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Distributed subunit interactions in CheA contribute to dimer stability: a sedimentation equilibrium study.

Laila Kott¹, Emory H Braswell, Anthony L Shrout

¹Department of Chemistry, University of Massachusetts, Amherst, MA 01003-9336, USA.

Biochimica Et Biophysica Acta

|January 17, 2004

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Escherichia coli CheA protein dimerization is influenced by its structural domains. The P domain remnant in CheA(S) unexpectedly enhances dimerization through hydrophobic interactions, improving CheZ binding.

Area of Science:

Biochemistry
Molecular Biology
Protein Structure and Function

Background:

The Escherichia coli CheA protein is crucial for chemotaxis signaling.
CheA possesses distinct structural domains: N-terminal phosphate-accepting (P), CheY/CheB-binding (B), and C-terminal dimerization/catalytic/regulatory domains.
Dimerization of CheA is essential for its trans-autophosphorylation activity.

Purpose of the Study:

To investigate the role of secondary interaction sites in CheA dimerization.
To compare the dimerization properties of full-length CheA (CheA(L)), truncated CheA (CheA(S)), and a P domain-deleted mutant (CheA(T)).
To elucidate the contribution of the P domain and its remnants to CheA subunit interactions and CheZ binding.

Main Methods:

Sedimentation equilibrium experiments were employed to analyze protein-protein interactions.

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Dimerization constants (K(1,2)) were determined for different CheA constructs.

Experiments were conducted with isolated P domains and P domain remnants.

Main Results:

CheA(T) exhibited weaker dimerization (K(1,2) = 3.1 µM) compared to CheA(L) (K(1,2) = 0.49 µM), indicating P domain-catalytic domain interactions in CheA(L).
CheA(S) displayed unexpectedly strong dimerization (K(1,2) ≈ 20 nM) due to interactions between P domain remnants.
P domain remnants showed a propensity for self-association via hydrophobic forces, unlike the full-length P domain.
The P domain remnant demonstrated enhanced binding to CheZ compared to the intact P domain.

Conclusions:

Secondary interaction sites significantly influence CheA dimerization.
The P domain remnant in CheA(S) drives strong dimerization through hydrophobic interactions.
These P domain remnant interactions enhance CheZ binding, suggesting a regulatory role in signal transduction.