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N-terminal extension of beta B1-crystallin: identification of a critical region that modulates protein interaction with beta A3-crystallin.

ABSTRACT: The human lens proteins beta-crystallins are subdivided into acidic (betaA1-betaA4) and basic (betaB1-betaB3) subunit groups. These structural proteins exist at extremely high concentrations and associate into oligomers under physiological conditions. Crystallin acidic-basic pairs tend to form strong heteromolecular associations. The long N-terminal extensions of beta-crystallins may influence both homo- and heteromolecular interactions. However, identification of the critical regions of the extensions mediating protein associations has not been previously addressed. This was studied by comparing the self-association and heteromolecular associations of wild-type recombinant betaA3- and betaB1-crystallins and their N-terminally truncated counterparts (betaA3DeltaN30 and betaB1DeltaN56) using several biophysical techniques, including analytical ultracentrifugation and fluorescence spectroscopy. Removal of the N-terminal extension of betaA3 had no effect on dimerization or heteromolecular tetramer formation with betaB1. In contrast, the level of self-association of betaB1DeltaN56 increased, resulting in homotetramer formation, and heteromolecular association with betaA3 was blocked. Limited proteolysis of betaB1 produced betaB1DeltaN47, which is similar to intact protein formed dimers but in contrast showed enhanced heteromolecular tetramer formation with betaA3. The tryptic digestion was physiologically significant, corresponding to protease processing sites observed in vivo. Molecular modeling of the N-terminal betaB1 extension indicates structural features that position a mobile loop in the vicinity of these processing sites. The loop is derived from residues 48-56 which appear to be critical for mediating protein interactions with betaA3-crystallin.

SUBMITTER: Dolinska MB

PROVIDER: S-EPMC2764403 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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N-terminal extension of beta B1-crystallin: identification of a critical region that modulates protein interaction with beta A3-crystallin.

Dolinska Monika B MB Sergeev Yuri V YV Chan May P MP Palmer Ira I Wingfield Paul T PT

Biochemistry 20091001 40

The human lens proteins beta-crystallins are subdivided into acidic (betaA1-betaA4) and basic (betaB1-betaB3) subunit groups. These structural proteins exist at extremely high concentrations and associate into oligomers under physiological conditions. Crystallin acidic-basic pairs tend to form strong heteromolecular associations. The long N-terminal extensions of beta-crystallins may influence both homo- and heteromolecular interactions. However, identification of the critical regions of the ext ...[more]

PMID: 19746987

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Project description:Interaction among crystallins is required for the maintenance of lens transparency. Deamidation is one of the most common post-translational modifications in crystallins, which results in incorrect interaction and leads to aggregate formation. Various studies have established interaction among the ?- and ?-crystallins. Here, we investigated the effects of the deamidation of ?A- and ?B-crystallins on their interaction with ?A3-crystallin using surface plasmon resonance (SPR) and fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET) methods. SPR analysis confirmed adherence of WT ?A- and WT ?B-crystallins and their deamidated mutants with ?A3-crystallin. The deamidated mutants of ?A-crystallin (?A N101D and ?A N123D) displayed lower adherence propensity for ?A3-crystallin relative to the binding affinity shown by WT ?A-crystallin. Among ?B-crystallin mutants, ?B N78D displayed higher adherence propensity whereas ?B N146D mutant showed slightly lower binding affinity for ?A3-crystallin relative to that shown by WT ?B-crystallin. Under the in vivo condition (FLIM-FRET), both ?A-deamidated mutants (?A N101D and ?A N123D) exhibited strong interaction with ?A3-crystallin (32±4% and 36±4% FRET efficiencies, respectively) compared to WT ?A-crystallin (18±4%). Similarly, the ?B N78D and ?B N146D mutants showed strong interaction (36±4% and 22±4% FRET efficiencies, respectively) with ?A3-crystallin compared to 18±4% FRET efficiency of WT ?B-crystallin. Further, FLIM-FRET analysis of the C-terminal domain (CTE), N-terminal domain (NTD), and core domain (CD) of ?A- and ?B-crystallins with ?A3-crystallin suggested that interaction sites most likely reside in the ?A CTE and ?B NTD regions, respectively, as these domains showed the highest FRET efficiencies. Overall, results suggest that similar to WT ?A- and WT?B-crystallins, the deamidated mutants showed strong interactionfor ?A3-crystallin. Variable in vitro and in vivo interactions are most likely due to the mutant's large size oligomers, reduced hydrophobicity, and altered structures. Together, the results suggest that deamidation of ?-crystallin may facilitate greater interaction and the formation of large oligomers with other crystallins, and this may contribute to the cataractogenic mechanism.

Dataset Information

N-terminal extension of beta B1-crystallin: identification of a critical region that modulates protein interaction with beta A3-crystallin.

Publications

N-terminal extension of beta B1-crystallin: identification of a critical region that modulates protein interaction with beta A3-crystallin.

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