Studies on a single immunoglobulin-binding domain of protein L from Peptostreptococcus magnus: the role of tyrosine-53 in the reaction with human IgG

Abstract

Chemical modification experiments with tetranitromethane (TNM) have been used to investigate the role of tyrosine residues in the formation of the complex between PpL (the single Ig-binding domain of protein L, isolated from P, magnus strain 3316) and the kappa light chain (?-chain). Reaction of PpL with TNM causes the modification of 1.9 equiv. of tyrosine (Tyr(51) and Tyr(53)) and results in an approx. 140-fold decrease in affinity for human IgG. Similar experiments with mutated PpL proteins suggest that nitration predominantly inactivates the protein by modification of Tyr(53). Reduction of the nitrotyrosine groups to aminotyrosine by incubation with sodium hydrosulphite does not restore high affinity for IgG, Modification of ?-chain by TNM resulted in the nitration of 3.1 +/- 0.09 tyrosine residues. When the PpL-?-chain complex was incubated with TNM, 4.1+/-0.04 tyrosine residues were nitrated, indicating that one tyrosine residue previously modified by the reagent was protected from TNM when the proteins are in complex with each other. The K-d for the equilibrium between PpL, human IgG and their complex has been shown by ELISA to be 112 +/- 20 nM. A similar value (153 +/- 33 nM) was obtained for the complex formed between IgG and the Tyr(64) ? Trp mutant (Y64W). However, the K-d values for the equilibria involving the PpL mutants Y53F and Y53F,Y64W were found to be 3.2+/-0.2 and 4.6+/-1 muM respectively. These suggest that the phenol group of Tyr(53) in PpL is important to the stability of the PpL-?-chain complex.

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