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

Immunoglobulins of human heavy chain subgroup III have a binding site for Staphylococcal protein A on the heavy chain variable domain ( V H ), 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 K a of 5.5 ± 0.5 × 10 5 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, K a = 2.0 ± 0.3 × 10 5 M –1 , and Δ H = –7.1 ± 0.4 kcal mol –1 . Similar binding thermodynamics are obtained for titration of the isolated V H domain with E‐domain indicating that the E‐domain binding site on Fab resides within V H . E‐domain binding to an IgG1 Fc yields a higher affinity interaction with thermodynamic parameters n = 2.2 ± 0.1, K a > 1.0 × 10 7 M –1 , and Δ H = –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 1 H and 15 N 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 V H domain implicated previously in protein A binding.