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The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis
19:16

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis

Published on: March 17, 2010

A corresponding tyrosine residue in the C2/factor B type A domain is a hot spot in the decay acceleration of the

Lisa A Kuttner-Kondo1, Megan P Dybvig, Lynne M Mitchell

  • 1Case Western Reserve University, Institute of Pathology, Cleveland, Ohio 44106, USA.

The Journal of Biological Chemistry
|October 17, 2003
PubMed

Insights

Regulators like decay-accelerating factor (DAF) control complement activation by dissociating C3 convertases. The alpha4/5 region of C2a is critical for this decay acceleration by DAF, complement receptor 1 (CR1), and C4-binding protein (C4BP).

Area of Science:

  • Immunology
  • Biochemistry
  • Molecular Biology

Background:

  • Complement activation is regulated by the dissociation of C3 convertases (C3bBb, C4b2a).
  • Key regulators include decay-accelerating factor (DAF), complement receptor 1 (CR1), factor H, and C4-binding protein (C4BP).
  • Previous work implicated the Bb alpha4/5 region in C3bBb decay acceleration.

Purpose of the Study:

  • To investigate the role of the C2 alpha4/5 region in the decay acceleration of the classical/lectin pathway C3 convertase (C4b2a).
  • To determine if this region is a critical interaction site for regulators like DAF, CR1, and C4BP.

Main Methods:

  • Site-directed mutagenesis was used to create C2 mutants, including C2 Y327A (homologous to Bb Y338A).
  • Functional C4b2a complexes were generated using these C2 mutants.
  • The resistance of these complexes to decay acceleration by DAF, CR1 (long homologous repeat A), and C4BP was assessed.

Main Results:

  • The C2 Y327A mutant exhibited high resistance to decay acceleration by DAF, C4BP, and CR1.
  • Mutations at nearby residues (N324A, L328A) resulted in partial resistance to decay acceleration.
  • These findings highlight the alpha4/5 region of C2a as crucial for regulator-mediated decay acceleration.

Conclusions:

  • The alpha4/5 region of C2a is essential for the decay acceleration of C4b2a by DAF, C4BP, and CR1.
  • Decay acceleration of both C4b2a and C3bBb likely involves interaction between the convertase alpha4/5 region and CCP2/CCP3 domains of DAF or homologous regions of CR1 and C4BP.

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