title: Disability for function: loss of Ca(2+)-binding is obligatory for fitness of mammalian βγ-crystallins.
abstract: Vertebrate βγ-crystallins belonging to the βγ-crystallin superfamily lack functional Ca(2+)-binding sites, while their microbial homologues do not; for example, three out of four sites in lens γ-crystallins are disabled. Such loss of Ca(2+)-binding function in non-lens βγ-crystallins from mammals (e.g., AIM1 and Crybg3) raises the possibility of a trade-off in the evolutionary extinction of Ca(2+)-binding. We test this hypothesis by reconstructing ancestral Ca(2+)-binding motifs (transforming disabled motifs into the canonical ones) in the lens γB-crystallin by introducing minimal sets of mutations. Upon incorporation of serine at the fifth position in the N/D-N/D-X-X-S/T(5)-S motif, which endowed a domain with microbial characteristics, a decreased domain stability was observed. Ca(2+) further destabilized the N-terminal domain (NTD) and its serine mutants profoundly, while the incorporation of a C-terminal domain (CTD) nullified this destabilization. On the other hand, Ca(2+)-induced destabilization of the CTD was not rescued by the introduction of an NTD. Of note, only one out of four sites is functional in the NTD of γB-crystallins responsible for weak Ca(2+) binding, but the deleterious effects of Ca(2+) are overcome by introduction of a CTD. The rationale for the onset of cataracts by certain mutations, such as R77S, which have not been clarified by structural means, could be explained by this work. The findings presented here shed light on the evolutionary innovations in terms of the functional loss of Ca(2+)-binding and acquisition of a bilobed domain, besides imparting additional advantages (e.g., protection from light) required for specialized functions.
authors.affiliation: Centre for Cellular and Molecular Biology (CCMB), CSIR , Uppal Road, Hyderabad 500007, India.
authors.forename: Shashi Kumar
authors.initials: SK
authors.lastname: Suman
authors.forename: Amita
authors.initials: A
authors.lastname: Mishra
authors.forename: Lahari
authors.initials: L
authors.lastname: Yeramala
authors.forename: Ishan Das
authors.initials: ID
authors.lastname: Rastogi
authors.forename: Yogendra
authors.initials: Y
authors.lastname: Sharma
mesh_terms: D001665:Binding Sites; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002151:Calorimetry; D008958:Models, Molecular; D055550:Protein Stability; D013050:Spectrometry, Fluorescence; D013696:Temperature; D038204:beta-Crystallins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D038222:gamma-Crystallins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D038204:beta-Crystallins; D038222:gamma-Crystallins; D002118:Calcium