PDBsum entry 1ofh

Hydrolase

PDB id: 1ofh

Contents

Protein chains

309 a.a. *

(+ 0 more) 174 a.a. *

Ligands

ADP ×3

PO4 ×3

Metals

_MG ×12

Waters ×80

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Structure and reactivity of an asymmetric complex between hslv and i-Domain deleted hslu, A prokaryotic homolog of the eukaryotic proteasome.

Authors

A.R.Kwon, B.M.Kessler, H.S.Overkleeft, D.B.Mckay.

Ref.

J Mol Biol, 2003, 330, 185-195. [DOI no: 10.1016/S0022-2836(03)00580-1]

PubMed id

12823960

Abstract

In the prokaryotic homolog of the eukaryotic proteasome, HslUV, the "double donut" HslV protease is allosterically activated by HslU, an AAA protein of the Clp/Hsp100 family consisting of three (amino-terminal, carboxy-terminal, and intermediate) domains. The intermediate domains of HslU, which extend like tentacles from the hexameric ring formed by the amino-terminal and carboxy-terminal domains, have been deleted; an asymmetric HslU(DeltaI)(6)HslV(12) complex has been crystallized; and the structure has been solved to 2.5A resolution, revealing an assembly in which a HslU(DeltaI) hexamer binds one end of the HslV dodecamer. The conformation of the protomers of the HslU(DeltaI)-complexed HslV hexamer is similar to that in the symmetric wild-type HslUV complex, while the protomer conformation of the uncomplexed HslV hexamer is similar to that of HslV alone. Reaction in the crystals with a vinyl sulfone inhibitor reveals that the HslU(DeltaI)-complexed HslV hexamer is active, while the uncomplexed HslV hexamer is inactive. These results confirm that HslV can be activated by binding of a hexameric HslU(DeltaI)(6) ring lacking the I domains, that activation is effected through a conformational change in HslV rather than through alteration of the size of the entry channel into the protease catalytic cavity, and that the two HslV(6) rings in the protease dodecamer are activated independently rather than cooperatively.

Figure 2.
Figure 2. Structure of the HslU(DI)[6]HslV[12] complex. (a) A drawing of one complex viewed perpendicular to the 6-fold axis, with a-helices represented as cylinders. Color scheme: HslU(DI), magenta with one subunit blue; HslU(DI)-complexed HslV, yellow with one subunit red; uncomplexed HslV, cyan with one subunit green. (b) View of one HslU(DI)[6] hexamer looking parallel with the 6-fold axis. (c) HslU(DI)-complexed HslV hexamer viewed from the top of the molecules. (d) Uncomplexed HslV hexamer viewed from the bottom of the molecule.

Figure 4.
Figure 4. NLVS inhibitor. (a) F[o] -F[c] electron density map contoured at 2.6s, computed using calculated phases from model without inhibitor, showing that inhibitor is present in upper hexamer only. The two pairs of crystallographically independent HslV subunits of the HslU(DI)[6]HslV[12] complex that is aligned along the 3-fold axis are shown; subunits from HslU(DI)-complexed HslV are red and yellow; from uncomplexed HslV, green and cyan. Thr1, with which the inhibitor reacts, shown in magenta in all subunits. (b) Ribbon drawing of superposition of HslU(DI)-complexed and uncomplexed HslV, with NLVS inhibitor. Where HslV protomers are similar in conformation, as originally defined by Sousa et al.,[15.] structure is green; where they differ, HslU(DI)-complexed HslV is blue; uncomplexed HslV is yellow. Inhibitor model is red. (c) Stereo view of superposition of complexed and uncomplexed HslV near inhibitor. Selected amino acid side-chains and segments of polypeptide backbone that hydrogen-bond the inhibitor are shown. For a detailed description of interactions with inhibitor, see Sousa et al.[23.] C^a backbone trace of HslU(DI)-complexed subunits, cyan; backbone trace of uncomplexed subunits, yellow; carbon atoms of inhibitor, magenta; carbon atoms of amino acid side-chains, green; oxygen atoms, red; nitrogen atoms, blue; sulfur atoms, yellow; iodine atoms, purple.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2003, 330, 185-195) copyright 2003.

Secondary reference #1

Title

Crystal structure of hsluv complexed with a vinyl sulfone inhibitor: corroboration of a proposed mechanism of allosteric activation of hslv by hslu.

Authors

M.C.Sousa, B.M.Kessler, H.S.Overkleeft, D.B.Mckay.

Ref.

J Mol Biol, 2002, 318, 779-785. [DOI no: 10.1016/S0022-2836(02)00145-6]

PubMed id

12054822

Figure 1.
Figure 1. (a) Stereo view of F[o] -F[c] electron density map showing inhibitor bound to one subunit of HslV. Phases were computed from HslUV model that resulted from one cycle of simulated annealing after all inhibitor atoms were omitted from the model. Contour levels, 5s (magenta), 12.5s (cyan). The Figure was prepared with BOBSCRIPT[25.] and RASTER3D. [26.] (b) Proposed structure of NLVS-HslV covalent complex with Thr1 of HslV. [14.] Inhibitor atoms and bonds are drawn bold; HslV atoms/bonds are drawn fine. The S1 and S3 HslV binding pockets are shown schematically above the moieties they bind. Orientation of inhibitor is similar to orientation in part (a).

Figure 2.
Figure 2. Stereo views of the HslUV-NLVS inhibitor complex. (a) View showing the hydrogen bonding interactions of the inhibitor with HslV polypeptide backbone in the HslUV-NLVS complex. Carbon atoms of HslV are green; carbon atoms of inhibitor are gray; nitrogen atoms, blue; oxygen atoms, red; sulfur atom, yellow. (b) Superposition of the substrate binding clefts of HslUV-NLVS and the yeast proteasome with epoxomycin (subunit K of PDB 1G65).[15.] Hydrogen bonds between protein and inhibitor are the same as shown in part (a). Selected residues are labeled with format "HslV#/proteasome#". Color scheme: HslV of HslUV-NLVS complex, green; NLVS inhibitor atoms, cyan; proteasome, red; epoxomycin inhibitor atoms, gold. (c) View showing the inhibitor (semi-transparent CPK model) and the binding pockets of HslUV. HslV protomer to which NLVS is covalently attached is green; adjacent HslV protomer, yellow; HslU, magenta; inhibitor, gray. Selected side chains are included. Carbon atoms are the same color as corresponding protomer; oxygen atoms, red; nitrogen atoms, blue; sulfur atoms, cyan. (d) View showing the displacement of upper strand of substrate binding cleft of uncomplexed HslV [9.] relative to HslUV-NLVS; when lower segments of polypeptides are superimposed, upper segment of uncomplexed HslV is displaced vert, similar 3-4 Å from its position in the HslUV-NLVS complex. For clarity, only selected peptide backbone and C^a atoms of the proteins and "backbone" atoms of the inhibitor are included. Color scheme: HslV of HslUV-NLVS complex, green; NLVS inhibitor atoms, cyan; uncomplexed HslV, magenta. Superpositions were computed with the program LSQMAN.[27.] The Figure was prepared with MOLSCRIPT [28.] and RASTER3D. [26.]

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

Secondary reference #2

Title

Crystal and solution structures of an hsluv protease-Chaperone complex.

Authors

M.C.Sousa, C.B.Trame, H.Tsuruta, S.M.Wilbanks, V.S.Reddy, D.B.Mckay.

Ref.

Cell, 2000, 103, 633-643. [DOI no: 10.1016/S0092-8674(00)00166-5]

PubMed id

11106733

Figure 1.
Figure 1. Representative Electron Density MapsStereo views of F[o] − F[c] simulated annealing omit maps, computed with phases calculated from models in which the atoms of interest were deleted from the model used in refinement.(A) the ATP binding site of HslU, contoured at 5σ. Protein is shown as a ribbon diagram; ATP from the final HslUV model (average B factor 29.3) is shown as a ball and stick representation.(B) Carboxy-terminal segment of HslU (average B factor 119.1), contoured at 3σ (magenta) and 6σ (cyan). Residues of HslU which were omitted are shown in green, oriented with the carboxy-terminal Leu-444 at the bottom of the figure; neighboring residues of HslV are shown in standard colors (oxygen, red; nitrogen, blue; carbon, gray). Figure was prepared with BOBSCRIPT ([7 and 8]). The rendering and stereo pair generation of all figures was done with RASTER3D ( [25]) and IMAGEMAGIK (http://www.wizards.dupont.com/cristy/ImageMagick.html).

Figure 6.
Figure 6. Conformational Changes around the Catalytic Site of HslVStereo ribbon drawing of the active site region. The HslUV structure is colored green. The segment of uncomplexed HslV that differs substantially from the complex (see Figure 3A) is colored magenta. Selected residue side chains and polypeptide backbone are shown in the ball and stick representation.

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