Group II archaeal chaperonin recognition of partially folded human γD‐crystallin mutants

Abstract

The features in partially folded intermediates that allow the group II chaperonins to distinguish partially folded from native states remain unclear. The archaeal group II chaperonin from Methanococcus Mauripaludis (Mm-Cpn) assists the in vitro refolding of the well-characterized bsheet lens protein human cD-crystallin (HcD-Crys). The domain interface and buried cores of this Greek key conformation include side chains, which might be exposed in partially folded intermediates. We sought to assess whether particular features buried in the native state, but absent from the native protein surface, might serve as recognition signals. The features tested were (a) paired aromatic side chains, (b) side chains in the interface between the duplicated domains of HcD-Crys, and (c) side chains in the buried core which result in congenital cataract when substituted. We tested the Mm-Cpn suppression of aggregation of these HcD-Crys mutants upon dilution out of denaturant. Mm-Cpn was capable of suppressing the off-pathway aggregation of the three classes of mutants indicating that the buried residues were not recognition signals. In fact, Mm-Cpn recognized the HcD-Crys mutants better than (wild-type) WT and refolded most mutant HcD-Crys to levels twice that of WT HcD-Crys. This presumably represents the increased population or longer lifetimes of the partially folded intermediates of the mutant proteins. The results suggest that MmCpn does not recognize the features of HcD-Crys tested-paired aromatics, exposed domain interface, or destabilized core-but rather recognizes other features of the partially folded b-sheet conformation that are absent or inaccessible in the native state of HcD-Crys.

Biochemical Characterization of Mutants in Chaperonin Proteins CCT4 and CCT5 Associated with Hereditary Sensory Neuropathy

“…This decrease was ϳ30% for WT human ␥D-crystallin and ϳ20% for Y92A/Y97A human ␥D-crystallin, but the amount of human ␥D-crystallin refolded by H147R CCT5 was not significantly different from background refolding in both cases. In general, Y92A/Y97A human ␥D-crystallin was refolded to lower levels compared with WT human ␥D-crystallin, contrary to what was seen for the archaeal Methanococcus marapaludis chaperonin previously (18).…”

“…In this case, the aggregating protein was human ␥D-crystallin carrying a double-alanine substitution of tyrosine residues, Y92A/Y97A (18,25). Suppression of aggregation by WT CCT5 was similar to that found with WT human ␥D-crystallin (Fig.…”

“…Interestingly, the crystallin mutant used herein, Y92A/Y97A, was refolded to higher levels by the archaeal M. marapaludis chaperonin (18). Here, both WT and H147R CCT5 refolded the mutant substrate chains to levels of about one-third of those of WT human ␥D-crystallin chains.…”

“…The human ␥D-crystallin aggregation suppression and refolding assay used in this study has been used for many other chaperonins (13,14,17,18). Interestingly, the crystallin mutant used herein, Y92A/Y97A, was refolded to higher levels by the archaeal M. marapaludis chaperonin (18).…”