Related Experiment Video

Updated: Jul 13, 2026

Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects
13:57

Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects

Published on: February 18, 2014

Asymmetric interactions of ATP with the AAA+ ClpX6 unfoldase: allosteric control of a protein machine

Greg L Hersch1, Randall E Burton, Daniel N Bolon

  • 1Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02319, USA.

Cell
|July 2, 2005
PubMed

Insights

The AAA+ ClpX hexamer uses asymmetric ATP binding sites to unfold and translocate proteins for degradation. This mechanism allows multiple attempts without releasing substrates, ruling out concerted hydrolysis models.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Structural Biology

Background:

  • AAA+ proteins, like ClpX, are ATP-dependent molecular machines crucial for cellular protein homeostasis.
  • ClpX hexamers utilize ATP hydrolysis to power protein unfolding and translocation for degradation by the ClpXP protease.
  • Understanding the nucleotide binding and hydrolysis mechanism is key to elucidating ClpX function.

Purpose of the Study:

  • To investigate the asymmetric nucleotide binding properties of the AAA+ ClpX hexamer.
  • To determine the functional states of ClpX during substrate recognition and processing.
  • To elucidate the mechanism of ATP hydrolysis and its role in protein unfolding and translocation.

Main Methods:

  • Biochemical assays to characterize ATP binding and hydrolysis kinetics at individual nucleotide-binding sites.
  • Structural studies to visualize nucleotide-bound states of the ClpX hexamer.
  • Analysis of substrate recognition and unfolding mechanisms in relation to nucleotide occupancy.

Main Results:

  • Identified three distinct classes of nucleotide binding sites within the ClpX hexamer, exhibiting asymmetric properties.
  • Demonstrated that substrate recognition requires ATP binding at specific sites and ATP/ADP at others, enabling repeated unfolding attempts.
  • Ruled out models of concerted ATP hydrolysis across all six sites, supporting functional states with three or four bound nucleotides.

Conclusions:

  • The ClpX hexamer operates through an asymmetric mechanism, utilizing distinct nucleotide binding sites for efficient protein processing.
  • The findings reveal a mechanism allowing for repeated substrate unfolding without release, crucial for degradation.
  • Highlights conserved mechanistic principles among diverse AAA+ proteins, including translocases and helicases.

More Related Videos

Measuring In Vitro ATPase Activity for Enzymatic Characterization07:38

Measuring In Vitro ATPase Activity for Enzymatic Characterization

Published on: August 23, 2016

Single-Molecule FRET Imaging for Observing the Conformational Dynamics of Dynamin-Like GTPase Atlastin10:19

Single-Molecule FRET Imaging for Observing the Conformational Dynamics of Dynamin-Like GTPase Atlastin

Published on: January 24, 2025

Related Concept Videos

Ligand Binding and Linkage00:49

Ligand Binding and Linkage

Allosteric proteins have more than one ligand binding site; the binding of a ligand to any of these sites influences the binding of ligands to the other sites. When a protein is allosteric, its binding sites are called coupled or linked.  In the case of enzymes, the site that binds to the substrate is known as the active site and the other site is known as the regulatory site. When a ligand binds to the regulatory site, this leads to conformational changes in the protein that can influence the...
Mechanical Protein Functions01:58

Mechanical Protein Functions

Proteins perform many mechanical functions in a cell. These proteins can be classified into two general categories- proteins that generate mechanical forces and proteins that are subjected to mechanical forces. Proteins providing mechanical support to the structure of the cell, such as keratin, are subjected to mechanical force, whereas proteins involved in cell movement and transport of molecules across cell membranes, such as an ion pump, are examples of generating mechanical force. 
ATP Synthase: Mechanism01:48

ATP Synthase: Mechanism

In animals, the mitochondrial F1F0 ATP synthase is the key protein that synthesizes ATP molecules through a complex catalytic mechanism. While the nuclear genome encodes the majority of ATP synthase subunits, the mitochondrial genome encodes some of the enzyme's most critical components. The formation of this multi-subunit enzyme is a complex multi-step process regulated at the level of transcription, translation, and assembly. Defects in one or more of these steps can result in decreased ATP...
ATP Synthase: Structure01:18

ATP Synthase: Structure

ATP synthase or ATPase is among the most conserved proteins found in bacteria, mammals, and plants. This enzyme can catalyze a forward reaction in response to the electrochemical gradient, producing ATP from ADP and inorganic phosphate. ATP synthase can also work in a reverse direction by hydrolyzing ATP and generating an electrochemical gradient. Different forms of ATP synthases have evolved special features to meet the specific demands of the cell. Based on their specific feature, ATP...
Allosteric Proteins-ATCase01:19

Allosteric Proteins-ATCase

Binding sites linkages can regulate a protein's function.  For example, enzyme activity is often regulated through a feedback mechanism where the end product of the biochemical process serves as an inhibitor.
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to  N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis pathway,...
Membrane Asymmetry Regulating Transporters01:19

Membrane Asymmetry Regulating Transporters

Enzymes like flippase, floppase, and scramblase transfer phospholipids from one layer to another in the membrane, thereby affecting membrane asymmetry.
Flippase
Eukaryotic flippases are type-IV P-type ATPases or P4-ATPases belonging to P-type ATPase family proteins that are membrane-bound pumps involved in the ATP-mediated transport of ions and molecules across the membrane. Flippases flip specific phospholipids from the outer to the inner leaflet of a membrane. All P4-ATPases have one...