X-ray crystallography reveals stringent conservation of protein fold after removal of the only disulfide bridge from a stabilized immunoglobulin variable domain

Abstract

Background:

Immunoglobulin domains owe a crucial fraction of their conformational stability to an invariant central disulfide bridge, the closure of which requires oxidation. Under the reducing conditions prevailing in cell cytoplasm, accumulation of soluble immunoglobulin is prohibited by its inability to acquire and maintain the native conformation. Previously, we have shown that disulfide-free immunoglobulins can be produced in Escherichia coli and purified from cytoplasmic extracts.

Results:

Immunoglobulin REI_v is the variable domain of a human κ light chain. The disulfide-free variant REI_v-C23V/Y32H was crystallized and its structure analyzed by X-ray crystallography (2.8 Å resolution). The conformation of the variant is nearly identical to that of the wild-type protein and the conformationally stabilized variant REI_v-T39K. This constitutes the first crystal structure of an immunoglobulin fragment without a disulfide bridge. The lack of the disulfide bridge produces no obvious local change in structure (compared with the wild type), whereas the Y32H mutation allows the formation of an additional hydrogen bond. There is a further change in the structure that is seen in the dimer in which Tyr49 has flipped out of the dimer interface in the mutant.

Conclusions:

Immunoglobulin derivatives without a central disulfide bridge but with stringently conserved wild-type conformation can be constructed in a practical two-step approach. First, the protein is endowed with additional folding stability by the introduction of one or more stabilizing amino acid exchanges; second, the disulfide bridge is destroyed by substitution of one of the two invariant cysteines. Such derivatives can be accumulated in soluble form in the cytoplasmic compartment of the E. coli cell. Higher protein yields and evolutionary refinement of catalytic antibodies by genetic complementation are among the possible advantages.