Calcium‐binding to lens βB2‐ and βA3‐crystallins suggests that all β‐crystallins are calcium‐binding proteins

Abstract

Crystallins are abundant proteins found in the eye lens of vertebrates that belong to two superfamilies named as a-crystallins and bc-crystallins [1]. a-Crystallins are known to play an important role as molecular chaperone [2]. On the other hand, bc-crystallins are thought to play structural role in the mammalian eye lens. Their nonstructural functions, which appear to be very important, have not been elucidated [3].b-Crystallins from vertebrate eye lens are a group of seven proteins broadly classified into four acidic (bA1 ⁄ A3, bA2 and bA4) and three basic b-crystallins (bB1, bB2, and bB3). b-Crystallins have high sequence similarity and identity [4]. Acidic b-crystallins have both N-and C-terminal extensions, whereas basic b-crystallins have only N-terminal extensions. All b-crystallins have four Greek key motifs organized into two crystallin domains. In this respect, b-crystallins are similar to c-crystallins, which also have a similar domain organization and structure [5,6]. The major difference between the b-and c-crystallins is their oligomeric state. c-Crystallins are monomeric, whereas b-crystallins exist as dimers to octamers in solution [7]. b-and c-crystallins are the prototype and founding members of the bc-crystallin superfamily [8,9]. Crystallins are the major proteins of a mammalian eye lens. The topologically similar eye lens proteins, b-and c-crystallins, are the prototype and founding members of the bc-crystallin superfamily. bc-Crystallins have until recently been regarded as structural proteins. However, the calciumbinding properties of a few members and the potential role of bc-crystallins in fertility are being investigated. Because the calcium-binding elements of other member proteins, such as spherulin 3a, are not present in bB2-crystallin and other bc-crystallins from fish and mammalian genomes, it was argued that lens bc-crystallins should not bind calcium. In order to probe whether b-crystallins can bind calcium, we selected one basic (bB2) and one acidic (bA3) b-crystallin for calcium-binding studies. Using calciumbinding assays such as 45 Ca overlay, terbium binding, Stains-All and isothermal titration calorimetry, we established that both bB2-and bA3-crystallin bind calcium with moderate affinity. There was no significant change in their conformation upon binding calcium as monitored by fluorescence and circular dichroism spectroscopy. However, 15 N-1 H heteronuclear single quantum correlation NMR spectroscopy revealed that amide environment of several residues underwent changes indicating calcium ligation. With the corroboration of calcium-binding to bB2-and bA3-crystallins, we suggest that all b-crystallins bind calcium. Our results have important implications for understanding the calcium-related cataractogenesis and maintenance of ionic homeostasis in the lens.Abbreviations AIM1, protein absent in melanoma 1; HSQC, heteronuclear single quantum correlation; ITC, isothermal titration calorimetry; PDB, protein databank; TCEP, Tris(2-carboxyethyl) phosphine hydrochloride.

Calcium Binds to Leptospiral Immunoglobulin-like Protein, LigB, and Modulates Fibronectin Binding

“…. Similar results have been noted in some members of the lens ␤␥-crystallin superfamily, such as microbial crystallins (37,46,47). There is a significant tertiary fold in apo-LigBCen2, suggesting that this protein is well folded.…”

“…The exact function of lens ␤␥-crystallins is not known. However, we have shown earlier that ␤␥-crystallins belong to a different superfamily of low affinity Ca 2ϩ -binding proteins (37,38). Based on the fold similarities, we predicted that these Lig proteins might bind Ca 2ϩ .…”

Ca2+-binding Motif of βγ-Crystallins

“…17). Some individual domains of larger proteins from the pathogenic bacterium Yersinia pestis and the extremophilic Caulobacter crescentus and Hahella chejuensis are intrinsically unstructured (or partly unstructured) in the apo form and gain significant structure upon binding Ca 2ϩ (38,39,83). Although ␤␥ domains do not undergo major structural change upon binding Ca 2ϩ , they assume a reduced hydrodynamic size and thermodynamically drift to a state of higher structural stabilization.…”

Decoding the Molecular Design Principles Underlying Ca2+ Binding to βγ-Crystallin Motifs

“…17,25 In the case of diverged or noncanonical motifs, Ca 2 + binding is either abolished or very poor (up to 200 μM), as in the case of lens crystallins. 26,27 Although many members of the βγ-superfamily have been shown to bind Ca 2+ using the N/D-N/D-X 1 -X 2 -S/T-S motif, a comprehensive understanding of its intricacies such as controlling factors and determinants of the wide-ranging response toward Ca 2+ is lacking. We have noted significant variations in the sequence of this six-amino-acid canonical Ca 2+ -binding motif of these proteins, but a careful analysis shows that the residue at the −x position (SC) is usually Asp or Asn in some exceptions.…”