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Evolutionary remodeling of ??-crystallins for domain stability at cost of Ca2+ binding.

ABSTRACT: The topologically similar ??-crystallins that are prevalent in all kingdoms of life have evolved for high innate domain stability to perform their specialized functions. The evolution of stability and its control in ??-crystallins that possess either a canonical (mostly from microorganisms) or degenerate (principally found in vertebrate homologues) Ca2+-binding motif is not known. Using equilibrium unfolding of ??-crystallin domains (26 wild-type domains and their mutants) in apo- and holo-forms, we demonstrate the presence of a stability gradient across these members, which is attained by the choice of residues in the (N/D)(N/D)XX(S/T)S Ca2+-binding motif. The occurrence of a polar, hydrophobic, or Ser residue at the 1st, 3rd, or 5th position of the motif is likely linked to a higher domain stability. Partial conversion of a microbe-type domain (with a canonical Ca2+-binding motif) to a vertebrate-type domain (with a degenerate Ca2+-binding motif) by mutating serine to arginine/lysine disables the Ca2+-binding but significantly augments its stability. Conversely, stability is compromised when arginine (in a vertebrate-type disabled domain) is replaced by serine (as a microbe type). Our results suggest that such conversions were acquired as a strategy for desired stability in vertebrate members at the cost of Ca2+-binding. In a physiological context, we demonstrate that a mutation such as an arginine to serine (R77S) mutation in this motif of ?-crystallin (partial conversion to microbe-type), implicated in cataracts, decreases the domain stability. Thus, this motif acts as a "central tuning knob" for innate as well as Ca2+-induced gain in stability, incorporating a stability gradient across ??-crystallin members critical for their specialized functions.

SUBMITTER: Suman SK

PROVIDER: S-EPMC3243557 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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Evolutionary remodeling of βγ-crystallins for domain stability at cost of Ca2+ binding.

Suman Shashi Kumar SK Mishra Amita A Ravindra Daddali D Yeramala Lahari L Sharma Yogendra Y

The Journal of biological chemistry 20110926 51

The topologically similar βγ-crystallins that are prevalent in all kingdoms of life have evolved for high innate domain stability to perform their specialized functions. The evolution of stability and its control in βγ-crystallins that possess either a canonical (mostly from microorganisms) or degenerate (principally found in vertebrate homologues) Ca2+-binding motif is not known. Using equilibrium unfolding of βγ-crystallin domains (26 wild-type domains and their mutants) in apo- and holo-forms ...[more]

PMID: 21949186

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Project description:BackgroundWe highlight an unrecognized physiological role for the Greek key motif, an evolutionarily conserved super-secondary structural topology of the βγ-crystallins. These proteins constitute the bulk of the human eye lens, packed at very high concentrations in a compact, globular, short-range order, generating transparency. Congenital cataract (affecting 400,000 newborns yearly worldwide), associated with 54 mutations in βγ-crystallins, occurs in two major phenotypes nuclear cataract, which blocks the central visual axis, hampering the development of the growing eye and demanding earliest intervention, and the milder peripheral progressive cataract where surgery can wait. In order to understand this phenotypic dichotomy at the molecular level, we have studied the structural and aggregation features of representative mutations.MethodsWild type and several representative mutant proteins were cloned, expressed and purified and their secondary and tertiary structural details, as well as structural stability, were compared in solution, using spectroscopy. Their tendencies to aggregate in vitro and in cellulo were also compared. In addition, we analyzed their structural differences by molecular modeling in silico.ResultsBased on their properties, mutants are seen to fall into two classes. Mutants A36P, L45PL54P, R140X, and G165fs display lowered solubility and structural stability, expose several buried residues to the surface, aggregate in vitro and in cellulo, and disturb/distort the Greek key motif. And they are associated with nuclear cataract. In contrast, mutants P24T and R77S, associated with peripheral cataract, behave quite similar to the wild type molecule, and do not affect the Greek key topology.ConclusionWhen a mutation distorts even one of the four Greek key motifs, the protein readily self-aggregates and precipitates, consistent with the phenotype of nuclear cataract, while mutations not affecting the motif display 'native state aggregation', leading to peripheral cataract, thus offering a protein structural rationale for the cataract phenotypic dichotomy "distort motif, lose central vision".

Dataset Information

Evolutionary remodeling of ??-crystallins for domain stability at cost of Ca2+ binding.

Publications

Evolutionary remodeling of βγ-crystallins for domain stability at cost of Ca2+ binding.

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