PDBsum entry 3e47

Hydrolase

PDB id: 3e47

Contents

Protein chains

250 a.a.

244 a.a.

241 a.a.

242 a.a.

233 a.a.

244 a.a.

243 a.a.

222 a.a.

204 a.a.

198 a.a.

212 a.a.

222 a.a.

233 a.a.

196 a.a.

Ligands

ESY ×2

Waters ×858

References listed in PDB file

Key reference

Title

Inhibitor-Binding mode of homobelactosin c to proteasomes: new insights into class i mhc ligand generation.

Authors

M.Groll, O.V.Larionov, R.Huber, A.De meijere.

Ref.

Proc Natl Acad Sci U S A, 2006, 103, 4576-4579. [DOI no: 10.1073/pnas.0600647103]

PubMed id

16537370

Abstract

Most class I MHC ligands are generated from the vast majority of cellular proteins by proteolysis within the ubiquitin-proteasome pathway and are presented on the cell surface by MHC class I molecules. Here, we present the crystallographic analysis of yeast 20S proteasome in complex with the inhibitor homobelactosin C. The structure reveals a unique inhibitor-binding mode and provides information about the composition of proteasomal primed substrate-binding sites. IFN-gamma inducible substitution of proteasomal constitutive subunits by immunosubunits modulates characteristics of generated peptides, thus producing fragments with higher preference for binding to MHC class I molecules. The structural data for the proteasome:homobelactosin C complex provide an explanation for involvement of immunosubunits in antigen generation and open perspectives for rational design of ligands, inhibiting exclusively constitutive proteasomes or immunoproteasomes.

Figure 1.
Fig. 1. Proteasomal proteolytically active sites involved in the generation of MHC class I ligands. (a) Surface representation of the yeast 20S proteasome in complex with propeptides, clipped along the cylindrical pseudo sevenfold symmetry axis. Accessible surfaces are depicted in blue, and the cutting surface is in white. Propeptides are shown as space-filling models in yellow and indicate the proteolytically different active sites. The various proteolytic active centers are marked in a specific color coding: blue, subunit 1; red, subunit 2; and green, subunit 5. Cleavage preferences, termed caspase-, tryptic-, and chymotryptic-like activity, are zoomed and illustrated as surfaces; propeptides are presented as ball-and-stick models. Surface colors indicate positive and negative electrostatic potential contoured from 15 kT/e (intense blue) to –15 kT/e (intense red). (b) Topology of the 28 subunits of the yeast 20S proteasome in ribbon presentation. IFN- -inducible mammalian subunits 1i, 2I, and 5i are modeled by the corresponding constitutive yeast subunits. (c Left) MHC class I molecule in complex with an antigen (c Right). Structural superposition of propeptides 1, 2, and 5 with NEF-HIV1 and GAG-HIV2 antigen bound to MHC class I molecules. (d) Standard orientation for peptide substrates bound to the proteasomal specificity pockets. Substrates are oriented from their N to their C terminus. The scissile peptide bond is shown in magenta, flanked by the nucleophilic water molecule, which is incorporated into the product during hydrolysis. Residues on the left side of the scissile peptide bond in substrates, generating the C-terminal part in the product, are termed P sites; residues on the right side are termed P' sites. Specificity pockets, which are responsible for ligand stabilization, are termed S and S' pockets, respectively (11). (e) Sequence alignment of the yeast and the human constitutive subunit and immunosubunit for subunit 1 (nonprimed S1 site, Upper) and subunit 5 (nonprimed S1 and primed substrate-binding channel, Lower). Conserved residues are marked by vertical green boxes, significant variations between human constitutive subunits and immunosubunits are highlighted by yellow boxes, and variations in residues in proximity to the specificity pockets are shown against a gray background. Secondary structure elements are indicated in green.

Figure 2.
Fig. 2. Homobelactosin C specifically binds to the chymotryptic-like active site by formation of an ester. (a) Chemical structures of omuralide and bisbenzyl-protected homobelactosin C in their native and bound conformation. The lead structure segments that in particular are involved in inhibitor binding are depicted in blue; Thr-1 of subunit 5 is in red. (b) Stereorepresentation of the chymotryptic-like active site of the yeast 20S proteasome (colored in gray) in complex with bisbenzyl-protected homobelactosin C (colored in green). Covalent linkage of the inhibitor with 5-Thr1O^ is drawn in magenta. The electron density map (colored in blue) is contoured from 1 around Thr-1 (colored in black) with 2F[o] – F[c] coefficients after twofold averaging. Temperature factor refinement indicates full occupancies of the inhibitor-binding site. The homobelactosin C derivative has been omitted for phasing. (c) Surface representation of the chymotryptic-like active site in complex with omuralide (depicted in brown, Left) and homobelactosin C (depicted in green, Right), covalently bound to Thr-1 (depicted in white). Note the overall similarity in the binding mode of both inhibitors but the different orientations of the generated C4-hydroxy group upon -lactone ring opening (indicated by a black arrow). Surface colors indicate positive and negative electrostatic potential contoured from 15 kT/e (intense blue) to –15 kT/e (intense red). (d) Stereorepresentation of the superposition of lactacystin and bisbenzyl-protected homobelactosin C, including Thr-1 with respect to subunit 5. Omuralide is shown in brown, bisbenzyl-protected homobelactosin C is drawn in green, and the active site Thr-1 is in black. The superposition indicates that both inhibitors occupy the S1 specificity pocket in a unique way. The bisbenzyl-protected homobelactosin C is prolonged to the primed site. Nonprimed and primed sites are indicated by a black arrow.