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Measuring In Vitro ATPase Activity for Enzymatic Characterization
07:38

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Published on: August 23, 2016

Nucleotide-dependent conformational changes in a protease-associated ATPase HsIU.

J Wang1, J J Song, I S Seong

  • 1Department of Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Avenue, New Haven, CT 06520, USA. wang@mail.csb.yale.edu

Structure (London, England : 1993)
|November 16, 2001
PubMed
Summary

This study explores how a bacterial ATPase called HslU changes shape depending on the nucleotides it binds. HslU is part of a protease complex that breaks down proteins. The researchers identified four distinct structural states of HslU, each linked to a specific nucleotide. These changes involve rotations in a key domain of HslU. The study also corrects a previously misassigned nucleotide state and reveals how ATP hydrolysis influences the HslVU complex. These findings provide a clearer picture of how HslU functions and may help in understanding similar proteins.

Keywords:
AAA(+) protein conformationHslU structural dynamicsATPase nucleotide bindingProtease function mechanism

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Area of Science:

  • Structural biology of ATPases
  • Protease function in bacterial proteolysis
  • AAA(+) protein conformational dynamics

Background:

AAA(+) ATPases are essential for many cellular processes, including protein degradation. Prior research has shown that these proteins undergo conformational changes driven by nucleotide binding and hydrolysis. However, the exact sequence of structural transitions in HslU remains unclear. It was already known that HslU forms a hexameric complex with HslV to create an ATP-dependent protease. No prior work had resolved the full range of conformational states in HslU. This gap motivated the current study to map the nucleotide-dependent structural changes in HslU. Understanding these changes could clarify how ATP hydrolysis influences protease activity. The study aimed to identify the sequence of conformational states and their functional implications. This work addresses a key uncertainty in AAA(+) protein mechanics.

Purpose Of The Study:

This study aimed to define the nucleotide-dependent conformational states of HslU and their role in protease function. The researchers sought to identify the sequence of structural transitions in HslU. They wanted to clarify how nucleotide binding affects HslU conformation. The study focused on how ATP hydrolysis influences HslU structural dynamics. They also aimed to correct misassigned nucleotide states in prior structures. The goal was to establish a framework for understanding AAA(+) protein mechanics. The researchers proposed that HslU conformational changes are directly linked to protease activity. This work provides a detailed map of HslU structural transitions.

Main Methods:

The researchers used structural analysis to identify conformational states of HslU. They examined four distinct states: empty, SO(4), ATP, and ADP-bound forms. The team analyzed the alpha/beta and alpha-helical domains in HslU. They focused on nucleotide binding at the domain interface. The study compared structural changes across the four states. They measured domain rotations and conformational shifts. The researchers corrected nucleotide identity in a previously misassigned structure. They linked ATP hydrolysis to ring rotation and C-terminal changes in HslU.

Main Results:

The study identified four sequential conformational states of HslU. The empty state is followed by SO(4), ATP, and ADP-bound forms. Nucleotide binding occurs at a cleft between two domains. The alpha-helical domain rotates in each conformational state. The ATP state shows a distinct ring rotation in the HslVU complex. The ADP state reveals a conformational change in the HslU C terminus. The corrected nucleotide identity in one structure improved structural accuracy. These findings suggest a revised mechanism for protein unfolding and translocation.

Conclusions:

The authors propose that nucleotide binding drives conformational changes in HslU. These changes are essential for HslVU protease function. The study clarifies the sequence of structural transitions in HslU. The corrected nucleotide identity improves understanding of HslU mechanics. The observed ring rotation and C-terminal changes are linked to ATP hydrolysis. The findings support an amended translocation mechanism for HslVU protease. The study provides a framework for understanding AAA(+) protein function. These results may inform future studies on ATPase-driven proteolysis.

The four states are empty, SO(4), ATP-bound, and ADP-bound forms of HslU.

Nucleotide binds at a cleft between an alpha/beta domain and an alpha-helical domain.

The rotation of the alpha-helical domain is a key feature distinguishing each conformational state.

ATP hydrolysis leads to ring rotation and a conformational change in the HslU C terminus.

The study corrects nucleotide identity in one previously misassigned HslU structure.

The authors suggest these changes support a revised translocation mechanism in HslVU protease.