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Journal of Molecular Biologyj ourna l homepage: ht tp : / /ees .e lsev ie r.com. jmb

doi:10.1016/j.jmb.2011.10.037 J. Mol. Biol. (2012) 415, 75–91

Decoding the Molecular Design Principles UnderlyingCa 2+ Binding to βγ-Crystallin Motifs

Amita Mishra†, Shashi Kumar Suman†, Shanti Swaroop Srivastava‡,Rajan Sankaranarayanan⁎ and Yogendra Sharma⁎Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research, Uppal Road,Hyderabad 500007, India

Received 30 June 2011;received in revised form19 October 2011;accepted 20 October 2011Available online31 October 2011

Edited by J. E. Ladbury

Keywords:βγ-crystallin;calcium binding;calcium-binding motif;N/D-N/D-X-X-S/T-S motif

*Corresponding authors. E-mail addsankar@ccmb.res.in; yogendra@ccm† Joint first authors.‡ Solved the crystal structures.Abbreviations used: ITC, isotherm

calorimetry; wt, wild type; PDB, Pro

0022-2836/$ - see front matter © 2011 E

Numerous proteins belonging to the recently expanded βγ-crystallinsuperfamily bind Ca2+ at the double-clamp N/D-N/D-X1-X2-S/T-S motif.However, there have been no attempts to understand the intricaciesinvolving Ca2+ binding, such as the determinants of Ca2+-binding affinityand their contributions to gain in stability. This work is an in-depth analysisof understanding the modes and determinants of Ca2+ binding to βγ-crystallin motifs. We have performed extensive naturally occurringsubstitutions from related proteins on the βγ-crystallin domains of flavollin,a low-affinity Ca2+-binding protein, and clostrillin, a moderate-affinityprotein. We monitored the consequences of these modifications on Ca2+

binding by isothermal titration calorimetry, thermal stability and confor-mational and crystal structure analyses. We demonstrate that Ca2+ bindingto the two sites of a βγ-domain is interdependent and that the presence ofArg at the fifth position disables a site. A change from Thr to Ser, or viceversa, influences Ca2+-binding affinity, highlighting the basis of diversityfound in these domains. A subtle change in the first site has a greaterinfluence on Ca2+ binding than a similar alteration in the second site. Thus,the second site is more variable in nature. Replacing an acidic orhydrophobic residue in a binding site alters the Ca2+-binding propertiesdrastically. While it appears from their binding site sequence that thesedomains have evolved randomly, our examination illustrates the subtlety inthe design of these modules. Decoding such design schemes would aid inour understanding of the functional themes underlying differential Ca2+

binding and in predicting these in emerging sequence information.

© 2011 Elsevier Ltd. All rights reserved.

Introduction

The functions of Ca2+ in a cell are regulated viabinding to specific classes of proteins, called Ca2+-binding proteins (CaBP), which takes place at a

resses:b.res.in.

al titrationtein Data Bank.

lsevier Ltd. All rights reserve

specialized motif. A number of specialized motifsfor Ca2+ binding have been studied in great detail,such as the EF-hand motif, the C2 domain of proteinkinase and so on. These proteins show a wide rangeof affinity for Ca2+, from very high (10− 10 M) to verylow (millimolar range), and there is a large variationin the geometry of Ca2+-binding sites.1 Based onenvironmental constraints to perform specializedfunctions, Ca2+-binding proteins are evolved formore suitable affinity rather than possessing ahighest possible affinity and thus demonstrategreat diversity in function.1 A newmotif for bindingCa2+ with a sequence of N/D-N/D-X1-X2-S/T-S has

d.

76 Determinants of Ca2+-Binding to βγ-Crystallins

been identified recently in members of βγ-crystallinsuperfamily2–4 forming a separate class of Ca2+-binding proteins. Certain microbial sequences foldto form a domain that comprises two structurallysimilar β/γ-like motifs that together form two suchCa2+-binding motifs.There is a close similarity in domain topology of

these members, which is formed by two Greek keymotifs, each motif composed of four antiparallel β-strands.5–7 The two βγ-crystallin-type Greek keymotifs pair to form a pseudosymmetric domain thatharbors two similar binding sites for Ca2+.2–4 Unlikethe EF-hand motifs where the components of a siteare a part of a linear sequence, a βγ-motif is uniquein the sense that it is formed by an interlock of aminoacids located at three discrete positions in the linearsequence.2–4 Structural studies have shown thatCa2+-binding sites are conserved not only in

Fig. 1. (a) Organization of the canonical double-clampmotif othe first and second binding sites, respectively. The broken avia side-chain (SC) oxygen and main-chain (MC) carbonystructural overlap of Ca2+-binding sites of clostrillin (green;Structure-based sequence alignment of clostrillin and flavollinpresent in both domains and the residues involved in Ca2+ coas blue and red, respectively.

prokaryotes4,8,9 , but also in a mycetazoan2 and aurochordate3 and are lost in eye lens βγ-crystallins.These structural data enable a Ca2+-binding se-quence fingerprint to be mapped onto pairs of βγ-crystallin motif sequences predicted to fold into aβγ-domain.2–4 Based on several crystal structures ofthis domain in the Ca2+-bound form, the pattern ofCa2+ coordination is depicted in Fig. 1a.2,4 The Ca2+

coordination number varies from 6 to 8 in the βγ-crystallins. At each site, four coordinations (+x, −x,+y and +z) are provided by protein atoms, whilethe remaining coordinations are satisfied by watermolecule. Of the four coordinations provided by theprotein atoms, two are main-chain carbonyls (+xand +y) and the other two are mediated by sidechains (−x and +z). Interestingly, the side chain atthe −x position lies in the second Greek key motif,thus making this as a double-clamp motif (Fig. 1).

fβγ-crystallin superfamily. Residues 1–4 and 5–8 representnd continuous lines represent residues coordinating Ca2+

l oxygen, respectively. (b) Cartoon depiction showingPDB ID: 3I9H) and flavollin (cyan; PDB ID: 3HZB). (c)showing the N/D-N/D-X1-X2-S/T-S Ca2+-binding motifsordination via backbone (BB) and side chain (SC) marked

Table 1. Frequency of amino acids occurring at differentpositions in the N/D-N/D-X1-X2-T/S-S motif of βγ-crystallin superfamily

N/D N/D X1 X2 S/T S

N-93 D-96 R-26 I-57 S-106 Most casesD-14 N-20 K-25 V-21 T-11S-3 H-2 D-13 A-12 R-2W-3 R-2 Q-8 M-10 I-2E-2 Q-1 T-8 L-8 Y-2F-2 G-1 S-8 T-8 V-1A-2 W-1 V-6 F-2 N-1Q-2 E-1 W-5 N-2 H-1G-2 Y-1 L-5 G-1 Q-1T-1 L-1 A-5 W-1L-1 K-1 N-4 P-1K-1 C-3 D-1

M-3 H-1Y-1 K-1

Y-1

A total of 127 canonical motifs from 63 different proteins from thedatabase were analyzed.

77Determinants of Ca2+-Binding to βγ-Crystallins

Members of this expanding structural superfamilycontaining diverse members from various organismshave structural features similar to those of lens β- andγ-crystallins10–12 and exhibit moderate to low affinitytoward Ca2+. Apart from lens βγ-crystallins, someearlier studied members are protein S from Myxococ-cus xanthus, 8,9,13 spherulin 3a from Physarumpolycephalum,2,14–16 Yersinia crystallin from Yersiniapestis,17 geodin from the sponge Geodia cydonium,18

Ci-βγ-crystallin from the urochordate Cionaintestinalis,3 absent in melanoma (AIM1),19–21 nitrollinfrom Nitrosospira multiformis,22 M-crystallin from anarchaea Methanosarcina acetivorans23 and so on. Themacroscopic binding constants of Ca2+ binding toprotein S are 27 and 76 μM and those to spherulin 3aare 9 and 200 μM,16,24 whereas Ca2+-binding affinitiesto Yersinia crystallin and caulollins are in the range of5–25 μM.17,25 In the case of diverged or noncanonicalmotifs, Ca2+ 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-superfamilyhave been shown to bind Ca2+ using the N/D-N/D-X1-X2-S/T-S motif, a comprehensive understanding ofits intricacies such as controlling factors and determi-nants of the wide-ranging response toward Ca2+ islacking. We have noted significant variations in thesequence of this six-amino-acid canonical Ca2+-bind-ingmotif of these proteins, but a careful analysis showsthat the residue at the−xposition (SC) is usuallyAsporAsn in some exceptions. Similarly, the +z position (SC)has Ser or, in a few cases, Thr (Table 1). Thus, the fourcoordinations mediated by the protein moleculecorrespond to two main-chain carbonyl oxygens andtwo highly conserved side-chain residues. In such ascenario, it would be interesting to know how affinityand Ca2+-induced responses vary among familymembers. Taking the clues from the significantsimilarities and variations present at different posi-tions, we have undertaken this study to ascertain thedesign themes of thismotif, such as the preference for acertain aminoacid residue at individual locations in theN/D-N/D-X1-X2-S/T-S motif and its relation to weakor tight binding or to Ca2+-dependent alteration instability or conformation.In our comprehensive efforts in this direction, we

have selected two homologous proteins sharing 38%sequence identity, namely, clostrillin (a βγ-crystallindomain-containing protein from Clostridium beijer-inckii) and flavollin (a βγ-crystallin domain-contain-ing protein from Flavobacterium johnsoniae), forunderstanding the complexities of the design ofCa2+ binding and Ca2+-induced gains in domainstability. We created a number of mutations in thecoordinating positions by replacing the amino acidsin the N/D-N/D-X1-X2-S/T-S sequence. The aminoacids selected as substituents are naturally presentin the sequence of other proteins, thus simulatingnaturally occurring environments. We describe thepossible consequences of Ser or Thr at the +z ligation

position or the polarity or hydrophobicity at the +yposition (X1 position in the N/D-N/D-X1-X2-S/T-Smotif) on Ca2+-binding affinity, binding site geom-etry and gain of thermal stability. Using structuralanalysis, we demonstrate the similarities and dis-similarities in the pattern of ion coordination indifferent motifs. This work addresses the complexityof the motif by demonstrating that, unlike the EF-hand motif, the effects of a replacement are notstraightforward and that even subtle modificationswould have profound effects on Ca2+-bindingproperties, thus contributing to the high diversityseen within this class of Ca2+-binding protein.

Results

Geometry of Ca2+ binding to the N/D-N/D-X1-X2-S/T-S motif of βγ-crystallins

ACa2+-binding site in a βγ-crystallin is formed bythe three components of a domain, viz., hairpin loop1/2, loop 1 and loop 2. These three components of aβγ-crystallin-type Ca2+-binding site are not linearlyarranged but are present at mutually discreteregions in the sequence (Fig. 1a). In βγ-crystallins,generally two Ca2+-binding sites are present, locat-ed at a distance of ∼12 Å relative to each other. Atboth sites, the four coordinations are provided byprotein atoms, that is, +x (main chain), +y (mainchain), +z (side chain) and −x (side chain), whereasthe rest of the coordinations are fulfilled by watermolecules (positions −y and −z). For example, at site1, the hairpin 1 provides the coordination at the +xposition (via main-chain carbonyl oxygen), loop 1provides +y (backbone) and +z coordinations (via

78 Determinants of Ca2+-Binding to βγ-Crystallins

side chain) and the −x coordination (via side chain)is provided by loop 2 (which is the part of the secondGreek key). Similarly, at the second site, hairpin 2provides +x coordination, loop 2 provides +y and+z coordinations and loop 1 from the first Greek keyprovides −x coordination (Fig. 1). In clostrillin, bothsites have a coordination number of 7, with apentagonal bipyramidal geometry where +x, −x, +yand two −y coordinations take the position at thevertices of a pentagonal plane and −z and +zpositions pair up perpendicular to the x/y pentag-onal plane. While a similar ligation pattern is seen inthe first site of flavollin, the second site has acoordination number of 8 with a square anti-prismgeometry.The canonical sequence of the Ca2+-binding motif

of βγ-crystallins is a stretch of six residues inrandom combinations of amino acids in additionto a residue from the first β-strand (hairpin loop)located next to the conserved aromatic amino acid(Fig. 1a and b). The first two residues of N/D-N/D-X1-X2-S/T-S are exchanged with each other and playa role in constituting the two interdependent Ca2+-binding sites. The first residue forms a hydrogenbond with the fifth residue of the other N/D-N/D-X1-X2-S/T-S motif (i.e., with Ser/Thr) and plays animportant role in stabilizing the pocket. While thesecond amino acid is generally a polar residuecoordinating with Ca2+ at the −x position via its sidechain, the third residue forms coordination usingmain-chain carbonyl at the +y position. The fourthresidue is a nonpolar amino acid forming ahydrophobic core of the protein together with theresidue at the same position in another motifconnecting the sites and might be one of the factorsfor determining the extent of interdependence of

Table 2. Sequence of the Ca2+-binding motifs and their mutat

Residues marked in red are the substituted one with respect to their w

both sites. The fifth amino acid is either Ser or Thr,though most proteins have Ser (e.g., flavollin,spherulin 3a, etc.) and coordinates via side chain(–OH) at the +z position (Fig. 1b).

The effect of mutations in the Ca2+-binding motif

To understand the features and determinants ofCa2+ binding to the N/D-N/D-X1-X2-S/T-S motif ofβγ-crystallins, we selected two structurally similar,single-domain proteins, clostrillin and flavollin withminor differences in the Ca2+-binding motif se-quence (NDWMTS and NDKMTS of clostrillin;NDDISS and NDKVSS of flavollin) (Fig. 1c; Table2 and Fig. S1). To examine if there is any interactionbetween the sites, we disabled a site by mutatingThr41 or Thr82 to Arg serially and concurrently(T41R, T82R) in clostrillin. Similarly, to understandthe preference of Ser or Thr, we mutated Thr41 (inthe first motif) or Thr82 (in the second motif) to Serin clostrillin and vice versa in flavollin (Table 2). Inaddition to this, we mutated Trp39 to Asp (at thethird position) in the NDWMTS sequence (first site)of clostrillin and Asp38 (third residue) to Val in thefirst Ca2+-binding motif (from NDDISS to NDVISS)of flavollin (Table 2). The apo conformation andchanges upon Ca2+ binding due to a mutation werelargely similar to that of wild-type (wt) proteins.

Interdependency of sites: Disabling of aCa2+-binding site by Ser/Thr to Arg

To understand if there is any interaction betweenbinding sites, we disabled the sites by mutatingThr41 (at the fifth position of N/D-N/D-X1-X2-S/T-S)to Arg (first site disabled, while the second remained

ions

t protein.

79Determinants of Ca2+-Binding to βγ-Crystallins

intact) and T82R (second site disabled, while first siteis intact), and a double mutant with both sitesdisabled. Comparative affinities (macroscopic bind-ing constants) of the two sites show that site 1 is astronger binder (Kd, first site of clostrillin T82R, is47 μM) than site 2 (Kd of T41R is 270 μM) (Fig. 2a andTable 3). Using these data, we infer that K1 (33 μM)and K2 (0.5 μM) of wt clostrillin belong to site 2 andsite 1, respectively4 (Table 3). The decrease inbinding affinity upon disabling one site suggeststhat the sites are not independent, and thus, there isan interaction between them. As expected, thedouble mutant of clostrillin (T41R, T82R mutant)does not bind Ca2+.

Disabling a site increases innate thermalstability

We next examined if disabling a binding site couldlead to an alteration in the innate and Ca2+-

Time (min) Tim(a)

(b)

µcal

/sec

µcal

/sec

0

3

-2

-1

Molar Ratio

kcal

mol

-1 o

f inj

ecta

nt

kcal

mol

-1 o

f inj

ecta

nt

-4

-3Clostrillin T41R Clo

Clostrillin wt No Ca2+

CaCl2

Clostrillin wt No Ca2+

CaCl2

No Ca2+

CaCl2

Clostrillin T41R No Ca2+

Clostrillin T82R

Fra

ctio

n U

nfo

lded

(i)

Temp (°C)

0 30 60 90 120 0 30 60150 180

-1.0

-0.5

0.0

-2.0

-1.5

-0.8

-2.4

-1.6

-3.20 1 2 3 4

Mola0 1 2

-0.4

0.0

-0.8

0.6

0.8

1.0

1.2

0.0

0.2

0.4

-0.230 40 50 60 70 80

Fra

ctio

n U

nfo

lded

Tem

0.6

0.8

1.0

1.2

0.0

0.2

0.4

-0.230 40 50

CaCl2

Fig. 2. (a) Isothermal titration calorigram of Ca2+ binding tomutants. The concentrations of protein and Ca2+ used in ITCprotein in the sample cell was titrated with Ca2+, which was lstock of 5 mMCaCl2. (b) Thermal stability of (i) clostrillin T41Rand its comparison with clostrillin wt. The Tm of mutants (Thrits wt. Protein concentrations used were 0.1 mg/ml.

dependent gain in stability. In the apo forms of allthree Thr-to-Arg mutants (site 1 disabled, site 2disabled or both sites disabled), the stability in-creased by about 8–15 °C, with maximum augmen-tation seen in the clostrillin T41R, T82R mutant (Tm,59.2 °C) (Tm of wt protein is 44.4 °C) (Fig. 2b andTable 4). wt clostrillin gained high thermal stabilityupon Ca2+ binding, with an increase in Tm from44 °C (apo form) to 64 °C for the holo form. This gainof stability by Ca2+ was decreased in all threemutants (Tm, between 57 and 60 °C) (Table 4),suggesting that disabling a site increases the thermalstability (of apoproteins), while gain in stability uponCa2+ binding decreased significantly.

Structural analysis

To examine the structural geometry of disabledbinding sites, we attempted to crystallize thesemutants. However, only clostrillin T82R could be

e (min)

0.0

-0.2

-0.1

kcal

mol

-1 o

f inj

ecta

nt

-0.3strillin T82R Clostrillin T41R,T82R

Clostrillin T41,82R

Clostrillin wt No Ca2+

CaCl2

No Ca2+

CaCl2

(ii) (iii)

90 120Time (min)

0 30 60 90 120

r Ratio3 4 5 6

Molar Ratio0 2 4 6 8

µcal

/sec

-0.4

0.0

0.4

-0.8

p (°C)60 70 80

Fra

ctio

n U

nfo

lded

Temp (°C)

0.6

0.8

1.0

1.2

0.0

0.2

0.4

-0.230 40 50 60 70 80

clostrillin T41R, clostrillin T82R and clostrillin T41R, T82Rexperiments were 100 μM and 5 mM, respectively. The

oaded into a syringe with 85 injections of 3 μl each from a, (ii) clostrillin T82R and (iii) clostrillin T41R, T82Rmutantsto Arg) increased significantly (∼10–15 °C) as compared to

Table 3. Dissociation constants and thermodynamic parameters of Ca2+ binding to clostrillin and flavollin (wt and theirmutants) calculated from ITC

Proteins Model KA (M−1)aMacroscopic dissociation

constant Kd (μM)a ΔH (kcal/mol) ΔS (cal/mol)

Clostrillin wtb Kd=4 μMClostrillin T41R One set of site 3.7×103±0.2×102 270 μM 270 −36.5±2.1 −105Clostrillin T82R One set of site 2.1×104±2.1×103 47 μM 47 −17.6±0.7 −38.5Clostrillin T41S Two set of sites 1.1×104±7.2×102 90 μM 22.5±6c −8.4±0.1 −3.7

1.8×105±2.3×104 5.6 μM −7.3±2.5 −5.7Clostrillin T82S Two set of sites 1.2×105±5.2×104 8.3 μM 10±3c −13.7±1.5 −4.5

8.0×104±6.20×103 12.5 μM −5.8±0.6 3.3Clostrillin T41S,

T82STwo set of sites 1.2×104±0.7×103 83 μM 12±4c −12.2±0.7 −19.3

5.6×105±2.3×104 1.7 μM −12.6±0.9 −15.4Clostrillin W39D Sequential two sites 8.6×104±3.7×103 11.6 μM 31 −8.6±0.1 −6.0

1.2×104±3.8×102 83 μM −13.6±0.2 −26.3Flavollin wtb Kd=33 μMFlavollin S40T Two set of sites 3.8×103±0.8×101 263 μM 637 5.5±1.0 34.8

6.46×102±0.7×102 1.5 mM −1.2±0.5 27.5Flavollin S81T Two set of sites

(sequential)5.7×103±5.9×102 175 μM 134 −2.1±0.1 10.29.6×103±6.0×102 104 μM −7.9±0.2 −7.83

Kd represents the average dissociation constant and was calculated as Kd=1/√K1K2). All fittings were performed using two independentsite models except for flavollin S81T.

a KA represents the macroscopic association constants.b Ca2+-binding-affinity data of clostrillin wt and flavollin wt are adopted from Ref. 4.c Error was deduced from three independent experiments.

80 Determinants of Ca2+-Binding to βγ-Crystallins

crystallized under the conditions tested. In thismutant, the second site (T82R) shows no Ca2+

(disabled for Ca2+ binding), although the overallstructure is not affected (Fig. 3). The +z ligationposition is replaced by the Cγ atom of Arg82, andthe −yw1 position is taken up by NE of Arg. Theside chain of Lys80 is at the position correspond-

Table 4. Melting temperature (Tm) of clostrillin, flavollinand their mutants in apo and holo forms, calculated by CD

ProteinsTm (°C),apo form

Tm (°C),holo form

ΔTm (°C)(holo–apo)

ΔTm (°C)(mutant–wt)

Clostrillin wt 44.4±0.3 64.7±0.2 20.3 —Clostrillin

T41R54.9±0.1 57.5±0.2 2.6 10.5

ClostrillinT82R

52.7±0.1 60.8±0.3 8.1 8.3

ClostrillinT41R, T82R

59.2±0.1 58.6±0.1 −0.6 15.2

ClostrillinW39D

32.1±0.5 52.1±0.1 20.0 −12.3

Clostrillin T41S 47.8±0.1 69.4±0.1 21.6 3.4Clostrillin T82S 48.2±0.1 66.9±0.1 18.7 3.8Clostrillin

T41S, T82S50.2±0.2 72.1±0.2 21.9 5.8

Flavollin wt 47.5±0.5 48.3±0.4 0.8 —Flavollin D38V 55.3±0.1 58.0±0.06 2.7 7.8Flavollin S40T 43.2±0.2 48.2±0.1 5.0 −4.3Flavollin S81T 49.7±0.1 60.1±0.05 10.4 2.2Flavollin S40T,

S81T39.8±0.4 44.3±0.1 4.5 −7.7

The gain in stability is demonstrated as ΔTm(Tm,holo−Tm,apo), andthe change in innate stability due to mutation (only apo form) isdescribed as ΔTm(Tm,mutant−Tm,wt).

ing to the −z water, whereas −yw1 water ispresent. A water molecule is seen in a positionnear the Ca2+ location. This distance of ligandpresent at +x, +y and −x positions from this watermolecule is similar to that of Ca2+ to liganddistance in the wt protein. As seen in the crystalstructure, Asp38 (second residue in the N/D-N/D-X1-X2-S/T-S motif), which directly ligates Ca2+ viaside chain in the wt protein, forms hydrogenbonds and ionic interaction with Arg82 in themutant and helps loop 1 to stabilize even in theabsence of Ca2+ (Fig. 3). Thus, positions −yw1, +zand −z have been occupied by Arg82 and Lys80without affecting other positions of Ca2+ coordi-nations and thus disabling the site for Ca2+

binding without changing the overall structure.

Conversion of Thr to Ser

To understand the intricacies of the motif byanalyzing the positional preference of an amino acidpresent naturally in a domain, such as Thr or Ser (atthe fifth S/T position in N/D-N/D-X1-X2-S/T-S),we analyzed the effect of such changes on Ca2+

binding and on the geometry, by replacing Thr bySer (in clostrillin) or vice versa (in flavollin).

Conversion of Thr to Ser (fifth position) and reducedaffinity for Ca2+ binding

We compared the Ca2+-binding isotherms ofclostrillin T41S mutant (the first site) and clostrillinT82S (the second site) with the wt, which has Thr

(+ x)

(–y)w1

Glu 9(+ x)(–y)

w1

Glu 55

(+ y)(+ z)

w2 w3

Trp 39Thr 41

(+ y)(–y) (+ z)

Asp 79(–x)

Asp 38

Lys 80

α1α1

β1

β2

β3

β4 β8

β6

β5

CN

(a) (b)

(c) (d)

Arg 82

(–z) (–y) (–z)

(–x)

Fig. 3. Crystal structure of clostrillin T82R mutant: (a) second and (b) first Ca2+-binding sites of clostrillin T82R. (c)Structural overlaps of clostrillin T82R (purple) with wt protein (light gray). (d) Structural overlap of site 2 of clostrillin T82Rwith wt protein. Pink spheres correspond to water, and purple spheres correspond to Ca2+ in wt protein, whereas red spherescorrespond towater in T82Rmutant. The overall structure is similar to that of wt with an rmsd range of 0.16–0.30 Å over 87 Cα

atoms. The crystals were obtained in the space group I422 with cell dimensions similar to that of wt clostrillin form 2 crystals.Asp38 (second residue in the N/D-N/D-X1-X2-S/T-S motif) ligates directly with Ca2+ via side chain and forms hydrogenbonds and ionic interaction with Arg82. These interactions help loop 1 to get stabilized even in the absence of Ca2+.

81Determinants of Ca2+-Binding to βγ-Crystallins

(Fig. 4). Macroscopic binding constants for wtclostrillin are 33 and 0.5 μM (average Kd, 4 μM),4

which changed to 90 and 5.6 μM, respectively, inT41S mutant (affinity of one site decreased by 12times, from 0.5 to 6 μM, which likely is the firstsite). The macroscopic binding constants for T82Smutant are 8.3 and 12.5 μM (average affinity isslightly lower to that of wt), whereas dissociationconstants for Ca2+ binding to the double mutantwere almost similar (kD1, 83 μM; kD2, 1.7 μM) tothat of one site of wt and one site of T41S mutant(Table 3). Mutating Thr to Ser compromises theCa2+-binding affinity by 12-fold at the first site,while there is no significant effect on mutating thesame in the second site (Table 3).Conformationally, all of the three clostrillinmutants

exhibit quenched fluorescence intensity upon Ca2+

binding and emission maximum (λmax) at 340 nmsimilar to that seen in wt protein (Fig. S2). Minor but

similar conformational changes in far- and near-UVCDspectroscopy ofwt clostrillin and itsmutantswereseen upon binding Ca2+ (Fig. S3a and b).

Thr-to-Ser conversion affects domain stability

We next studied if Thr-to-Ser mutation causes anyalteration in the Ca2+-induced changes in stability(Fig. 4b). In the apo forms, the stability of all mutantsincreases by about 4–6 °C with maximum change inthe case of clostrillin T41S, T82S mutant (Tm, 50 °C)than wt (Tm, 44 °C) (Table 4). wt clostrillin gainshigh thermal stability upon Ca2+ binding, with anincrease in Tm from 44 °C (for apo form) to 64 °C forthe holo form. Almost similar thermal transitionswere followed by all the three mutants of clostrillin(Tm of apo from 44 to 50 °C and that for holo from 64to 70 °C), with greater gain seen in the doublemutant (Tm, 72 °C) (Table 4), suggesting that certain

000.0

Time (min)(a)

(b)

-4

-2

μca

l/sec

c

-2

-1

μca

l/sec

-1.5

-1.0

-0.5

μca

l/sec

4

-2

0

-2.0

-8

-6

-4

Clostrillin T41S Clostrillin T82S Clostrillin T41S,T82S

Clostrillin wt No Ca2+

0 3 6 9Molar Ratio

0 3 6 9Molar Ratio

0 3 6 9Molar Ratio

kcal

mol

-1 o

f inj

ecta

nt

kcal

mol

-1 o

f inj

ecta

nt

kcal

mol

-1 o

f inj

ecta

nt

Clostrillin T82SCaCl2

Clostrillin wt No Ca2+

CaCl2

Clostrillin wt No Ca2+

CaCl2

No Ca2+

CaCl2 No Ca2+

CaCl2 No Ca2+

CaCl2

Clostrillin T41S Clostrillin T41S,T82S

Fra

ctio

n U

nfo

lded

Temp (°C)

(i) (ii) (iii)

0 30 60 90 120 150 180Time (min)

0 30 60 90 120 150 180Time (min)

0 30 60 90 120 150 180

-4

0

-16

-12

-8

-3

0-3

-12

-9

-6

1.0

1.2

0.2

0.4

0.6

0.8

-0.2

0.0 Fra

ctio

n U

nfo

lded 1.0

1.2

0.2

0.4

0.6

0.8

-0.2

0.0 Fra

ctio

n U

nfo

lded 1.0

1.2

0.2

0.4

0.6

0.8

-0.2

0.0

30 40 50 60 70 80Temp (°C)

30 40 50 60 70 80Temp (°C)

30 40 50 60 70 80

Fig. 4. (a) Isothermal titration calorigram of Ca2+ binding to clostrillin T41S, clostrillin T82S and clostrillin T41S, T82S.The affinity can be best described in the order clostrillin T41Sbclostrillin T41S, T82Sbclostrillin T82S≤clostrillin wt. Ca2+

binding to all three mutants was enthalpically favorable as indicated by the negative sign of ΔH. The concentrations ofprotein and Ca2+ used in ITC experiments were 100 μM and 2 mM, respectively. The protein in the sample cell wastitrated with Ca2+, which was loaded into a syringe with 85 injections of 3 μl each from a stock of 2 mMCaCl2. The best-fitvalues of thermodynamic parameters are listed in Table 3. (b) Thermal stability of (i) clostrillin T41S, (ii) clostrillin T82Sand (iii) clostrillin T41S, T82S compared with that of wt clostrillin. Mutant proteins show significant increase in thermalstability (Tm increased by 18–20 °C) in the holo protein. Protein concentrations used were 0.1–0.2 mg/ml, and spectrawere recorded in the absence or presence of 5 mM CaCl2.

82 Determinants of Ca2+-Binding to βγ-Crystallins

Thr-to-Ser mutations increase the thermal stabilityof apo and holo clostrillins significantly (Fig. 4b).

Structural analysis

To assess the structural changes as a consequenceof mutations, we obtained crystals of clostrillin T41Sand clostrillin T41S, T82S mutants (Fig. 5a). Therewere eight monomers in the asymmetric unit of thecrystal of T41S mutant similar to that of wt protein[Protein Data Bank (PDB) ID: 3IAJ] (Fig. 5b), but thecell dimensions and packing of the moleculeschanged. Out of eight monomers present in theasymmetric unit of the crystal, six monomers showsimilarity with the wt protein in terms of Ca2+-to-ligand distance at both Ca2+-binding sites. Themajordifference was in terms of the number of watermolecules (varied from zero to three) coordinating

with Ca2+. Though the second site has not beenmutated in T41S, interestingly, none of the proto-mers shows the presence of all the three Ca2+-coordinating waters in the site without any muta-tion. The average B-factor of Ca2+ (36 Å2) wassignificantly higher in comparison to that of coordi-nating residues, indicating a weak binding. In onlyone monomer (chain A) at site 1, the B-factor of Ca2+

was 17.91Å2 (similar to that of residues participatingin ligation). In the second site of chain F, the B-factorof Ca2+ (55.57 Å2) was significantly high ascompared to that of its interacting residues. Inchain G, a water molecule was present at the placeof Ca2+ at the second site. We could not see thedensity for the residues 30–36 and 73–79 inmonomerH, which is pseudotranslationally related to mono-mer G. These stretches are the part of loop 1 and loop2 that are involved in the Ca2+-binding site

(a)

(b)

(c)

Glu 55(i) (ii) (iii)

(+ x)(–y)(–z)

(+ z)

w1w3

Glu 9 (+ x)

(+ y)

(–z)(+ z)

w1(–y)

w3Thr 82

ββ1

β2

β3

β4β8

β6

β5

α1α1

(–x)(–y)w2 Trp 39

Asp 79T41S (site 1)

(–x)

Asp 38 Lys 80

T41S (site 2)T41SC

N

1

β2

β3β4

β8

β6

β5

α1α1

(+ x)(–y)

(–y)

(–z)w2

w1

w3

Glu 9

(+ x)(–y)(–z)

w1w3

Glu 55

(i) (ii) (iii)

CN

(–x)(+ y)(+ z)

Trp 39 Asp 79

Thr 41

wt (site 1)

(–x)

(+ y)(–y)

(+ z)w2

Thr 82

Asp 38

Lys 80

(–y)w1 Glu 9

(+ x)(–y)

–z

w1

w3

Glu 55

2β8

β5

α1α1

Overlap of T41S and T41S,T82S over wt

(i) (ii) (iii)

(+ x)

(–x)

(+ y)(–y)

(–z)

(+ z)

w2w3

Trp 39Ser 41 (–x)

(+ y)

( )

(+ z)Ser 82

Lys 80

β1 β3 β6

Asp 79T41S, T82S (site 1) T41S, T82S (site 2)CN T41S,T82S

(+ y)Ser 41

β

wt (site 2)

Asp 38

β4β4β

Fig. 5. Crystal structures of clostrillin T41S and clostrillin T41S, T82S double mutant compared to that of wt protein. (a)Crystal structure of T41S. The crystals were formed in P212121 space group, and the structure was solved at a resolution of1.93 Å. The structure was similar to wt with an rmsd range of 0.22–0.62 Å for 85–87 Cα atoms: (i) first Ca2+-binding site,(ii) cartoon representation of the crystal structure and (iii) second Ca2+-binding site. (b) Clostrillin wt structure is shownfor the sake of comparison with mutants: (i) first Ca2+-binding site; (ii) structural overlap of wt protein (light gray) withT41S (pink) and T41S, T82S (green) mutants and (iii) second Ca2+-binding site of wt protein. (c) Crystal structure ofclostrillin T41S, T82S mutant: (i) first Ca2+-binding site, (ii) cartoon representation of the crystal structure and (iii) secondCa2+-binding site. The first site in all the monomers of clostrillin T41S, T82S is similar to that of wt. Though the secondCa2+-binding site appears to be similar to wt, a water molecule in place of Ca2+ is seen. Residue numbers in this figure aremarked as per amino acid sequence. In all figures, purple spheres represent Ca2+, and light-pink spheres represent watermolecules.

83Determinants of Ca2+-Binding to βγ-Crystallins

formation. The electron density was observed forsimilar regions in chain G, but the B-factors of theresidues were higher as compared to those of the restof the residues, indicating that this region is flexibleand further stabilized upon Ca2+ binding. The chainH gives some glimpses where the coordinatingresidues corresponding to +x, +y and +z positionsare present but no density for the −x residue,indicating that this site is partly formed.The crystal structure of the double mutant

clostrillin T41S, T82S solved at 1.85 Å resolutionshows high similarity with the wt protein with an

rmsd range of 0.14–0.31 Å over 87 Cα atoms.There were eight monomers per asymmetric unitsimilar to that of wt. The first site in all themonomers was similar to that of wt, and we couldobserve the three water molecules around the Ca2+

(Fig. 5c). The second Ca2+-binding site appears tobe similar to the wt except that we notice a watermolecule sitting in place of Ca2+, which could bedue to the reduced affinity of the site for Ca2+. Thedistance of this water molecule, from the ligatingresidue, is more when compared to that of Ca2+ inwt protein.

84 Determinants of Ca2+-Binding to βγ-Crystallins

In summary, Thr-to-Ser replacement affects theCa2+-binding affinity in a site-dependent manner inclostrillin.

Conversion of Ser to Thr in flavollin

Alteration in affinity depends on the environment ofbinding site

We compared the affinity of wt flavollin forCa2+ with that of the S40T and S81T mutants. Themacroscopic dissociation constants for wt flavollinare 9 and 111 μM (average Kd, 33 μM),4 whichdecreases drastically to 263 μM with a positive ΔHfor the first site and further low affinity to anothersite in the S40T mutant (Fig. 6a and Table 3). Thepositive ΔH suggests that the binding is enthalpi-cally unfavorable and entropically favorable and,thus, might not overcompensate the interactionwith the solvent. In S81T mutation, the affinity isdecreased but to a less extent than S40T mutant

0.0

0 40 80 120 160

Time (min)(a)

-0.4

-0.2

µcal

/sec

0.0

-0.6

-0.6

-0.4

Flavollin S40T

0.8

1.0 Flavollin wt No Ca2+

CaCl2

Flavollin wt No Ca2+

CaCl2

0 2 4 6 8 10 12Molar Ratio

kcal

/mol

e of

inje

ctan

t

(b)

0.4

0.6Flavollin S40T

No Ca2+

CaCl2 No Ca2+

CaCl2

Flavollin S81T

30 40 50 60 70 80

0.0

0.2

Fra

ctio

n U

nfo

lded

Temp (°°C)

0.8

1.0

0.4

0.6

30 40 50

0.0

0.2

Fra

ctio

n U

nfo

lded

Tem

(i)

-0.2

Fig. 6. (a) ITC calorigram of Ca2+ binding to flavollin S40Tbinding more than does in the second site. Thr at both sites togein 50 mM Tris–Cl (pH 7.5) and 100 mM KCl was loaded in sasame buffer. The best-fit thermodynamic parameters are listeflavollin S40T, (ii) flavollin S81T and (iii) flavollin S40T, S81T aexcept flavollin S81T mutant, almost insignificant gain in TmS81T mutant increased significantly (≈10 °C) in the presence

(kA1=104 μM and kA2=175 μM; average Kd,134 μM), but the binding is enthalpically drivenas reported in wt protein. Clearly seen from Fig. 6a(the extent of enthalpy change), the affinity is inthe sequence flavollin wtN flavollin T81S≫ flavol-lin S40T. The alteration in affinity upon Ser-to-Thrmutation is not residue dependent but is depen-dent on several factors including the possibleinteractions of methyl group of Thr in the bindingpocket. This is true in flavollin since –CH3 groupmight interfere sterically with the Cys at the firstsite. We further analyzed if Ser-to-Thr mutationsaffect the Ca2+-induced conformational changes inflavollin.Flavollin undergoes significant increase in fluo-

rescence intensity upon binding Ca2+ (up to 300%change), the intensity increased to 10-fold in all themutants, but exhibited quenched intensity in com-parison with wt (Fig. S4). This suggests that themicroenvironment of Trp was altered, likely due tothe change in hydrophobicity caused by the methyl

0 40 80 120 160

Time (min)

0

-2

-1

µcal

/sec

-1

0

-3

-3

-2

Flavollin S81T

No Ca2+

CaCl2

No Ca2+

CaCl2

Flavollin wt

0 2 4 6 8 10 12Molar Ratio

kcal

/mol

e of

inje

ctan

t

Flavollin S40T,S81T

60 70 80p (°°C)

0.8

1.0

0.4

0.6

30 40 50 60 70 80

0.0

0.2

Fra

ctio

n U

nfo

lded

Temp (°°C)

(ii) (iii)

and flavollin S81T. Ser or Thr in the first site affects thether reduces Ca2+ binding drastically. The protein (66 μM)mple cell and titrated with Ca2+ (10 mM) prepared in thed in Table 3. (b) Thermal stability monitored by CD: (i)nd its comparison with flavollin wt. In all mutant proteins,was observed in the presence of Ca2+. The Tm of flavollinof CaCl2. Protein concentrations used were 0.1 mg/ml.

85Determinants of Ca2+-Binding to βγ-Crystallins

group of Thr. In all three mutants, significantconformational changes were seen by near- andfar-UV CD spectroscopy upon Ca2+ binding (Fig.S5a and b).

Changes in Ca2+-dependent gain in thermal stabilityof Ser-to-Thr mutants

Flavollin unfolds with a Tm of 47.5 °C, which,unlike clostrillin, does not gain stability uponbinding Ca2+ (Tm, 48 °C). Tm of apo form of allthree mutants varies in between 40 and 47 °C(with low thermal stability of double mutant, Tm,40 °C) (Fig. 6b and Table 4). The Tm values ofCa2+-bound S40T and S81T and double mutant(S40T, S81T) of flavollins are 48, 60 and 44 °C,respectively (Ca2+ increases the stability of S81Tmutant by 10 °C). The properties of the apo form ofthe mutants changed significantly after mutatingSer to Thr residues, and Ca2+ binding changes inthe conformation (as seen by large increase influorescence intensity) in these mutants (Fig. S4).

(b)

2000

2500

3000

(a)

0

0 40 80 120Time (min)

1000

1500

F.U

.

-2

300 325 30

500

W0

-4

µcal

/sec

0.8

1.0

1.2

(c)

-4

-2

0.4

0.6

0 3 6 9-8

-6

kcal

mol

-1 o

f inj

ecta

nt

30 40-0.2

0.0

0.2

Fra

ctio

n U

nfol

ded

Molar Ratio

Fig. 7. (a) ITC thermogram of Ca2+ binding to clostrillin W3100 μM and 5 mM, respectively. (b) Change in the fluorescconcentration used was 0.1 mg/ml and titrated in the rangeW39D monitored at 218 nm by CD. Tm increased in the Ca2+-bCrystal structure of clostrillin W39D: (i) first Ca2+-binding sitesecond Ca2+-binding site. Purple sphere represents Ca2+, and

Environment around binding site

Hydrophobic or polar residue at X1 position?

We have noticed that the X1 (third) position in theCa2+-binding motif N/D-N/D-X1-X2-S/T-S is morevariable in nature and is occupied by either polar,hydrophobic, basic or a neutral amino acid (Table 1).We explored the effects of the nature of this residueon the properties of proteins upon Ca2+ binding.

A polar residue at X1 position reduces the affinity andthermal stability of clostrillin. In the first case, wegenerated a W39D mutant (Trp to polar residue) inclostrillin. As seen by isothermal titration calorim-etry (ITC) data, this mutation affected the Ca2+-binding isotherm, where a biphasic pattern was seenwith macroscopic binding constants of 11.6 and83 μM (the affinity of second site decreaseddrastically) (Fig. 7a and Table 3). The change inconformation of W39D mutant by Ca2+, as moni-tored by fluorescence spectroscopy, was drastic with

(d)

Glu 9(i)

No Ca2+

(+x)

(+y)

(-y) (-z)

(+z)Th 41

w1

Ca2+

(-x)

Asp 79

Thr 41

W39D (site 1)

50 375 400 425 450avelength (nm)

β2 β4

β8β5

α1α1(ii)

W39D

β

CN

Clostrillin wt No Ca2+ CaCl2

Clostrillin W39D

(+x)

Glu 55(iii)

50 60 70 80

No Ca2+ CaCl2

Temp (°C)(-x)

(+y)(+z)

w2(-y)

Thr 82

LysAsp 38 Lys 80

W39D (site 2)

1β3 β6

Asp 39

9D mutant. The concentrations of protein and CaCl2 wereence spectra of the protein upon Ca2+ binding. Proteinof 10 μM–5 mM CaCl2. (c) Thermal stability of clostrillinound protein (∼20 °C) as compared to the apoprotein. (d), (ii) cartoon representation of the crystal structure and (iii)light-pink spheres represent water molecules.

86 Determinants of Ca2+-Binding to βγ-Crystallins

an ∼6-nm blue shift in λmax from 343 nm in apoform to 337 nm, a change not observed in Sermutants (Fig. 7b and Fig. S2). The far-UV CD spectraof W39D were similar to that of wt, except that theLa transition of Tyr along with the contribution of Bbband of Trp at 230 nm was lost, suggesting that, ofthe three Trp residues, Trp39 is responsible for thisband (Fig. S6a). In near-UV CD, the 1Lb band of Trpat 293 nm was less prominent and disappearedupon binding Ca2+ (Fig. S6b). Another observationwas a drastic reduction in thermal stability (Tmdecreases by ∼12 °C of the apoprotein in compar-ison to wt) (Table 4), though the gain in stabilityupon Ca2+ binding (from a Tm of 32 °C of apo to52 °C of holo) was nearly the same (∼20 °C) as seenin clostrillin wt (Fig. 7c and Table 4).

Structural analysis of clostrillin W39D

The disruption in hydrophobic interaction and anegatively charged residue in binding site ofclostrillin W39D are likely to destabilize the pocket.The structure solved at a resolution of 2.1 Å wasnearly identical with wt protein with an rmsd rangeof 0.19–0.28 Å over 87 Cα atoms (Fig. 7d). The sidechain of this Asp also interacts with Ca2+, which isunusual in terms of binding. The −z water positionhas been taken over by the side chain of Asp39. Site2 remained unaffected except that we could not seewater present at −yw1 and −z positions. In site 1 offlavollin, corresponding to Glu8, Lys7 interactswith Asp by forming a salt bridge. In clostrillinW39D, Glu9 comes closer to Asp39 and forms ahydrogen bond, unlike in wt, thus stabilizing theinteraction.

(b)

0 40 80 120 160Time (min)

300

400(a)

-0.3

0.0

100

200F.U

.

-0.9

-0.6

-0.3

0.0

-0.9

-0.6

µcal

/sec

300 330 3600

Waveleng

(c)

0.8

1.0 Flavollin Wt No Ca2+

CaCl2

0 2 4 6 8 10 12

0.4

0.6 No Ca2+

CaCl2

Molar Ratio

kcal

/mol

e of

inje

ctan

t

30 40 50

0.0

0.2Fra

ctio

n U

nfol

ded

Temp

Flavollin D38V

A hydrophobic residue in flavollin increases the Ca2+-induced gain in thermal stability. Taking a clue fromthe high gain in stability of spherulin 3a by Ca2+,16

where X1 residue is Val, we replaced D38 to V inflavollin. The binding isothermwas atypical, and thedata fitting in any binding models was not proper(thus not presented) (Fig. 8a). The overall change inenthalpy was very low, with a descending nature,suggesting weak binding (the nature of thermogramwas similar to that of the flavollin S40T mutant). Trpfluorescence of apoprotein was quenched as seen inother Ser to Thr (Fig. 8b). The changes in secondaryand tertiary structures upon binding Ca2+ weresignificant, suggesting that the D38V mutant has amore flexible conformation (Fig. S6).The D38V mutation affected domain stability in

the absence of Ca2+ (Tm increased from 47.5 °C forwtto 55 °C for mutant; Table 4). In the Ca2+-boundform, the Tm obtained was marginally higher (58 °C)(Fig. 8c). Structurally, this Asp-to-Val substitutionmay lead to the formation of a strong hydrophobicinteraction with Leu34 and Phe14, which are in closeproximity in the three-dimensional structure. Al-though the side-chain chemistry of residue at X1 doesnot dictate Ca2+ binding directly, through this study,it is seen to play a vital but indirect role by alteringthe microenvironment of the binding site withconcomitant effects on the stability of the domain.

Discussion

βγ-Crystallins form a separate class of Ca2+-binding proteins as an addendum to the otherwell-known classes of Ca2+-binding motifs, such as

Ca2+

No Ca2+

390 420 450th (nm)

60 70 80(°C)

Fig. 8. (a) ITC thermogram ofCa2+ binding to flavollin D38Vmutant. The concentrations of pro-tein and Ca2+ used were 100 μMand 10 mM, respectively. (b)Changes in the fluorescence spec-trum of proteins (0.1 mg/ml) uponbinding Ca2+ (10 μM–5 mM CaCl2).(c) Thermal stability of flavollinD38V mutant. Tm of Ca2+-boundprotein increased moderately(∼3 °C) as compared to apoprotein.

87Determinants of Ca2+-Binding to βγ-Crystallins

EF-hand motif and C2 domain.3,28,29 The physio-logical implications of Ca2+ binding by these pro-teins are unclear, and the degree to which bindingoccurs varies among the family members. The N/D-N/D-X1-X2-S/T-S motif of βγ-crystallins, which, bycasual analysis, appears to be a random combinationof amino acids, is in fact a well-designed motifcontaining a nonlinear double clamp, in which oneamino acid from two different Greek key motifsinterlocks for Ca2+ ligation. This feature makes itmore complicated than sequentially linear motifs,such as the EF-hand motif. In this effort, we haveattempted to decipher the intricacies involved inCa2+ binding by focusing on the positional prefer-ences of amino acids in this motif.

Diversity in amino acid sequence of thecanonical N/D-N/D-X1-X2-S/T-S motif

There have been only limited studies without anycomprehensive analysis of the motif. Our analysis ofsequences from 65 domains (127 motifs) containingthe canonical sequence demonstrates that thesequence of the N/D-N/D-X1-X2-S/T-S motif isdiverse in terms of the occurrence of an amino acidsat the six positions (Table 1). The last Ser residue islargely conserved and does not participate directlyin Ca2+ coordination but is important for maintain-ing the protein scaffold.5,6,30

The presence of Thr in place of Ser brings moresubtleties to the binding site because of the addi-tional methyl group of Thr. When Ser in the bindingpocket was mutated to Thr in flavollin, the affinityfor Ca2+ binding was reduced, the effect being moreprominent in the first site. In the crystal structure, themethyl group of Thr comes closer to the otherwisedistant Cys10 and Cys71 (Fig. S7). This proximitycould interferewith disulfide bond formation duringfolding, which would result in altered bindingaffinity of flavollin mutants due to disruption ofsite by steric effect. Domains lacking this disulfidebridge could likely accommodate the Ser-to-Thrmutation without major effects. The serine in thesecond site in flavollin does not lie in the vicinity ofany such local interactions, and therefore, flavollinS81T shows a similar isotherm but has reducedaffinity as in the case of wt protein. The influences ofThr on binding would depend on local interactionsinvolving the –CH3 group. Thus, the Ca2+-bindingaffinity of this motif is a consequence of additionalinteractions in the domain via the –CH3 of the sidechain.The protein T81S undergoes a significant Ca2+-

dependent gain in stability. This demonstrates that adomain could have attained higher stability at thecost of little change in affinity even though naturehas not selected Thr in this domain. This may benecessary for its function in the context of thatprotein in flavobacterium.

Decoding the N/D-N/D-X1-X2-S/T-S motif

There are various factors that control the Ca2+-binding properties of a βγ-crystallin domain. Thepresence of a hydrophobic residue or a polar residueat X1 position plays a very important role indetermining the Ca2+-dependent gain in proteinthermal stability. The residue at this position co-ordinates with Ca2+ at the +y position via thebackbone carbonyl, and therefore, it could be apolar, hydrophobic or charged (positive or negative)residue (Table 1). In the crystal structure of flavollin,water forms the fifth coordination with Ca2+ and isheld strongly in place with Asp38 at the first site (Fig.S7). Replacing this water via a nonpolar residueperturbs the water shell around Ca2+, which, in turn,affects the electrostatic interaction. A gradual increasein exothermic heat is seen, which does not reachsaturation in ITC (Fig. S7). Trp39 in clostrillin plays arole in filling the hydrophobic space that is created byPhe7, Ile35 and Val18 (Fig. 5). Replacing Trp39 withAsp disrupts the strong hydrophobic interactionbetween loop and hairpin and is even involved inCa2+ ligation via side chain in place of a watermolecule (Fig. 7). In this mutant, binding affinity andthermal stability are drastically reduced. The pres-ence of a polar residue might not affect the Ca2+-dependent stability, while a hydrophobic residue atthis position could lead to increased stability in theholo form. This can also be corroborated fromspherulin 3a, which has V in both motifs andwhose stability in the presence of Ca2+ is enhancedsignificantly.16 Some key features of the motif aresummarized as follows: (i) sites are not independent:Aminor change in one site affects the binding affinityat both sites. (ii) The first site may be less amenable tomodification than the second site. (iii) Even thoughbinding isotherms may change, there is no apparentchange in the geometry of a binding site. Ligationpattern to Ca2+ changes when polarity of a site isaltered (such as seen in clostrillin W39D structure).(iv) Microenvironment around a binding site isresponsible for Ca2+-dependent gains in thermalstability. Polarity/hydrophobicity of a residue in thecore of a binding site modulates Ca2+-bindingproperties and domain thermal stability.Though drawing a global conclusion would not be

appropriate since any effect on Ca2+ binding is due tothe accumulated contributions from many residues,this work sets up a beginning in our understandingon the intricacies of ion binding in this diverse motif.Wide divergence is seen in this motif, and severalproteins with a disabled motif do not bind Ca2+ orhave very low affinity for it. More efforts are neededto understand the causative factors in βγ-crystallindomain that are intrinsically unstructured in the apoform, which readily adopt a β-sheet conformationupon binding Ca2+,17,31 as seen in Yersinia crystallinand in hahellin from Hahella. Dynamic features of a

88 Determinants of Ca2+-Binding to βγ-Crystallins

motif, such as intrinsically unstructured βγ-domains,cannot be predicted from sequence comparisons.In conclusion, this is a comprehensive study in

understanding this highly divergent Ca2+-bindingmotif present in nature. We have demonstrated howthe nature of amino acids and microenvironmentalfactors present in the βγ-crystallin domain around abinding site govern its Ca2+-binding affinity, Ca2+-induced changes in conformation and domainstability.

Experimental Procedures

Cloning of crystallin domains from Clostridium andFlavobacterium species

Genomic DNA of F. johnsoniae was a kind gift from Dr.Mark J. McBride, University of Wisconsin, USA. Thegenomic DNA from C. beijerinckii (obtained from DeutscheSammlung vonMikroorganismen und Zellkulturen, Germa-ny) was isolated using the hot phenol method. The region ofinterest (from residue 500 to residue 591, named flavollin) ofprotein ZP_01247028 (annotated as Ricin B lectin:Carbohy-drate binding family 6) from F. johnsoniae genome32 andresidue 118 to 212 amino acids (called clostrillin) of proteinZP_00908234 (annotated as β- and γ-crystallins) from C.beijerinckii were amplified by using gene-specific primers.33

The PCR products were cloned in pET21a expression vector(Novagen) at NdeI and BamHI sites as described earlier.4

Site-directed mutagenesis for generatingvarious clostrillin and flavollin mutants

Mutations were generated at desired positions in wtclostrillin and flavollin by site-directed mutagenesis PCR.About 11 mutants (clostrillin T41R; clostrillin T82R;clostrillin T41R, T82R; clostrillin T41S; clostrillin T82S;clostrillin T41S, T82S; clostrillin W39D; flavollin S40T;flavollin S81T; flavollin S40T, S81T and flavollin D38V)were constructed (Table 2). All recombinant plasmidswere confirmed by DNA sequencing.

Over-expression of untagged clostrillin andflavollin proteins

wt flavollin and its recombinant mutants wereexpressed in the bacterial strain Escherichia coli BL21(DE3) (Invitrogen) grown in LB medium after inductionwith 1 mM IPTG for 10 h at 37 °C. Over-expression ofclostrillin and its mutant was performed by growing E. coliBL21(DE3) in LBmedia at 18 °C for 16–18 h after inductionwith 1 mM IPTG.

Purification of recombinant proteins

wt clostrillin and its mutants were purified from thesoluble fraction on a Q-Sepharose column using 50 mMTris buffer, pH 9.9. The final purification of these proteinswas performed using appropriate gel-filtration columns.

wt flavollin and its mutants were purified from inclusionbodies using an on-column refolding procedure.34 Briefly,inclusion bodies {washed with 1 M urea and 0.1% 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonicacid} were solubilized in 50 mM Tris buffer (pH 8.5), 3.5 Murea and 1 mM DTT (buffer A) and were loaded on a pre-equilibrated Q-Sepharose column with buffer A. Theseproteins were refolded by setting a gradient betweenbuffer A and buffer A without urea. After the refoldingstep, proteins were eluted using a linear gradient of 0–1.5 M NaCl. The concentrated proteins were finallypurified by a Superdex-75 gel-filtration column on a Bio-Rad Duo-Flow purification system.

Isothermal titration calorimetry

Ca2+-binding isotherms of proteins were determinedusing a Microcal VP-ITC (Microcal Inc., USA). The proteinsolutions and calcium chloride were prepared in a chelex-treated 50 mM Tris (pH 7.0) and 100 mM KCl buffer. Weused 1.4 ml of protein solutions in the concentration rangeof 50–100 μM for the binding experiment at 30 °C.Aliquots of 3 μl of CaCl2 as ligand (2–10 mM) wereinjected for each titration. Blanks were obtained bytitrating the buffer with identical concentrations of Ca2+.The curve fitting was performed using the Origin software(version 7.0), supplied by Microcal after subtraction withthe appropriate buffer blank.

Fluorescence spectroscopy

Intrinsic fluorescence emission spectra were recordedon an F-4500 fluorescence spectrofluorimeter (Hitachi Inc.,Japan) with proteins in 0.1- to 1.0-mg/ml concentrationrange in 50 mM Tris–HCl (pH 7.0) and 100 mM KCl. Thespectra were recorded from 300 to 450 nm at an excitationwavelength of 295 nm in correct spectrum mode of theinstrument using excitation and emission band passes of5 nm each. The effect of Ca2+ on protein conformation wasstudied by titrating a protein solution with aliquots ofstandard CaCl2 solution.

CD spectroscopy

Far- and near-UV CD spectra of all proteins wererecorded at room temperature, on a Jasco-815 spectro-polarimeter using 0.01-cm- and 1-cm-path-length cu-vettes, respectively. The change in protein conformationupon binding Ca2+ was studied by titrating the proteinsolutions with aliquots of standard CaCl2 solution.

Thermal unfolding

Thermal unfolding experiments were performed bymonitoring the change in ellipticity in far-UV range (208,216 or 218 nm) on a Jasco J-815 spectropolarimeter as afunction of temperature. Protein solutions [12–24 μg in200 μl of 50 mM Tris–HCl (pH 7.0) and 100 mM KCl] weretaken in 1-mm-path-length cuvette, and CD was moni-tored between 25 and 85 °C using a Jasco peltier systemattached to a sample holder. The rate of increment oftemperature was 1 °C/min, and data were recorded at

89Determinants of Ca2+-Binding to βγ-Crystallins

0.2 °C intervals. Data obtained were plotted and best fittedusing the Origin 7.0 program.

Crystallization

Purified proteins (mutants of clostrillin) with anapproximate concentration of 10 mg/ml in buffer contain-ing 10 mM Tris (pH 7.2), 20 mMNaCl, 3–5 mM CaCl2 and0.02% sodium azide were used for setting the expansionunder the conditions of wt protein [26% polyethyleneglycol 3350, 0.1 M Hepes (pH 7.3) and 0.2 M lithiumsulfate]. Screens were set on Greiner 96-well plates bysitting-drop (1 μl+1 μl) vapor diffusion methods and on24-well formats in Linbro plates by hanging-drop (2 μl+2 μl) vapor diffusion methods. Crystals were obtained in25–30% polyethylene glycol 3350 at pH 7.0–7.5 maintainedusing 0.1 M Hepes buffer with 0.1–0.2 M lithium sulfate.Clostrillin W39D and clostrillin T41S were obtained insitting-drop experiments, whereas well-diffracting crys-tals of clostrillin T41S, T82S and clostrillin T82R wereobtained by the hanging-drop method.

Table 5. Crystallographic data and refinement statistics of clo

Clostrillin T41S Clost

Data statisticsX-rays CuKαSpace group P212121Cell dimensions (Å) a=64.6, b=89.5,

c=101.6a=

Resolution (Å) 50.0–1.93 (2.00–1.93) 50–Number of observations 190,597Number of unique reflections 44,875Mosaicity range 0.61–0.68Completion (%) 99.7 (98.7)Redundancy 4.2 (4.1)I/σI 14.2 (2.6)Rsym (%) 9.9 (45.9)Number of molecules per asymmetric

unit8

Refinement statisticsNumber of reflections 42,558Rwork (%) 19.8Rfree (%) 23.8rmsd bond lengths 0.007rmsd bond angles 0.99Number of atoms 5704Protein 5310Water 381Calcium 13Sulfate —B-factorsProtein 20.6Water 30.2Calcium 36.3Sulfate —Ramachandran plot (%)Allowed region 507 (88.0)Additional and generously allowed 69 (12.0)PDB code 3SO0

Values in parentheses are for the highest-resolution shell.Rsym=∑|I(h)− ⟨I(h)⟩|/∑I(h), where I(h) is the observed intensity, and ⟨of I(h).Rwork and Rfree=∑h||F(h)obs|−|F(h)calc||/∑h|F(h)obs|. Throughout

Data collection and processing

X-ray data of all the mutant proteins were collected in-house using a Rigaku R-AXIS IV image plate detectorattached to a FR-E+ SuperBright microfocus rotatinganode generator with VariMax optics and operated at45 kV/55 mA. All the data sets were processed and scaledby HKL2000.35 Conversion of reflection formats andmerging were performed using the CCP4 suite of pro-grams version 6.1.2.36

Structure determination

Structures of clostrillin mutants were solvedby molecular replacement using MOLREP andBALBES.37,38 The structure of wt clostrillin (3I9H) wasused as a template to solve the structures. Similar to thewt clostrillin (form 1, 3I9H), clostrillin T41S, T82S andclostrillin T41S also had two sets of four protomers (eightmolecules per asymmetric unit) related by a pseudotran-slation vector of (−0.00, 0.35, 0.50) that resulted in a non-

strillin mutants

rillin T41S, T82S Clostrillin W39D Clostrillin T82R

CuKα CuKα CuKαP212121 I432 I422

67.6, b=103.7,c=105.7

a=110.1, b=110.1,c=110.1

a=77.3, b=77.3,c=77.7

1.85 (1.92–1.85) 50.0–2.00 (2.07–2.00) 50.0–1.85 (1.92–1.85)443,910 181,397 136,04663,524 7993 10,2420.6–0.7 0.54–0.58 0.53–0.65

99.1 (95.0) 99.9 (100.0) 98.8 (96.9)7.0 (6.6) 22.7 (22.7)) 13.3 (12.7)24.5 (3.2) 68.53 (6.32) 84.1 (31.8)7.8 (52.3) 4.9 (52.7) 2.9 (8.1)

8 1 1

60,228 7608 974721.6 20.8 18.624.6 24.6 22.00.006 0.008 0.0060.90 1.09 0.876143 727 8085492 679 689633 46 1088 2 110 — 10

21.0 44.6 14.629.7 52.6 31.329.8 59.2 28.352.4 — 34.4

518 (85.8) 67 (89.3) 66 (88)86 (14.2) 8 (10.7) 9 (12)3SO1 3SNZ 3SNY

I(h)⟩ is the mean intensity of the reflection h over all measurements

refinement, 5% of the total reflections were held aside for Rfree.

90 Determinants of Ca2+-Binding to βγ-Crystallins

origin peak in the Patterson map whose intensity was 34%of the origin peak. Though the space groups of clostrillinT41S, T82S and clostrillin T41S were similar with wt, thatis, P212121, the cell dimensions and arrangement ofmolecules in the crystal lattice of clostrillin T41S werealtered. Clostrillin T82R and clostrillin W39D crystallizedin I422 and I432 space groups, respectively.

Refinement and structural analysis

Repeated cycles of refinements were performed usingCNS andREFMAC (Table 5).39,40 For clostrillinW39D, TLSand restrained refinementwere performed in defaultmodewith weighting term 0.05 using REFMAC. For modelbuilding in required regions, structural alignment/super-position and distance calculations, we used Coot.41 All therefined structures were validated using PROCHECK, andfigures were rendered using PyMOL.42

Accession numbers

Coordinates and structure factors have been depositedin the PDB for clostrillin T82R, clostrillin W39D, clostrillinT41S and clostrillin T41S, T82S with accession numbers3SNY, 3SNZ, 3SO0 and 3SO1, respectively.

Acknowledgements

We are grateful to Dr. Mark J. McBride, Universityof Wisconsin–Milwaukee for genomic DNA ofF. johnsoniae. We thank Syed Sayeed Abdul for hisassistance in the laboratory and Lora Narayana forITC instrumentation. A.M. was supported by a post-doctoral fellowship of the Department of Biotech-nology. S.K.S. and S.S.S. are the recipient of seniorresearch fellowships from the Council of Scientificand Industrial Research, Government of India. Thisresearch was supported by the Department ofScience and Technology (Science and EngineeringResearch Council) and the Department of Biotech-nology, Government of India, grants to Y.S. and aSwarnjayanti grant of Department of Science andTechnology to R.S.

Supplementary Data

Supplementary data associated with this articlecan be found, in the online version, at doi:10.1016/j.jmb.2011.10.037

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