3 antibody and anti-rabbit HRP-conjugated antibody. The right lane (C) contains lysate from untransfected cells as a control. b: Arl3Q71L binds specifically to PDE δ in transfected 293 kidney cells. Flag-tagged PDE δ was immunoprecipitated from 293 cells transfected with Flag-PDE δ-pcDNA3 and ARL3-pcDNA3 or its mutants, respectively. Lysates were immunoprecipitated using anti-Flag antibodies. Immunoprecipitates were prepared for Western blotting and PDE δ and Arl3 were detected by anti-Flag or anti-ARL3 antibodies, respectively. PDE δ expression in lysates form 293 cells after 48 h (lane 1), PDE δ after immunoprecipitation (lane 2), PDE δ after immunoprecipitation in the presence of WT-Arl3 (lane 3), PDE δ after immunoprecipitation in the presence of Arl3T31N (lane 4) or in the presence of Arl3Q71L (lane 5). Only Arl3Q71L was co-immunoprecipitated with PDE δ. This indicates a GTP specific Arl3-PDE δ interaction under physiological conditions. The lysate in lane 6 was treated like in lane 5 but the antibodies were omitted to account for unspecific binding to protein G-Sepharose.

The above figures are reproduced from the cited reference with permission from the Federation of European Biochemical Societies