PDBsum entry 1e94

Chaperone

PDB id: 1e94

Contents

Protein chains

174 a.a. *

408 a.a. *

Ligands

ANP ×2

Waters ×286

* Residue conservation analysis

PDB id:

1e94

Name:

Chaperone

Title:

Hslv-hslu from e.Coli

Structure:

Heat shock protein hslv. Chain: a, b, c, d. Synonym: hslv. Engineered: yes. Heat shock protein hslu. Chain: e, f. Synonym: hslu. Engineered: yes

Source:

Escherichia coli. Organism_taxid: 469008. Strain: bl21(de3). Cellular_location: cytoplasm. Expressed in: escherichia coli. Expression_system_taxid: 469008.

Biol. unit:

Homo-Dodecamer (from PDB file)

Resolution:

2.80Å R-factor: 0.254 R-free: 0.304

Authors:

H.K.Song,C.Hartmann,R.Ravishankar,M.Bochtler

Key ref:

H.K.Song et al. (2000). Mutational studies on HslU and its docking mode with HslV. Proc Natl Acad Sci U S A, 97, 14103-14108. PubMed id: 11114186 DOI: 10.1073/pnas.250491797

Date:

07-Oct-00 Release date: 17-Nov-00

Supersedes:

1doo

Protein chains

P0A7B8  (HSLV_ECOLI) -  ATP-dependent protease subunit HslV from Escherichia coli (strain K12)

Seq: 176 a.a.

Struc: 174 a.a.

Protein chains

P0A6H5  (HSLU_ECOLI) -  ATP-dependent protease ATPase subunit HslU from Escherichia coli (strain K12)

Seq: 443 a.a.

Struc: 408 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: Chains A, B, C, D: E.C.3.4.25.2 - HslU--HslV peptidase.

DOI no: 10.1073/pnas.250491797

Proc Natl Acad Sci U S A 97:14103-14108 (2000)

PubMed id: 11114186

Mutational studies on HslU and its docking mode with HslV.

H.K.Song, C.Hartmann, R.Ramachandran, M.Bochtler, R.Behrendt, L.Moroder, R.Huber.

ABSTRACT

HslVU is an ATP-dependent prokaryotic protease complex. Despite detailed crystal and molecular structure determinations of free HslV and HslU, the mechanism of ATP-dependent peptide and protein hydrolysis remained unclear, mainly because the productive complex of HslV and HslU could not be unambiguously identified from the crystal data. In the crystalline complex, the I domains of HslU interact with HslV. Observations based on electron microscopy data were interpreted in the light of the crystal structure to indicate an alternative mode of association with the intermediate domains away from HslV. By generation and analysis of two dozen HslU mutants, we find that the amidolytic and caseinolytic activities of HslVU are quite robust to mutations on both alternative docking surfaces on HslU. In contrast, HslVU activity against the maltose-binding protein-SulA fusion protein depends on the presence of the I domain and is also sensitive to mutations in the N-terminal and C-terminal domains of HslU. Mutational studies around the hexameric pore of HslU seem to show that it is involved in the recognition/translocation of maltose-binding protein-SulA but not of chromogenic small substrates and casein. ATP-binding site mutations, among other things, confirm the essential role of the "sensor arginine" (R393) and the "arginine finger" (R325) in the ATPase action of HslU and demonstrate an important role for E321. Additionally, we report a better refined structure of the HslVU complex crystallized along with resorufin-labeled casein.

Selected figure(s)

Figure 2.
Fig. 2. Representation of the electrostatic potential surfaces of HslV (Left) and HslU (Center) involved in the EM mode of docking. Negatively charged regions are in red, and positively charged regions are in blue. Sites of mutations in the HslU (Right). Numbers 1 (green) and 3 (pink) mark sites of pentaglycine insertions after residues 264 and 387 as well as changes of surface charges (E266Q; E266Q/E385K), 2 (blue) marks the site of introduction of a bulky side chain (I312W), and 4 (red) marks the site of a charge reversal (E436K/D437K). The hexamer pore is colored in yellow. This figure was drawn by using GRASP (28).

Figure 3.
Fig. 3. Sites of mutations in the hexamer pore. Side-chain atoms (yellow) are shown only in one subunit for clarity. Mutation sites in the hexamer pore are colored in pink. Top view of HslU (Left). Side view of the central pore of HslU hexamer (Right). Two subunits from the ring nearest to the reader are removed to expose the interior. This figure was drawn by using GRASP (28).

Figures were selected by an automated process.