Crystal structure of a dimeric chymotrypsin inhibitor 2 mutant containing an inserted glutamine repeat

Abstract

We have constructed mutants of chymotrypsin inhibitor 2 with short glutamine repeats inserted into its inhibitory loop. These mutants oligomerize when expressed in Escherichia coli. The dimer of a mutant with four glutamines now has been crystallized, and its structure has been solved by molecular replacement by using the wild-type monomer as a search model. The structure of each half of the dimer is found to be the same as that of the wild-type monomer, except around the glutamine insertion. It was proposed that the components of the oligomers are held together by hydrogen bonds between the main-chain and side-chain amides of the glutamine repeats. Instead, they appear to form by swapping domains on folding in E. coli, and the glutamine repeats connecting the components of the dimers are disordered.

Domain swapping is a consequence of minimal frustration

“…To date, there is no detectable evidence of domain-swapping of CI2 in its wild-type form. We note, however, that artificial insertion of a long glutamine repeat does lead to domain swapping (33). In our simulated annealing runs of CI2 in its wild-type form, we found not a unique structure but rather multiple, distinct sets of dimeric conformations.…”

Solution Studies of Chymotrypsin Inhibitor-2 Glutamine Insertion Mutants Show No Interglutamine Interactions

“…relationships to neighbouring insertions (6). In the event, however, no electron density could be detected for any of the inserted residues.…”

“…However, the dimer and trimer were so stable that they could only be dissociated by protein denaturation, and further experiments showed that the same was true of dimers and trimers formed by CI2 mutants containing glutamine insertions as short as four residues. It was therefore clear that another mechanism must be responsible for oligomerisation, and indeed it has now been demonstrated that they are formed by domain swapping (6). Each "pseudo-monomer" in a domain-swapped dimer is formed from the N-terminal part (here residues 1-57 of wild type CI2) of one chain and the C-terminal part (here residues 61-83 of wild type CI2) of the other chain; the two polypeptide chains are thus held together by a multitude of native-like interactions within the structure of each "pseudo-monomer", while the two "pseudo-monomers" are held together covalently because the two polypeptide chains run between them.…”

An expanded glutamine repeat destabilizes native ataxin-3 structure and mediates formation of parallel β-fibrils

“…When inserted between two domains of a protein, expanded glutamine repeats may promote domain swapping by partially destabilizing the interactions between those domains. Indeed, it has been shown that the insertion of a small glutamine repeat into chymotrypsin inhibitor 2 causes dimerization by means of domain swapping (28). Second, the propensity of isolated polyglutamine stretches to assemble into hydrogen-bonded polar zippers is well documented (29).…”