Mutational analysis of the interaction between albumin-binding domain from streptococcal protein G and human serum albumin

Abstract

Streptococcal protein G (SpG) is a bacterial cell surface receptor exhibiting affinity to both human immunoglobulin (IgG) and human serum albumin (HSA). Interestingly, the serum albumin and immunoglobulin-binding activities have been shown to reside at functionally and structurally separated receptor domains. The binding domain of the HSA-binding part has been shown to be a 46-residue triple alpha-helical structure, but the binding site to HSA has not yet been determined. Here, we have investigated the precise binding region of this bacterial receptor by protein engineering applying an alanine-scanning procedure followed by binding studies by surface plasmon resonance (SPR). The secondary structure as well as the HSA binding of the resulting albumin-binding domain (ABD) variants were analyzed using circular dichroism (CD) and affinity blotting. The analysis shows that the HSA binding involves residues mainly in the second alpha-helix.

Fig. 1.

An overview of the streptococcal protein G (SpG). The IgG and albumin-binding parts are separated. Both parts consist of three homologous domains. Nomenclature according to Ståhl and Nygren (1999).

Fig. 2.

Graphic comparison of the changes in free binding energy of the analyzed ABD variants compared to the parental ABD (δδG in kcal/mole). The values are calculated using BIAevaluation 3.02.

Fig. 3.

The binding pattern for the three triple mutants of ABD*, E3A, V6A, L7A; R10A, E11A, K14A; and K35A, I38A, D39A. The concentration was 100 nM. The signal from a nonimmobilized surface was subtracted. The mean values from at least two different measurements are shown.

Fig. 4.

The three-dimensional structure of ABD where the effect of different mutations is displayed. (A) Change in k_on upon mutation. Orange color means a decreased and green color shows an increased on-rate when the original residue is exchanged to alanine. Residues shown in yellow denote an unchanged behavior in the on-rate despite a change to alanine. (B) How the off-rate changes while replacing certain residues for alanine. Orange shows a position where the off-rate increases and green color positions where the off-rate decreases upon mutation. Yellow means positions where a mutation does not affect the k_off.