Abstract: βγ-Crystallin-type double clamp (N/D)(N/D)XX(S/T)S motif is an established but sparsely investigated motif for Ca2+ binding. A βγ-crystallin domain is formed of two Greek key motifs, accommodating two Ca2+-binding sites. βγ-Crystallins make a separate class of Ca2+-binding proteins (CaBP), apparently a major group of CaBP in bacteria. Paralleling the diversity in βγ-crystallin domains, these motifs also show great diversity, both in structure and in function. Although the expression of some of them has been associated with stress, virulence, and adhesion, the functional implications of Ca2+ binding to βγ-crystallins in mediating biological processes are yet to be elucidated. βγ-Crystallin-type double clamp (N/D)(N/D)XX(S/T)S motif is an established but sparsely investigated motif for Ca2+ binding. A βγ-crystallin domain is formed of two Greek key motifs, accommodating two Ca2+-binding sites. βγ-Crystallins make a separate class of Ca2+-binding proteins (CaBP), apparently a major group of CaBP in bacteria. Paralleling the diversity in βγ-crystallin domains, these motifs also show great diversity, both in structure and in function. Although the expression of some of them has been associated with stress, virulence, and adhesion, the functional implications of Ca2+ binding to βγ-crystallins in mediating biological processes are yet to be elucidated. Ca2+ binding, already a very abundant physiological process, has a repertoire of associated Ca2+-binding proteins (CaBP) 4The abbreviation used is: CaBPCa2+-binding proteins. , including numerous proteins involved in signaling events (a few reviews, see Refs. 1.Clapham D.E. et al.Calcium signaling.Cell. 2007; 131: 1047-1058Abstract Full Text Full Text PDF PubMed Scopus (2952) Google Scholar2.Burgoyne R.D. et al.Neuronal calcium sensor proteins: Generating diversity in neuronal Ca2+ signaling.Nat. Rev. Neurosci. 2007; 8: 182-193Crossref PubMed Scopus (418) Google Scholar, 3.Dominguez D.C. et al.Calcium signalling in bacteria.Mol. Microbiol. 2004; 54: 291-297Crossref PubMed Scopus (268) Google Scholar4.Marks A.R. et al.Calcium cycling proteins and heart failure: mechanisms and therapeutics.J. Clin. Invest. 2013; 123: 46-52Crossref PubMed Scopus (268) Google Scholar). Ca2+-binding proteins are grouped according to the nature of their binding motifs. The well studied all-α-helical EF-hand motif and all-β-sheet-containing C2 domains, which possess diverse functions, predominate sensory CaBP (5.Lewit-Bentley A. Réty S. et al.EF-hand calcium-binding proteins.Curr. Opin. Struct. Biol. 2000; 10: 637-643Crossref PubMed Scopus (419) Google Scholar6.Gifford J.L. Walsh M.P. Vogel H.J. et al.Structures and metal-ion-binding properties of the Ca2+-binding helix-loop-helix EF-hand motifs.Biochem. J. 2007; 405: 199-221Crossref PubMed Scopus (651) Google Scholar, 7.Rizo J. Südhof T.C. et al.C2-domains, structure and function of a universal Ca2+-binding domain.J. Biol. Chem. 1998; 273: 15879-15882Abstract Full Text Full Text PDF PubMed Scopus (708) Google Scholar8.Südhof T.C. et al.Calcium control of neurotransmitter release.Cold Spring Harb. Perspect. Biol. 2012; 4: a011353Crossref PubMed Scopus (267) Google Scholar). Many CaBP with extracellular EGF domains, β-propeller-like domains, and cadherins are also known (9.Stenflo J. Stenberg Y. Muranyi A. et al.Calcium-binding EGF-like modules in coagulation proteinases: Function of the calcium ion in module interactions.Biochim. Biophys. Acta. 2000; 1477: 51-63Crossref PubMed Scopus (107) Google Scholar10.Fu S. Sun J. Qian L. et al.Effect of Ca2+ on β-propeller phytases.Protein Pept. Lett. 2008; 15: 39-42Crossref PubMed Scopus (14) Google Scholar, 11.Nagar B. Overduin M. Ikura M. Rini J.M. et al.Structural basis of calcium-induced E-cadherin rigidification and dimerization.Nature. 1996; 380: 360-364Crossref PubMed Scopus (564) Google Scholar12.van Roy F. Berx G. et al.The cell-cell adhesion molecule E-cadherin.Cell Mol. Life Sci. 2008; 65: 3756-3788Crossref PubMed Scopus (893) Google Scholar). These independent structural units perform their Ca2+-dependent roles either as complete proteins or as modules in multidomain proteins. Ca2+-binding proteins. The Ca2+-binding motif of the βγ-crystallin type is a recently established motif with a wide prevalence. Here, we lay out our understanding of this motif with respect to the geometry of binding sites, modes of Ca2+ coordination, and prediction of functional, disabled, or degenerate (nonfunctional) motifs. The well characterized founding members of the βγ-crystallin superfamily are lens β- and γ-crystallins, which are major constituents of the vertebrate eye lens, rendering it with a high refractive index and transparency (for reviews, see Refs. 13.Wistow G.J. Piatigorsky J. et al.Lens crystallins: the evolution and expression of proteins for a highly specialized tissue.Annu. Rev. Biochem. 1988; 57: 479-504Crossref PubMed Scopus (675) Google Scholar14.van Rens G.L.M. de Jong W.W. Bloemendal H. et al.A superfamily in the mammalian eye lens: the β/γ-crystallins.Mol. Biol. Rep. 1992; 16: 1-10Crossref PubMed Scopus (21) Google Scholar, 15.Jaenicke R. Slingsby C. et al.Lens crystallins and their microbial homologs: structure, stability, and function.Crit. Rev. Biochem. Mol. Biol. 2001; 36: 435-499Crossref PubMed Scopus (135) Google Scholar, 16.Bhat S.P. et al.Crystallins, genes and cataract.Prog. Drug Res. 2003; 60: 205-262Crossref PubMed Scopus (78) Google Scholar, 17.Bloemendal H. de Jong W. Jaenicke R. Lubsen N.H. Slingsby C. Tardieu A. et al.Ageing and vision: structure, stability and function of lens crystallins.Prog. Biophys. Mol. Biol. 2004; 86: 407-485Crossref PubMed Scopus (702) Google Scholar18.Slingsby C. Wistow G.J. Clark A.R. et al.Evolution of crystallins for a role in the vertebrate eye lens.Protein Sci. 2013; 22: 367-380Crossref PubMed Scopus (103) Google Scholar). As in the case of some other Ca2+-binding proteins (C2 domains, EGF domains, and cadherins), βγ-crystallins possess an all-β fold, made of strand exchanged Greek key motifs (19.Blundell T. Lindley P. Miller L. Moss D. Slingsby C. Tickle I. Turnell B. Wistow G. et al.The molecular structure and stability of the eye lens: x-ray analysis of γ-crystallin II.Nature. 1981; 289: 771-777Crossref PubMed Scopus (388) Google Scholar, 20.Wistow G. Turnell B. Summers L. Slingsby C. Moss D. Miller L. Lindley P. Blundell T. et al.X-ray analysis of the eye lens protein γ-II crystallin at 1.9 Å resolution.J. Mol. Biol. 1983; 170: 175-202Crossref PubMed Scopus (280) Google Scholar). The βγ-crystallin domain is an ancient protein fold, and several proteins across different domains of life are found to have this fold, a majority of them being expressed in bacterial species (15.Jaenicke R. Slingsby C. et al.Lens crystallins and their microbial homologs: structure, stability, and function.Crit. Rev. Biochem. Mol. Biol. 2001; 36: 435-499Crossref PubMed Scopus (135) Google Scholar, 17.Bloemendal H. de Jong W. Jaenicke R. Lubsen N.H. Slingsby C. Tardieu A. et al.Ageing and vision: structure, stability and function of lens crystallins.Prog. Biophys. Mol. Biol. 2004; 86: 407-485Crossref PubMed Scopus (702) Google Scholar, 21.Kappé G. Purkiss A.G. van Genesen S.T. Slingsby C. Lubsen N.H. et al.Explosive expansion of βγ-crystallin genes in the ancestral vertebrate.J. Mol. Evol. 2010; 71: 219-230Crossref PubMed Scopus (30) Google Scholar). Many βγ-crystallin domains have been studied in the recent past, and insights obtained from ion binding in these βγ-crystallins have led to the proposition that proteins of this superfamily possess a universal Ca2+-binding motif (22.Clout N.J. Kretschmar M. Jaenicke R. Slingsby C. et al.Crystal structure of the calcium-loaded spherulin 3a dimer sheds light on the evolution of the eye lens βγ-crystallin domain fold.Structure. 2001; 9: 115-124Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 23.Aravind P. Mishra A. Suman S.K. Jobby M.K. Sankaranarayanan R. Sharma Y. et al.βγ-Crystallin superfamily contains a universal motif for binding calcium.Biochemistry. 2009; 48: 12180-12190Crossref PubMed Scopus (55) Google Scholar). Notwithstanding that βγ-crystallins have been established as a superfamily of Ca2+-binding proteins, their involvement in the cellular Ca2+ metabolism or signaling is far from being recognized yet. We summarize a critical analysis of the Ca2+-binding motif present in this superfamily of proteins. Although the very first non-lens protein belonging to the βγ-crystallin superfamily was Protein S from the bacterium Myxococcus xanthus (24.Teintze M. Inouye M. Inouye S. et al.Characterization of calcium-binding sites in development-specific protein S of Myxococcus xanthus using site-specific mutagenesis.J. Biol. Chem. 1988; 263: 1199-1203Abstract Full Text PDF PubMed Google Scholar), it took almost two decades to classify these proteins as a separate group of CaBP. In the absence of structural information, Protein S was earlier speculated to possess extensively modified EF-hand motifs (25.Inouye S. Franceschini T. Inouye M. et al.Structural similarities between the development-specific protein S from a gram-negative bacterium, Myxococcus xanthus, and calmodulin.Proc. Natl. Acad. Sci. U.S.A. 1983; 80: 6829-6833Crossref PubMed Scopus (57) Google Scholar). Weak binding of Ca2+ to lens β-crystallins was reported on the basis of equilibrium dialysis and interaction with the Ca2+ mimic dye Stains-all (26.Sharma Y. Rao C.M. Narasu M.L. Rao S.C. Somasundaram T. Gopalakrishna A. Balasubramanian D. et al.Calcium ion binding to δ- and to β-crystallins: the presence of the "EF-hand" motif in δ-crystallin that aids in calcium ion binding.J. Biol. Chem. 1989; 264: 12794-12799Abstract Full Text PDF PubMed Google Scholar, 27.Sharma Y. Rao C.M. Rao S.C. Krishna A.G. Somasundaram T. Balasubramanian D. et al.Binding site conformation dictates the color of the dye Stains-all: a study of the binding of this dye to the eye lens proteins crystallins.J. Biol. Chem. 1989; 264: 20923-20927Abstract Full Text PDF PubMed Google Scholar). Another decade passed before Spherulin 3a, a protein from slime mold Physarum polycephalum that was predicted to be a single domain βγ-crystallin (28.Wistow G. et al.Evolution of a protein superfamily: relationships between vertebrate lens crystallins and microorganism dormancy proteins.J. Mol. Evol. 1990; 30: 140-145Crossref PubMed Scopus (127) Google Scholar), was finally shown to bind Ca2+, with clues from Ca2+ binding to lens β-crystallin (29.Rosinke B. Renner C. Mayr E.M. Jaenicke R. Holak T.A. et al.Ca2+-loaded spherulin 3a from Physarum polycephalum adopts the prototype γ-crystallin fold in aqueous solution.J. Mol. Biol. 1997; 271: 645-655Crossref PubMed Scopus (39) Google Scholar, 30.Kretschmar M. Jaenicke R. et al.Stability of a homo-dimeric Ca2+-binding member of the βγ-crystallin superfamily: DSC measurements on spherulin 3a from Physarum polycephalum.J. Mol. Biol. 1999; 291: 1147-1153Crossref PubMed Scopus (19) Google Scholar31.Kretschmar M. Mayr E.M. Jaenicke R. et al.Kinetic and thermodynamic stabilization of the βγ-crystallin homolog spherulin 3a from Physarum polycephalum by calcium binding.J. Mol. Biol. 1999; 289: 701-705Crossref PubMed Scopus (32) Google Scholar). Based on Ca2+ binding to a peptide corresponding to a Greek key motif of γ-crystallin (32.Rajini B. Shridas P. Sundari C.S. Muralidhar D. Chandani S. Thomas F. Sharma Y. et al.Calcium binding properties of γ-crystallin: calcium ion binds at the Greek key βγ-crystallin fold.J. Biol. Chem. 2001; 276: 38464-38471Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), a proposition that βγ-crystallin-type Greek key could form the motif for Ca2+ binding was formulated. Concurrent structural studies shed light on the coordination pattern of Ca2+ binding to Spherulin 3a and Protein S (22.Clout N.J. Kretschmar M. Jaenicke R. Slingsby C. et al.Crystal structure of the calcium-loaded spherulin 3a dimer sheds light on the evolution of the eye lens βγ-crystallin domain fold.Structure. 2001; 9: 115-124Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 33.Wenk M. Baumgartner R. Holak T.A. Huber R. Jaenicke R. Mayr E.M. et al.The domains of protein S from Myxococcus xanthus: structure, stability and interactions.J. Mol. Biol. 1999; 286: 1533-1545Crossref PubMed Scopus (44) Google Scholar). The recognition of βγ-crystallins as a distinct superfamily of Ca2+-binding proteins was still in its infancy. The factors responsible for this were: (i) undefined motif of Ca2+ binding, (ii) lack of information about the role of Ca2+ in protein functions, and (iii) no substantial addition of novel members to this superfamily. For a long time, this superfamily was considered a sparsely distributed family with only a few scattered members, although some more proteins (e.g. WmKT, SKLP, and SMPI) were identified based on structural similarity (34.Antuch W. Güntert P. Wüthrich K. et al.Ancestral βγ-crystallin precursor structure in a yeast killer toxin.Nat. Struct. Biol. 1996; 3: 662-665Crossref PubMed Scopus (74) Google Scholar, 35.Ohno A. Tate S. Seeram S.S. Hiraga K. Swindells M.B. Oda K. Kainosho M. et al.NMR structure of the Streptomyces metalloproteinase inhibitor, SMPI, isolated from Streptomyces nigrescens TK-23: Another example of an ancestral βγ-crystallin precursor structure.J. Mol. Biol. 1998; 282: 421-433Crossref PubMed Scopus (37) Google Scholar36.Ohki S.Y. Kariya E. Hiraga K. Wakamiya A. Isobe T. Oda K. Kainosho M. et al.NMR structure of Streptomyces killer toxin-like protein, SKLP: further evidence for the wide distribution of single-domain βγ-crystallin superfamily proteins.J. Mol. Biol. 2001; 305: 109-120Crossref PubMed Scopus (23) Google Scholar); these did not belong to the βγ-crystallins lineage and may have arisen from convergent evolution (37.Clout N.J. Slingsby C. Wistow G.J. et al.Picture story. An eye on crystallins.Nat. Struct. Biol. 1997; 4: 685Crossref PubMed Scopus (21) Google Scholar). With the advent of genomic sequence information, many members from diverse species (bacteria, archaea, and urochordate) were added to the superfamily and also confirmed to be Ca2+-binding proteins (22.Clout N.J. Kretschmar M. Jaenicke R. Slingsby C. et al.Crystal structure of the calcium-loaded spherulin 3a dimer sheds light on the evolution of the eye lens βγ-crystallin domain fold.Structure. 2001; 9: 115-124Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 23.Aravind P. Mishra A. Suman S.K. Jobby M.K. Sankaranarayanan R. Sharma Y. et al.βγ-Crystallin superfamily contains a universal motif for binding calcium.Biochemistry. 2009; 48: 12180-12190Crossref PubMed Scopus (55) Google Scholar, 38.Jobby M.K. Sharma Y. et al.Calcium-binding crystallins from Yersinia pestis: characterization of two single βγ-crystallin domains of a putative exported protein.J. Biol. Chem. 2005; 280: 1209-1216Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar39.Jobby M.K. Sharma Y. et al.Calcium-binding to lens βB2- and βA3-crystallins suggests that all β-crystallins are calcium-binding proteins.FEBS J. 2007; 274: 4135-4147Crossref PubMed Scopus (49) Google Scholar, 40.Shimeld S.M. Purkiss A.G. Dirks R.P. Bateman O.A. Slingsby C. Lubsen N.H. et al.Urochordate βγ-crystallin and the evolutionary origin of the vertebrate eye lens.Curr. Biol. 2005; 15: 1684-1689Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 41.Barnwal R.P. Jobby M.K. Devi K.M. Sharma Y. Chary K.V. et al.Solution structure and calcium-binding properties of M-crystallin, a primordial βγ-crystallin from archaea.J. Mol. Biol. 2009; 386: 675-689Crossref PubMed Scopus (49) Google Scholar, 42.Vergara A. Grassi M. Sica F. Pizzo E. D'Alessio G. Mazzarella L. Merlino A. et al.A novel interdomain interface in crystallins: structural characterization of the βγ-crystallin from Geodia cydonium at 0.99 Å resolution.Acta Crystallogr. D Biol. Crystallogr. 2013; 69: 960-967Crossref PubMed Scopus (7) Google Scholar43.Aravind P. Suman S.K. Mishra A. Sharma Y. Sankaranarayanan R. et al.Three dimensional domain swapping in nitrollin, a single-domain βγ-crystallin from Nitrosospira multiformis, controls protein conformation and stability but not dimerization.J. Mol. Biol. 2009; 385: 163-177Crossref PubMed Scopus (18) Google Scholar), leading to the recognition of a common motif for ion binding, and thus prompting the organization of these proteins as a separate set of Ca2+-binding proteins (23.Aravind P. Mishra A. Suman S.K. Jobby M.K. Sankaranarayanan R. Sharma Y. et al.βγ-Crystallin superfamily contains a universal motif for binding calcium.Biochemistry. 2009; 48: 12180-12190Crossref PubMed Scopus (55) Google Scholar, 44.Mishra A. Suman S.K. Srivastava S.S. Sankaranarayanan R. Sharma Y. et al.Decoding the molecular design principles underlying Ca2+-binding to βγ-crystallin motifs.J. Mol. Biol. 2012; 415: 75-91Crossref PubMed Scopus (20) Google Scholar). The domain topology of βγ-crystallins is based on a pair of Greek key motifs (19.Blundell T. Lindley P. Miller L. Moss D. Slingsby C. Tickle I. Turnell B. Wistow G. et al.The molecular structure and stability of the eye lens: x-ray analysis of γ-crystallin II.Nature. 1981; 289: 771-777Crossref PubMed Scopus (388) Google Scholar, 28.Wistow G. et al.Evolution of a protein superfamily: relationships between vertebrate lens crystallins and microorganism dormancy proteins.J. Mol. Evol. 1990; 30: 140-145Crossref PubMed Scopus (127) Google Scholar). The Greek key motif, a terminology based on the pattern and supersecondary features observed in proteins, is a basic theme of many all-β proteins (45.Richardson J.S. et al.Handedness of cross over connections in β sheets.Proc. Natl. Acad. Sci. U.S.A. 1976; 73: 2619-2623Crossref PubMed Scopus (166) Google Scholar, 46.Richardson J.S. et al.β-Sheet topology and the relatedness of proteins.Nature. 1977; 268: 495-500Crossref PubMed Scopus (465) Google Scholar). Greek keys are quite diverse in topology and hence were further classified as (4,0), (3,1), and (2,2) patterns, based on the arrangement and connectivity of strands (46.Richardson J.S. et al.β-Sheet topology and the relatedness of proteins.Nature. 1977; 268: 495-500Crossref PubMed Scopus (465) Google Scholar). According to the above classification, proteins of the βγ-crystallin superfamily fall into the (3,1) category (47.Hutchinson E.G. Thornton J.M. et al.The Greek key motif: extraction, classification and analysis.Protein Eng. 1993; 6: 233-245Crossref PubMed Scopus (78) Google Scholar), where one strand (third strand, c and c′ of the respective Greek key motif) out of the four is shared by a partner motif (Fig. 1). βγ-Crystallin-type Greek key motif has a distinct signature sequence of residues "(F/Y/W)XXXX(F/Y)XG" (28.Wistow G. et al.Evolution of a protein superfamily: relationships between vertebrate lens crystallins and microorganism dormancy proteins.J. Mol. Evol. 1990; 30: 140-145Crossref PubMed Scopus (127) Google Scholar) in the β-hairpin loop between the first and the second strands (19.Blundell T. Lindley P. Miller L. Moss D. Slingsby C. Tickle I. Turnell B. Wistow G. et al.The molecular structure and stability of the eye lens: x-ray analysis of γ-crystallin II.Nature. 1981; 289: 771-777Crossref PubMed Scopus (388) Google Scholar) (Fig. 1). Along with this signature sequence, ∼24–30 residues downstream, a conserved Ser is located (on the fourth β-strand), which plays a structural role by stabilizing this β-hairpin (19.Blundell T. Lindley P. Miller L. Moss D. Slingsby C. Tickle I. Turnell B. Wistow G. et al.The molecular structure and stability of the eye lens: x-ray analysis of γ-crystallin II.Nature. 1981; 289: 771-777Crossref PubMed Scopus (388) Google Scholar, 20.Wistow G. Turnell B. Summers L. Slingsby C. Moss D. Miller L. Lindley P. Blundell T. et al.X-ray analysis of the eye lens protein γ-II crystallin at 1.9 Å resolution.J. Mol. Biol. 1983; 170: 175-202Crossref PubMed Scopus (280) Google Scholar, 48.Slingsby C. Norledge B. Simpson A. Bateman O.A. Wright G. Driessen H.P.C. Lindley P.F. Moss D.S. Bax B. et al.X-ray diffraction and structure of crystallins.Prog. Retin. Eye Res. 1997; 16: 3-29Crossref Scopus (42) Google Scholar). The third strand of each Greek key is swapped and becomes a part of the partner Greek key motif ((3,1) arrangement of β-strands) (Fig. 1b). The fourth strand folds back to the previous sheet via a connecting loop, which is variable in length and occasionally has a small helical segment. This loop (named loop1 in the first Greek key and loop2 in the second Greek key motif) occupies the top of the βγ-crystallin domain (Fig. 1c). A βγ domain has two juxtaposed Ca2+ binding sites, which are mainly formed of the loops (loop1 and loop2) with an (N/D)(N/D)XX(T/S)S sequence stretch, running in opposite directions in three-dimensional space, along with the residues from β-hairpins (22.Clout N.J. Kretschmar M. Jaenicke R. Slingsby C. et al.Crystal structure of the calcium-loaded spherulin 3a dimer sheds light on the evolution of the eye lens βγ-crystallin domain fold.Structure. 2001; 9: 115-124Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 23.Aravind P. Mishra A. Suman S.K. Jobby M.K. Sankaranarayanan R. Sharma Y. et al.βγ-Crystallin superfamily contains a universal motif for binding calcium.Biochemistry. 2009; 48: 12180-12190Crossref PubMed Scopus (55) Google Scholar, 33.Wenk M. Baumgartner R. Holak T.A. Huber R. Jaenicke R. Mayr E.M. et al.The domains of protein S from Myxococcus xanthus: structure, stability and interactions.J. Mol. Biol. 1999; 286: 1533-1545Crossref PubMed Scopus (44) Google Scholar, 40.Shimeld S.M. Purkiss A.G. Dirks R.P. Bateman O.A. Slingsby C. Lubsen N.H. et al.Urochordate βγ-crystallin and the evolutionary origin of the vertebrate eye lens.Curr. Biol. 2005; 15: 1684-1689Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar) (Fig. 1, c and d). Thus, each Ca2+-binding site is formed by four residues located at three different regions in the primary sequence. The first Ca2+-binding site is formed via one residue from β-hairpin1, two residues from loop1, and one residue from loop2, and in a similar way, the second site is formed by one residue from β-hairpin2, two residues from loop2, and one residue from loop1 (22.Clout N.J. Kretschmar M. Jaenicke R. Slingsby C. et al.Crystal structure of the calcium-loaded spherulin 3a dimer sheds light on the evolution of the eye lens βγ-crystallin domain fold.Structure. 2001; 9: 115-124Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 23.Aravind P. Mishra A. Suman S.K. Jobby M.K. Sankaranarayanan R. Sharma Y. et al.βγ-Crystallin superfamily contains a universal motif for binding calcium.Biochemistry. 2009; 48: 12180-12190Crossref PubMed Scopus (55) Google Scholar) (Fig. 1d). This arrangement is common in the βγ domains studied structurally: Protein S (33.Wenk M. Baumgartner R. Holak T.A. Huber R. Jaenicke R. Mayr E.M. et al.The domains of protein S from Myxococcus xanthus: structure, stability and interactions.J. Mol. Biol. 1999; 286: 1533-1545Crossref PubMed Scopus (44) Google Scholar), Spherulin 3a (22.Clout N.J. Kretschmar M. Jaenicke R. Slingsby C. et al.Crystal structure of the calcium-loaded spherulin 3a dimer sheds light on the evolution of the eye lens βγ-crystallin domain fold.Structure. 2001; 9: 115-124Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), Ci-βγ-crystallin (40.Shimeld S.M. Purkiss A.G. Dirks R.P. Bateman O.A. Slingsby C. Lubsen N.H. et al.Urochordate βγ-crystallin and the evolutionary origin of the vertebrate eye lens.Curr. Biol. 2005; 15: 1684-1689Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), Clostrillin, Flavollin, M-crystallin (23.Aravind P. Mishra A. Suman S.K. Jobby M.K. Sankaranarayanan R. Sharma Y. et al.βγ-Crystallin superfamily contains a universal motif for binding calcium.Biochemistry. 2009; 48: 12180-12190Crossref PubMed Scopus (55) Google Scholar, 41.Barnwal R.P. Jobby M.K. Devi K.M. Sharma Y. Chary K.V. et al.Solution structure and calcium-binding properties of M-crystallin, a primordial βγ-crystallin from archaea.J. Mol. Biol. 2009; 386: 675-689Crossref PubMed Scopus (49) Google Scholar), and Geodin (42.Vergara A. Grassi M. Sica F. Pizzo E. D'Alessio G. Mazzarella L. Merlino A. et al.A novel interdomain interface in crystallins: structural characterization of the βγ-crystallin from Geodia cydonium at 0.99 Å resolution.Acta Crystallogr. D Biol. Crystallogr. 2013; 69: 960-967Crossref PubMed Scopus (7) Google Scholar). In βγ-crystallins, the Ca2+ coordination number varies from five to eight, four being provided by protein ligands, the rest being satisfied by water molecules (22.Clout N.J. Kretschmar M. Jaenicke R. Slingsby C. et al.Crystal structure of the calcium-loaded spherulin 3a dimer sheds light on the evolution of the eye lens βγ-crystallin domain fold.Structure. 2001; 9: 115-124Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 23.Aravind P. Mishra A. Suman S.K. Jobby M.K. Sankaranarayanan R. Sharma Y. et al.βγ-Crystallin superfamily contains a universal motif for binding calcium.Biochemistry. 2009; 48: 12180-12190Crossref PubMed Scopus (55) Google Scholar, 33.Wenk M. Baumgartner R. Holak T.A. Huber R. Jaenicke R. Mayr E.M. et al.The domains of protein S from Myxococcus xanthus: structure, stability and interactions.J. Mol. Biol. 1999; 286: 1533-1545Crossref PubMed Scopus (44) Google Scholar, 40.Shimeld S.M. Purkiss A.G. Dirks R.P. Bateman O.A. Slingsby C. Lubsen N.H. et al.Urochordate βγ-crystallin and the evolutionary origin of the vertebrate eye lens.Curr. Biol. 2005; 15: 1684-1689Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 42.Vergara A. Grassi M. Sica F. Pizzo E. D'Alessio G. Mazzarella L. Merlino A. et al.A novel interdomain interface in crystallins: structural characterization of the βγ-crystallin from Geodia cydonium at 0.99 Å resolution.Acta Crystallogr. D Biol. Crystallogr. 2013; 69: 960-967Crossref PubMed Scopus (7) Google Scholar). The +x position of the coordination sphere is provided by the main chain carbonyl of the first X residue of (F/Y)XXXX(Y/F)XG stretch, which is next to the first conserved aromatic residue of β-hairpin1 (Fig. 2) . The second coordination (+y position) is provided by the main chain carbonyl oxygen of the third residue and the third coordination (+z position) by the side chain oxygen of the fifth residue of the (N/D)(N/D)XX(T/S)S stretch of the same Greek key motif (22.Clout N.J. Kretschmar M. Jaenicke R. Slingsby C. et al.Crystal structure of the calcium-loaded spherulin 3a dimer sheds light on the evolution of the eye lens βγ-crystallin domain fold.Structure. 2001; 9: 115-124Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 23.Aravind P. Mishra A. Suman S.K. Jobby M.K. Sankaranarayanan R. Sharma Y. et al.βγ-Crystallin superfamily contains a universal motif for binding calcium.Biochemistry. 2009; 48: 12180-12190Crossref PubMed Scopus (55) Google Scholar, 40.Shimeld S.M. Purkiss A.G. Dirks R.P. Bateman O.A. Slingsby C. Lubsen N.H. et al.Urochordate βγ-crystallin and the evolutionary origin of the vertebrate eye lens.Curr. Biol. 2005; 15: 1684-1689Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar) (Fig. 2). The involvement of the Ser or Thr hydroxyl group to coordinate Ca2+ is distinctive to this motif as most Ca2+-binding sites do not involve these residues in Ca2+ ion coordination (49.McPhalen C.A. Strynadka N.C.J. James M.N.G. et al.Calcium-binding sites in proteins: a structural perspective.Adv. Protein Chem. 1991; 42: 77-144Crossref PubMed Scopus (258) Google Scholar). The fourth coordination (−x position) is provided by the side chain oxygen of the second residue (mostly Asp or sometime Asn) of the (N/D)(N/D)XX(T/S)S stretch from the partner Greek key motif. The −y and −z positions are satisfied by water molecules. Both sites of a domain exhibit similar or slightly altered coordination geometry. The coordination number is seven, with pentagonal bipyramidal geometry, but octahedral geometry (coordination number: 6) and square anti-prismatic (coordination number: 8) are also seen. A coordination number of five has also been observed in the first Ca2+ binding site of Protein S (33.Wenk M. Baumgartner R. Holak T.A. Huber R. Jaenicke R. Mayr E.M. et al.The domains of protein S from Myxococcus xanthus: structure, stability and interactions.J. Mol. Biol. 1999; 286: 1533-1545Crossref PubMed Scopus (44) Google Scholar). The average coordination radius at each of these sites varies from 2.4 to 2.8 Å. An analysis of >100 sequences of canonical motifs available demonstrated that the amino acid residues forming the (N/D)(N/D)XX(S/T)S fingerprint vary in various proteins of the superfamily (44.Mishra A. Suman S.K. Srivastava S.S. Sankaranarayanan R. Sharma Y. et al.Decoding the molecular design principles underlying Ca2+-binding to βγ-crystallin motifs.J. Mol. Biol. 2012; 415: 75-91Crossref PubMed Scopus (20) Google Scholar). The first residue of the fingerprint is involved in stabilizing the pocket through hydrogen bonding with the hydroxyl side chain of Ca2+ coordinating Ser/Thr and in some cases supports a water molecule present at the −y position. The second residue, also a polar amino acid (mostly Asp), directly coordinates Ca2+ by providing monodentate ligation at the −x position. These first two residues act in trans, i.e. the first two residues of loop1 of the first Greek key, become part of the second Ca2+-binding site and vice versa; thus, the motif gets its name as a "double clamp" motif (23.Aravind P. Mishra A. Suman S.K. Jobby M.K. Sankaranarayanan R. Sharma Y. et al.βγ-Crystallin superfamily contains a universal motif for binding calcium.Biochemistr