Cavity-Creating Mutation at the Dimer Interface of Plasmodium falciparum Triosephosphate Isomerase: Restoration of Stability by Disulfide Cross-Linking of Subunits

Abstract

Disulfide engineering across subunit interfaces provides a means of inhibiting dissociation during unfolding of multimeric enzymes. Two symmetry-related intersubunit disulfide bridges were introduced across the interface of the dimeric enzyme triosephosphate isomerase from Plasmodium falciparum. This was achieved by mutating a tyrosine residue at position 74 at the subunit interface to a cysteine, thereby enabling it to form a covalent cross-link with a pre-existing cysteine at position 13 of the ether subunit. The wild-type enzyme (TIMWT) and the oxidized (Y74Cox) and reduced (Y74Cred) forms of the mutant have similar enzymatic activity, absorption, and fluorescence spectra. All three proteins have similar far-UV CD spectra. The Y74Cred shows a distinct loss of near-UV CD. Thermal precipitation studies demonstrate that TIMWT and Y74Cox have very similar T-m values (T-m similar to 60 degrees C) whereas Y74Cred is surprisingly labile (T-m similar to 38 degrees C). The Y74C mutant results in the creation of a I,uge cavity (similar to 100 Angstrom(3)) at the dimer interface. The crystal structure for the oxidized form of Y74C mutant, crystallized in the presence of low concentrations of dithiothreitol, reveals an asymmetric dimer containing a disulfide bridge at one site and a reduced dithiol cysteine at the other. The crystal structure of the mutant offers insights into the destabilization effects of the interfacial cavities and the role of disulfide tethering in restoring protein stability