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Antibody variable region binding by Staphylococcal protein A: Thermodynamic analysis and location of the Fv binding site on E-domain

Published online by Cambridge University Press:  01 July 1999

MELISSA A. STAROVASNIK ,
MARK P. O'CONNELL ,
WAYNE J. FAIRBROTHER  and
ROBERT F. KELLEY
MELISSA A. STAROVASNIK
Affiliation:
Department of Protein Engineering, Genentech, Inc., One DNA Way, South San Francisco, California 94080
MARK P. O'CONNELL
Affiliation:
Department of Protein Engineering, Genentech, Inc., One DNA Way, South San Francisco, California 94080
WAYNE J. FAIRBROTHER
Affiliation:
Department of Protein Engineering, Genentech, Inc., One DNA Way, South San Francisco, California 94080
ROBERT F. KELLEY
Affiliation:
Department of Protein Engineering, Genentech, Inc., One DNA Way, South San Francisco, California 94080
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Abstract

Immunoglobulins of human heavy chain subgroup III have a binding site for Staphylococcal protein A on the heavy chain variable domain (VH), in addition to the well-known binding site on the Fc portion of the antibody. Thermodynamic characterization of this binding event and localization of the Fv-binding site on a domain of protein A is described. Isothermal titration calorimetry (ITC) was used to characterize the interaction between protein A or fragments of protein A and variants of the hu4D5 antibody Fab fragment. Analysis of binding isotherms obtained for titration of hu4D5 Fab with intact protein A suggests that 3–4 of the five immunoglobulin binding domains of full length protein A can bind simultaneously to Fab with a Ka of 5.5 ± 0.5 × 105 M−1. A synthetic single immunoglobulin binding domain, Z-domain, does not bind appreciably to hu4D5 Fab, but both the E and D domains are functional for hu4D5 Fab binding. Thermodynamic parameters for titration of the E-domain with hu4D5 Fab are n = 1.0 ± 0.1, Ka = 2.0 ± 0.3 × 105 M−1, and ΔH = −7.1 ± 0.4 kcal mol−1. Similar binding thermodynamics are obtained for titration of the isolated VH domain with E-domain indicating that the E-domain binding site on Fab resides within VH. E-domain binding to an IgG1 Fc yields a higher affinity interaction with thermodynamic parameters n = 2.2 ± 0.1, Ka > 1.0 × 107 M−1, and ΔH = −24.6 ± 0.6 kcal mol−1. Fc does not compete with Fab for binding to E-domain indicating that the two antibody fragments bind to different sites. Amide 1H and 15N resonances that undergo large changes in NMR chemical shift upon Fv binding map to a surface defined by helix-2 and helix-3 of E-domain, distinct from the Fc-binding site observed in the crystal structure of the B-domain/Fc complex. The Fv-binding region contains negatively charged residues and a small hydrophobic patch which complements the basic surface of the region of the VH domain implicated previously in protein A binding.

Keywords

calorimetryIgG binding domainsNMR chemical shift mappingStaphylococcal protein A
Type
Research Article
Information
Protein Science , Volume 8 , Issue 7 , July 1999 , pp. 1423 - 1431
Copyright
© 1999 The Protein Society

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