Mutational studies on HslU and its docking mode with HslV

Hyun Kyu Song, Claudia Hartmann, Ravishankar Ramachandran, Matthias Bochtler, Raymond Behrendt, Luis Moroder, Robert Huber

Research output: Contribution to journalArticle

131 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)14103-14108
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume97
Issue number26
DOIs
Publication statusPublished - 2000 Dec 19
Externally publishedYes

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Maltose-Binding Proteins
Caseins
Mutation
Arginine
ATP-Dependent Proteases
Adenosine Triphosphate
Chromogenic Compounds
Molecular Structure
Fingers
Adenosine Triphosphatases
Electron Microscopy
Proteins
Hydrolysis
Binding Sites
Peptides
resorufin

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Mutational studies on HslU and its docking mode with HslV. / Song, Hyun Kyu; Hartmann, Claudia; Ramachandran, Ravishankar; Bochtler, Matthias; Behrendt, Raymond; Moroder, Luis; Huber, Robert.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 97, No. 26, 19.12.2000, p. 14103-14108.

Research output: Contribution to journalArticle

Song, HK, Hartmann, C, Ramachandran, R, Bochtler, M, Behrendt, R, Moroder, L & Huber, R 2000, 'Mutational studies on HslU and its docking mode with HslV', Proceedings of the National Academy of Sciences of the United States of America, vol. 97, no. 26, pp. 14103-14108. https://doi.org/10.1073/pnas.250491797
Song, Hyun Kyu ; Hartmann, Claudia ; Ramachandran, Ravishankar ; Bochtler, Matthias ; Behrendt, Raymond ; Moroder, Luis ; Huber, Robert. / Mutational studies on HslU and its docking mode with HslV. In: Proceedings of the National Academy of Sciences of the United States of America. 2000 ; Vol. 97, No. 26. pp. 14103-14108.
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