REENGINEERING IMMUNOGLOBULIN DOMAIN INTERACTIONS BY INTRODUCTION OF CHARGED RESIDUES

Citation
R. Raffen et al., REENGINEERING IMMUNOGLOBULIN DOMAIN INTERACTIONS BY INTRODUCTION OF CHARGED RESIDUES, Protein engineering (Print), 11(4), 1998, pp. 303-309
Citations number
40
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biothechnology & Applied Migrobiology",Biology
Journal title
ISSN journal
0269-2139
Volume
11
Issue
4
Year of publication
1998
Pages
303 - 309
Database
ISI
SICI code
0269-2139(1998)11:4<303:RIDIBI>2.0.ZU;2-9
Abstract
The formation of the antibody variable domain binding unit (Fv) is the net result of three competing assembly reactions. The affinities of c oncurrent homologous interactions of heavy and light chain variable do mains limits the heterologous interaction leading to productive format ion of the Fv. To address the possible role of light chain dimerizatio n in this phenomenon, the Gln38 residue at the dimer interface of an i mmunoglobulin light chain variable domain (V-L) was replaced by charge d amino acids. The effects of these mutations on V-L homodimer formati on were monitored by small-zone size exclusion HPLC and the affinities of interaction were determined by computer simulation. Reduced VL hom odimerization was observed in three of the four mutants, Q38R, Q38D an d Q38K, The association constants for the Q38R and Q38D homodimers wer e 1.2x10(4) and 3.2x10(3) M-1, respectively. This corresponded to a 20 -75-fold reduction in the homodimer association constant relative to t he wild-type V-L, which had an association constant of 2.4x10(5) M-1. Surprisingly, the fourth charge mutant, Q38E, had a higher association constant than the wild-type V-L. The potential for charged residues t o facilitate heterodimeric assembly of immunoglobulin domains was also tested. Heterodimerization was observed between the Q38D and Q38R V(L )s, but,vith an association constant of 4,7x10(4) M-1, approximately f ivefold lower than that obtained for homodimerization of the native V- L. In addition, replacement of the neutral, solvent-accessible Gln38 r esidue with either Asp or Arg was found to be significantly destabiliz ing, These results suggest that charged residues could be introduced a t immunoglobulin domain interfaces to guide heterodimer formation and to minimize unfavorable competing homologous associations. Nonetheless , these apparently simple modifications may also result in unintended consequences that are likely to depend upon structural features of par ticular variable domains.