The linker of calmodulin - to helix or not to helix R.H. KRETSINGER

Department of Biology, University of Virginia, Charlottesville, Virginia, USA Abstract - The linker regions of the central helices of calmodulin and of troponh C are observed to be a-helices in crystal and in solution. However,these linkers are pre&ted to be non-helical by standard algorithms. Further,there is strong evidence that when cstmodulin interacts with some of its targets this linker helix bends. The linker appwrs to be delicately balanced between helical and non-helicalconformations.A review of this subject suggests that one can anticipate more unpredictedconformationsfor the central helices of the score of other proteins that have four EFhand domains.

Posingthe question The crystal structure of troponin C (TnC), a close homolog of calmodulin (CaM), revealed a completely unanticipated central helix that separates domains 1 and 2 from domains 3 and 4 by 12 8, [l-4]. This bizarre structure might have been attributed to strange crystal packing forces until the crystal structure of calmodulin [5-71 was seen to have a similar central helix comprised of a-helix F of the second El?-hand, the eight residue interdomain linker, and cl-helix E of the third EF-hand domain. Kretsinger and Barry [8] had made the masonable and generally accepted prediction that the structure of TnC consisted of two pairs (domains 1 and 2. and domains 3 and 4) of EF-hands, both similar to the CD, EF pair of parvalbumin. They noted that both pairs of domains would have extensive, exposed hydrophobic patches and that the 11 linking residues had a region predicted to be nonhelical. They proposed that the linker is bent and that the two hydrophobic patches touch one another producing a spherical molecule (Fig. 1). This prediction was proven to be incorrect by the crystal structure determinations. Yet, elements of

this proposed structure have been incorporated into a model of calmodulin’s interactions with its multiple targets. A chronological review of these studies provides a fascinatiug insight into the development of scientific models. Further, this review cautions that more surprises am forthcoming as we examine the structures and functions of 20 other homologs of CaM and TnC that contain four EF- hand domains, Table 1.

Prior to the crystal structures of troponin C and cahnodnlin, 1985 Wang et al. [9] examined the ‘Kinetics of Ca*’ &Xi!+e...’from troponin C and conchxled, ‘...that binding of Tb3’ to the C.a*‘- ific sites reduces the rate of dissociation of CaP from, and thereby enhances the affiity for, the Ca2’-Mga+sites; this in turn, suggests interactions between the two halves of the TnC molecule.’ Newton et al. [lo] found different sites of trypsinolysis in CaM in the presence and ab!%nceof calcium Fragment 78-148 blocks CaM activation of cyclic nucleotide phosphodiesterase but, ‘. . .

363

364

Fig. 1 The Kretsinger and Barry [E] model of troponin C is shown in a-carbontrace. The four EF-hand domains em numbered. The N- and C-&mini are so indicated. The ninth nridue of each domain is indicated. The a-wbons 2,5,6,9,22 $5, 26 and 29 of the four helices E and of the four helices F (excepting a& 29 of domains 2 tmd 4) are indicated with bold circles. These hydmpbobii residues, as indicated in Figure 2. form the cams of both lobes. The hydrophobicpat&s of Cah4 are illustratedin Figure 3

fully activates phosphorylase kinase but with a lower affiity than calmodulin. The attractive notion that calmoduliu-activated enzymes share a common binding domain is therefore, at best, an over-simplification.’ Aulabaugh et al. [ll] applied two dimensional proton NMR to tiagment 78-148 of CaM and confirmed ‘. . . domains III and IV as the high affinity calcium binding sites and further indicated that the fragments maintained most of the tertiary structum observed in the intact protein . . .’

CELLcAm

Klevit et al. [12] used their [‘HJ-NMR spectral assignments derived from studies of kypsin fragments of CalvI to assign resonances to the iutact protein. ‘Briefly, the two tryptic fragments, each of which spans two complete EF-hand domains, retain the conformations which they assume in whole calmodulht. This property obtains for both the Ca2’-fme and the Ca2’-bound forms.’ They found that, ‘The binding [of two Ca2’ ions to domains 3 and 41 affects the disposition of helices E and H (lettered from the N temiinus) and these helical movements transmit effects to the N-Wmiual half of the molecule, especially by interaction between helices A and D’. Calcium binding to the lower affinity domains 1 aud 2 exerts no discernible effect on the C-half of GM. They contrasted this result with earlier work from their laboratory [13], ‘High afftity Ca2’ binding to troponin C, however, results in an increase in tertiary structure in domains III and IV with no discernible effects on domains I and II’. They noted that the linker of TnC is three residues longer than that of CahI and conclude, ‘This difference in length may account for the difference in communication between the two halves of the proteins observed in these studies.’ Thulin et al. [14] also showed that domains 3 and 4 of CaM have higher affinity for calcium and for cadmium and that one equivalent of trifluoperazine binds both to the l-77 aud to the 78-148 fragments. They ‘. . .conclude that the majority of the conformational marrangements detected in the [lH]-NMR spectra must be localized to the half of the protein containing the [fust two] calcium binding sites involved in Ca2’ binding*. However after reviewing their own research and that of other; they caution ‘. . . that the two independently folded halves of cahuodulin do interact with one another.’ Bayley et al. 1151derivatixed calmodulin with 8-anilinonaphthalene sulfate in order to do stopped flow fluorescence studies. ‘These experiments provide confirmation that the calcium induced conformation change cannot be resolved kinetically from the calcium binding or dissociation, and by inference this conformational change is not a rate-limiting process in the function of calmoduliu.’ Martin et al. [16] extending the kinetic studies of Bayley et al. 1151found that, ‘One of the two high affinity uifluoperaxine-binding sites was found to be

THE LINKER

OF CALh4ODULJN-TO

HELIXOR NOT TO HEW

365

Table 1 Subfamilies of EF-hand homolog proteins. Nakayama et al. [59] classified the EF-hand homolog proteins into 29 distinct subfamilies. The tit 22 contain four EF-hand domains. ‘Ike kirst eight, ealmodulin through calcium dependent protein kinase, am inkned to be congruent; that is all eight evolved fium a common four domain piecursor. This implies that their linkers sham a common precursor. The other 14 have more complex histories of gene duplications, tmnsloeations, and splicings. Of the remaining seven (29-22) subfamilies, there are single examples of eight, six, and three domain subfamilies end four different subfamilies with two domains. Little is known of the linkers between pairs of domains in LPS and iu CLBN. The symbol + indieates calcium binding is known or inferxed in that domain, - not; f indicates that some members of the subfamily do and some do not bid calcium. The ‘...’ indicates that the W-hand domains a~ preceded or followed by regions that are not EF-hands

Abbreviation

Name

Calcium binding by domain

I-

2

t t

CaM

calmcdulin

t

TnC

WpMiIlC

ELC

essential light chain myoain

RLC

regulatory light chain myoain

CAL

call (caenorhabditis)

SQUD

SquiduIin @o&o)

f * t + t

CDC

CDC3 11%caltaactin

t

CDPK

Ca dep prot kinsse

CALP SARC

calpaia & sorcin sarcqlasm Ca bind pmt

. . .t . . .t +

EFHS

EFHS (Trypanosoma)

TPNV

troponin, non-vertebrate

CLNB

calcineurin B

AEQ

aequorin & luciferin BP

TPP

p24 thyroid pmt (Canfs)

IF8

IF8 & TB17 (Trypanosoma)

CVP

calcium vector protein

SPEC

StrongyloceMus CaBP

LAV

LAVI (Physarum)

VIS

visinin & recoverin

CMSE

CaBP (Strepkmzyces)

TCBP

Tetrahymena CaBP

LPS

Lytechbtus pictus

CLBN

calbindin 28 IrD

PARV DGK

parvalbumin a-a&in diglycerolkinese

Sloe

S 100, intestinal CaBP

CRGP

CAM related gene product

ACTN

t t t t f ...t t t t t t f . . .t ‘f t

3

4

t

f t

f

f f

t t

t t

f t t

f t

f t t t

t t

f + t

t t t t t t

f t t t t t t t t t t t t

f

t

t

t

t

t t t

f ... t t t

f t t t t

t 7 t t * t t t t

366

cELLcALciuM

A

En CaM 1

TnC

nn

n X Y ZG IF

5 n

nn

(n)

ADQLTEEQIA F&KE&FS&; DKDODGTITTKE LGTV&tS& ASMTDQQAEAMFLSEEMIA EFKAAFDMF DADGGGDISTKE LGTVMBML

CUM 2 TnC CaM 3 TnC CaM 4 TnC

GQNPTEA ELQDWNEY DADGNGTIDFPE FLUKE GQNPTKE ELDAIIEEV DEDGSGTIDFEE mmvw 76 &4D---TDSEE E~RlUJE'R~~DKDGNGYI!lAAE VN; 86 MKEDAKGKSEE ELANCE’RIP DKNADGPIDIEE KiEILEAT

39 49 75 a5 112 125

GEKLTDE EVDE&jIREQ DIDGDGQVNYEE FV-AK 148 GEHVIEE DIEDLMKDS DKNNDGKIDFDE FLKMUEGVQ 162

CAM

CAM83

TNC Fig. 2 The amino acid sequences of mammalian calmodulin and of chicken tqonin C am shown aligned by domains, Panel 2A. The insides of helices E and of helices F are. indicated by ‘n’. Position 9 is numbered as it is in Figne 1. Tht side chains that coordinate calcium am indicated by X (position 10) , Y (12). Z (14). ‘jI (18) and ? (21). Gly is usually found at 15. The side chain of Be, Val or Leu at 17 contributes to the hydrophobic core of the pair of domains, or lobe. Tbxe residues, shown in Figae 3 as comprising the hydrophobic patches of CaM, am underlined. The eight amino acids of CaM or 11 of TnC preceding domain 3, am nfermd to as the linker region of the central helix. In Panel 2B am shown the helix form& tendencies of the central hehccs of TnC, CaM and desGh184Cah4 as computed after Gamier 1611. Helices F2 and E3 am aligned by homology, the spacings of residues in the linker are varied to maintain alignment of F2 and of E3

located on the N-terminal ‘half and the other on the C-terminalhalf of calmoduliu.’ Their results ‘. . .

lend support to the notion that CaM is made up of two independent domains that may be identified

with these [tryptic] fragments.’ Suko et al. [17] in a study published a few months after that of Martin et al. [16], identified by stopped flow fluorescence two non-interacting

367

THELINKEiROFCALMODULIN-'l'OHELIXORNOTTOHELIX

2-ptoh&linylnaphthaIene sulfonate binding sites in CaM. Further, their aualyses of TNS fluorescence &om the l-77 and 78-148 fragments indicated that ‘ . . * the rate of the conformational change associated with calcium release from the high-affinity calcium-binding sites of the C-terminal half of calmodulin is not influenced by the N-terminal half of the molecule.’ Tsalkova and Privalov 1181 published an important and seldom cited study submitted before the crystal structure determinations of the the& denaturations of TnC and of CaU They concluded that for both them is a positive interaction between domainsland2aswellasbetweendomains3aud 4. However, ‘It appears that there is some repelling stress between these pairs of domains, which results in their mutual destabilization.’ They concluded that ‘The negative interaction between the domain pairs . . . prevents integration of all four domains into a single co-operative system (and) is a strong argument against the existence of a hydrophobic core in these molecules.’ By 1985, it was generally accepted that domains 3 and 4 which we refer to as lobe 3,4 of both cahnodulin and troponin bind calcium with higher affinity than do domains 1 and 2. Both lobes of C&I bind one equivalent of presumed analogs, such as phenothiaxine, of targets of CalK Yet, concerns were expressed about the generality of any binding site of CaM for its numerous targets. Many of the spectral and binding properties of CaM can be interpreted as the sum of the properties of the two fragnxzits, l-77 and 78-148. Yet numerous groups expressed caution about the extent and nature of the interactions between the two lobes.

The crystal structures - answers and questions The crystal structures, fust of troponin C and then of calmodulin (Fig. 3) inverted the question. ‘How could there be so little interaction between the two lobes of a spherical model?’ became ‘How could there be so much interaction when the two lobes are separated by 11 or by 8 residues of a-helix?’ This concern was compounded by suspicions that the structums in solution might be different from those

seenintheaystals.

Whyarethelinkenbelical?

How does CaM interact with its targets? Again, this story is best told by its actors. SundaraBngam et al. 131noted a hend of 16’ at theGlyinthelh&rofTnCaswellasacurvein the straighter linker of CaM. They suggested that the linker is stabilixed by salt bridges, polar hydrogen bonds, of amino acid side chains in this region. They noted the presence of proline in some helices homologous to this linker region in other subfamilies and suggested that, “Ihe conservation of the bend in the related calcium biuding proteins suggests that it may have an important biological function.’ Sundamlingam et al. [19] noted that ‘The amino acid sequence of the D/E linker indeed displays strong helix forming potential with the exception of the Gly-Lys-Ser segment Apparently, the latter segment was induced to assume a helix by the flanking D aud E helices.’ Figure 2 shows C&i and TnC sequences aligned by domains. Babu et al. [5] had argued that ‘Because this helix [of CaMI is shared by Ca-binding domains 2 and 3, it may have a role in the co-operative binding of Ca.’ They continued ‘Thus, the central helix in troponin-C and calmodulin may be buried in the absence of Ca and exposed when the ion is bound. O’Neil and DeGrado [20] published a predicted structure of cahuodulin, supported by the TnC crystal structure, with a helical linker. They suggested a ‘. . . binding site for basic amphiphilic &helical peptides located between the last E and F helices in the second domain [lobe] of calmodulin.’ Seaton et al. 1211 measured the small angle X-ray scattering (SAXS) of calmodulin ?d determined a maximum vector length of 62 A in the presence of calcium and 58 A in its absence. ‘The parameters determined for calmodulin are consistent with the solution structure of the protein being the same as that of the crystal, i.e. a dumbbell structure.’ Klevit et al. [22] evaluated the calcium dependent binding to CaM of the synthetic peptide Ml3 (KRRWKKNFIAVSASSGAL), representing the C-terminus of skeletal muscle myosin light chain kinase. The ‘. . . observed ellipticity of . an equimolar mixture of Ml3 and cah&ulm is much greater than the sum of the elliptic&s of the two isolated proteins.’ Most, if not all of this

368

change in the mixture is due to M13’s going from -0% to -100% &helix. The extensive changes in NMR spectra of the complex, relative to the sum of CaM plus Ml3 was attributed to ‘. . . only one molecule of peptide...being bound in the complex . . . in only one way . . .’ Ghan ed resonances include trimethyllysine-115 and 55 1 away in the crystal structure the aromatic cluster Phe-16, Phe-65 and Phe-68. ‘If Ml3 is entirely a-helical, it would span a distance of 39 A.’ They considered the possibility that Ml3 is partially extended, partially helical and concluded that ‘. . . it is still difficult to account for the remainder of the observed spectral change8 without invoking some changes in calmodulin tertiary structum.’ Putkey et al. 1231made three mutant calmodulins having 3, 16 and 19 changes in amino acid sequence distributed over the molecule. ‘The data demonstrate that the nature of the interaction of CaM with myosin light chain kinase is different from its interaction with calcineurin, CaM-dependent multiprotein kinase, and phosphorylase kinase and may involve different functional domains in CaM.’ Yazawa et al. [24] used [‘I-&NMR to study the binding of calcium to CaM in the presence of mastoparan or a fragment of caldesmon. They concluded that ‘The two domains of [ape]-calmodulin have no communication with each other, but the calmodulin opens the communication between the N-domain and the C-domain by the association with target proteins. The Ca2+ binding to C-domain induces co-operative Ca2’ binding to N-domain and Ca2’ binding to N-domain produces an active C-domain-targetcomplex. Small and Anderson [25] cross-linked Tyr-99 and Tyr-138 of CaM by UV radiation to form the 2,2’-biphenolderivative, which has a unique absorption (320 nm) and emission (400 nm) spectrum. The Tyrs are at domain 3, position 8 and at 4-19; both side chains are exposed to solvent and their a-carbons are 7.3 8, apart From dynamic fluomscence anisotropy measurements they concluded that calcium*CaM ‘. . . is elongated and has a length equal or nearly equal to that predicted by X-ray crystallographic results. In the absence of calcium, the molecule becomes highly compact and exhibits significant segmental motion.’ They continued ‘. . .

CELLCALCIUM

the binding of melittin and mastoparan . . . cause no major changes in the elongation of the molecule.’ Sundaralingam et al. [26] evaluated published structures and found that Glu or Asp/Lys or Arg pairings occurred at twice the expected frequency, based on random distribution, for pairs i to i f 3 or i f 4 residues along the helix for a-helices five turns or longer. The geometry of pairing might involve either a water bridge or polar hydrogen bond. They suggested that such ion pairs contribute to the stability of the central helices of TnC and of CaM. Satyshur et al. [4] noted in their refinement of chicken skeletal muscle TnC that ‘. . . water molecules have penetrated the screen of charges and are found to be hydrogen bonded to the backbone carbonyl oxygen atoms around the helix handle at residues 86,87, 88 and 90, giving almost a full turn of the helix.’ ‘, . .the summation of distortions produced by the hydrogen bonds from the water molecules surrounding the helix results in only a minor perturbation of the helix direction.’ Her&erg and James [2] described the crystal structure of turkey TnC refined using 2.0 8, data. They calculated that the radius of curvature of the central helix is 137 A. The temperanne factor is high in the linker region but not so high as in loops 1 and 2, which in the crystal at pH 5.0 do not bind calcium. Strynadka and James [27] used the refined crystal structure of TnC to predict two trifluoperazine binding sites on CaM, one on each lobe on its hydrophobic patch. This model did not posit any bending of the central helix. Babu et al. [6] described the crystal structure of calmodulin refined with X-ray diffraction data to 2.2 A resolution. CaM has an overall length of 65 A and ‘There am no contacts between the lobes.’ They noted that ‘Residue8 76 to 82 have some of the highest temperature factors in the molecule’ and that the ‘dihedral angles of residues 79 to 81 show significant deviations from ideal a-helical geometry . . . suggesting that the helix is somewhat strained in the middle.’ They proposed that target binding consists of the binding of calcium exposing hydrophobic regions (Fig. 3) and that ‘It is likely that the hydrophobic clefts located in the two halves of calmodulin are responsible for the interaction with hydrophobic regions of these compounds.’

THE LINKFiR OF CALMODULIN -

TO HE&IX OR NOT TO HELIX

369

Lys148

sp

Glu 7

80

Ng. 3 The crystal structure of calmodulin [61 ia shown in two views (a) Lobe I.2 is viewed down its local 2-fold axis showing the hydmphobic patch. Near white indicates the van der Waals smfaces of side chains of helices E; dark gmy indicates helices F; light grey indicates hydrophilic side chains or hydrophobica not coutiguous with the patch The helical &dues Tht~ through Ala10 am au exteusion of helix Fl. Me& the first residue of the linker, is also indicated after domain 2, which nominally ends at Lys~ to illustrate that it contriiutes to the patch. As indicated below, and ahown in Figure 2A. two nsiduea, not part of the canonical core, Levis and Met76 contriiute to the patch. Conversely seven11hydrophobic side chaim of the core do not contribute to the surface patch. The hydrophobic side chains illustrated as forming the patches anz domain 2 or 4

domain 1 or 3 111111111122 22222222 123456789 012345678901 23456789 n nnnn n nn n patch 1,2

F

A

LF

ML

patch 3,4

I

A

VF

N

L

111111111122 22222222 123456789 012345678901 23456789 n ml n n nnn n

v

MMM

M

A

MM

(b) ‘l%e hydrophobic patch of lobe 3,4, is viewed down its approximate Zfold axis as it is iu the cartoon of Figure 4c. The pmceding eight msiduea of the linker -&lKDTDSEEs~ are also shown to illustrate orientatiou. The residues, sTEEQlAto, shown pnoeding domain 1 have the same relative orieutation to lobe 12, ILp&es the linker to lobe 3,4. In the view of lobe 12, peoel38, tk link7 is viewed parallel to its axis and has been truncated at residue Lym. Patch 3.4. appears mom sccessible than dces patch lf, even though the homologous residues comprise both patches. In fact patch 1,2, has rme mom contributcr, Metes the first residue of the linker. Glu41 covem Me& more than its homolog Glul14 covers Metios of patch 3.4. Lyms covers Mctn of patch lf; the C-terminal Lyslrs is not identified in the crystal structure and is shown symbolically as the last residue of helix F4 with its side chain not covering MetIs,

The flexible tether

After living with the dumbbell for 3 years several

have suggested that confom~tional changes are associated with calmodulin’s binding targets. Yet there were no specific proposals as to what

groups

370

be. Persechini and Kretsinger [28] fast described the abilities of the three deletion mutants of CaM desGlu84, desGlu83Glu84, and desSer81Glu82Glu83Glu84- to activate skeletal myosin light chain kinase. They then presented their model in which the linker region of the central helix of CaM is bent thereby permitting the hydrophobic patches on lobe 1.2 and on lobe 3.4 to enfold the helical target, the Ml3 peptide of MLCK (Pig. 4). Persechiui and Kretsinger 1291 prepared an engineered CaM, Gln3CysEhr146Cys. This Q3C/T146C CaM was irreversibly cross-linked with bismaleimidohexane, thereby forcing a-carbons 3 and 146, which in the native CaM am 37 A apart to at least 19 A of one another. The proximity of hydrophobic patches of this bent BMH*CaM was inferred to be similar to that postulated in their model. This BMH*CaM fully activates MLCK, as does BMH*CaM in which the central helix has been selectively cleaved at Lys77 by trypsin. The two halves of wild type CaM are inactive following the analogous trypsin cleavage. These BMH cross-linking experiments were presented as strong supporting evidence that ‘The Central Helix of Calmoduliu Functions as a Flexible Tether.’ Persechim et al. [30] showed that the three mutant CaM’s - d84, d8384 and d81-84 activate the targets myosin light chain kinase, NAD kinase, and the protein phosphatase, calcineurin, and that these enzymes have Kms and Vmaxssimilar to those observed when activated by wild type CaM. CaM, d84, d8384 and d81-84 all bind acrylodan M-13 with similar affinities. They argued that the flexible tether model applied to many CaM target interactions. Subsequently Persechini et al. [31] showed that in Saccharomyces, which has an absolute requirement for CaM, the d84, d8384 and d81-84 CaM genes can restore full growth to yeast cells whose CaM encoding gene had been removed. Putkey et al. 1321made five C&M’s- CaMPM Thr79Pr0, CaMIM with Pro*Ser*Thr*Asp inserted before Pro79 of CaMPM, and CamMlMs with the PSTD insertion and Pro79 changed to Thr*Ser*Thr*Asp*Pro or to Thr*Ser*Thr*Asp*Gln or to Lys*Ser*Thr*Asp*Gln. ‘caLmoduliu, CaMPM, and CaMIM were indistinguishable in their ability to activate calcineurin and Ca2’ATPase. All mutated calmodulius would also max-

CsLLcALcluM

these changes might

Fig. 4 The cartoon of panel a, a&r Kxetsiogeret al. m, chows the relationshipof lobe 1.2, the linker, and lobe 3,4 supe-rimposed onthecWarbontlace. Ihe hatched areas symbol&e the hydrophobicpatches of lobe 1,2 and of lobe 3.4. Strynadka and James (621 reviewed the hydrophobic patches of TIC, CaM, pamalbumin and intestinal calcium binding protein. In the flexible tether model of Pemchini and Kretsinger [29] the linker region of the central helix bends permitting the hydrophobicpatches on the two lobes to enfold a helical pox&n of the targetprotein, panel b. The predicted structure of desCilu84 calmodulio coo&a of rotatinglobe 3,4 closer to lobe 1,2, along the linker by +lOO’and tmnslatingit by 1.5 A, panel c. In panel d, is shown the bent helix and the relative orientationof the two lobes as seen in the crystal structureof des01u84 Cah4 WI

imally activate cGMP-phosphodiesterase and myosin light chain kiuase; however, the concentrations of CaMPM and CaMIM necessary for halfmaximal activation (I&) were 2- and g-fold greater

THE LINKER OF CALMODULJN -

respectively,

TO HELIX OR NOT TO HELIX

than [wild type].’ The three CamMIMs ‘. . . did not restore normal calmodulin activity. These observations am consistent with a model in which the length but not composition of the central helix is more important for the activation of certain enzymes. The data also support the hypothesis that calmodulin contains multiple sites for protein-protein interaction that are differentially recognized by its multiple target proteins.’ Reinach and Karlsson [33] expressed TnCs in which Gly92 was replaced by Ala and by Pro aud found that both ‘. . . were able to mediate the calcium regulation of [myosin] ATPase. Since both the Ala and Pm mutants probably increase the rigidity of the ceutral helix it is unlikely that large rotations around this part of the molecule are involved in the function of TnC.’ Xu and Hitchcock-Degregori [34] engineered avian TnC with three residues (Lys91-Gly-Lys93) deleted from the linker region of the central helix. ‘The weaker affinity of the mutant TnC-TnI complex for actin-tropomyosin and the failure of the reconstituted ternary complex to inhibit the actomyosin ATPase in the absence of calcium could both be explained by a higher affinity of the mutant TnC for TnI in the absence of calcium.’ They noted the similarity of des91-93TnC with CaM and noted that in both the rotation of lobe 3.4 relative to lobe 1,2 is shifted by -60” relative to the relationship in TnC. ‘It is likely that the long range interactions between the two halves of TnC are transferred at least partially via the central helix and this deletion may alter communication between the two domains upon calcium binding to the high (or low) affinity sites.’ Heidom and Trewhella [35] measured the SAXS of both CaM and TnC. They showed that calcicalmodulin is extended and has a dumbbell!hape at pH 7.4. Further, the calcium form is 4 A longer than the apo form measured at either pH 7.4 or pH 5.5. The calcium form aggregates at pH 5.5. Magnesi-troponin C is also dumbbell shaped at pH 7.4. The calcium form aggregates. They reviewed the literature indicating that the magnesium and calcium forms have similar, though not identical, conformations. Magnesi-TnC is longer (d-, 70 f 3 A) than is calci-CaM (dma, 63 f 2 A) in solution. This is consistent with TnC’s linker being three

371

residues longer than that of CaM. SAXS is very sensitive to differences in length and in conformation. They note that their models am several A shorter than the maximum lengths observed in the crystal structures. However, they indicated the crystal structure of CaM to have a maximal length of 70 A. Babu et al. [6] stated that in the refined crystal structure of CaM the overall length is 65 A. Heidom and Trewhella [35] suggested that the solution structures am shorter because the linker regions are bent about 65’ from linearity. They concluded ‘, . . that the crystal structures do not predict the solution scattering data on the basis of comparisons of the P(r) {distribution of length vectors} functions.’ They considered the possibility ‘ . . . that in solution there are a number of conformational substates and that the distances between the globular domains could vary over a range of values and the solution scattering retlects the ‘average’ conformation.’ Hubbard et al. [36] concluded from their SAXS studies that TnC is dumbbell shaped in solution. They suggested that any deviation of vector length distribution P(r) observed from that based on the crystal structure is due to a layer of bound water between the two domains. Tsuda et al. [37] concluded from their NMR studies that the two lobes of calci-TnC do not interact; however, ‘. . . Tyr 10 of apo-TnC interacts with the C-terminal-halfdomain.’ Kataoka et al. [38] found that CaM undergoes a calcium dependent change in conformation upon binding melittin, GIGAVLKVLTTGLPALISWIKRKRQQ,a 26 residue analog of helical targets of CaM. ‘Upon binding n&tin, the radius of gyration decreases from 20.9 to 18.0 A and the largest dimension decreases from 60 to 47.5 A.’ From these SAXS studies they suggested, ‘. . . that the conformational change that occurs may be even mom extreme than that suggested by the model [of Persechini and Kretsinger].’ Yoshino et al. [39] and Matsushima et al. [40] studied the calcium dependent binding of mastoparan to CaM by SAXS. In the latter manuscript they concluded ‘. . . that the two globular domains of the calmodulin complex with Ca2’ and mastoparan come closer together by 8.0-9.5 A on average, if the size and the overall shape of the

372

globular domains are the same in Ca2+-calmodulinmastoparan complex as in calmodulin or Ca2+calmodulin complex.’ Heidorn et al. [41] studied the calcium dependent binding of the synthetic peptide, MLCK-1 which corresponds to residues 577-603 of skeletal muscle myosin light chain kinase, with CaM. SAXS indicated that the complex is ‘. . . considerably more compact than the uncomplexed calmodulin’ and that the dmaxdecreases from 67 to 49 A. ‘The solvent contrast dependence of Rs for neutron scattering indicates that the peptide is located more toward the center of the complex . . .’ They concurred that ‘ . . the helix region . . . is quite flexible in solution . . .’ Wachtell et al. [42] measured SAXS and concluded that turkey TnC ‘. . . at low pH in the presence of Mg2+ is an elongated molecule with maximum dimension and radius of gyration similar to that of the crystal structure, and with a deposition of domains similar to that found in solution at neutral PH. Fujisawa et al. [43] carefully investigated the dimerization of TnC, a serious source of artifact in SAXS studies, in terms of the concentration of protein and of free Ca2’ ion. They concluded that non-lyophilized TnC at 2 mg/ml shows little dimerization with Ca2’ ion concentrations as high as lOA M. ‘The maximum dimension of the molecule decreases from 111 to 98 A with increasing Ca2’ concentration. These results indicate that the troponin C molecule shrinks remarkably as Ca2’ ions bind to the high affinity sites of the molecule. Ca2’ binding to the low affinity sites on the other hand, leads to a less pronounced change.’ The distance between the centres of the two domains decreases from 46 to 35 A. The conformation of the two domains also changes upon Ca2’ binding; they become more compact, especially in the change from the Ca2’-free state to pCa 6.5 . . .’ They acknowledged that the length of TnC in the crystal structum is 75 8, and that their SAKS results differ from those of Hubbard et al. [36] and Heidom and Tmwhella [35]. They attributed these differences to the use of NaCl instead of KC1 by Hubbard et al. [36] and to the use of Mg2’ by Heidom and Trewhella [35] as well as to several differences in instrumentation and data analysis. They concluded ‘The 14.8 A of Rs(dom) [lobe of apo-TnC]

CEUCALCIUM

corresponds [to] a radius of a uniform sphere of 19.1 A so that the separation of the two domains may not be so clear as in the crystalline state when the high affiity sites am occupied. This structure could render it possible for the N- and C-domains to interact [with] each other . . .’ In 1990, Fujisawa et al. [44] extended their SAXS studies in the presence of 2.0 mM Mg2’ and concluded that ‘. . . the strnctural behaviour of tro onin C molecule is essentially the same when Cazp+ /Mg2+ ions bind to its high affinity sites.’ They also examined tryptic fragments ‘. . . the C-domain shrinks, with the radius of gyration changing from 17.0 to 14.9 8, while the Ndomain swells from 13.9 to 15.0 A upon Ca2’ binding [to apo-fragments].’ O’Neill et al. [45] and O’Neil and DeGrado [46] synthesized a series of analogs of the Ml3 helix of skeletal myosin light chain kinase that binds to CaM. They inserted Trp at many sites to determine by fluorescence spectroscopy which sites of MLCK arc buried when bound to CaM. Based on these results they put the photoaffinity label, p-benzoylphenylalanine, at selected sites and determined the sites of cross-linking to CaM. In the latter manuscript they conclude that ‘. . . when complexed with basic, amphiphilic peptides, calmodulin can adopt a conformation in which its two domains are significantly closer than in the crystal structure of the uncomplexed protein. By introduction of a kink in the central helix linking the two domains of the protein as described by Persechini and Kretsinger [28, 291, a conformation can be obtained for calmodulin which is consistent with both the labelling and the fluorescent studies described herein.’ Jkura et al. [47] made [113Cd]-NMRmeasurements of CaM complexed with mastoparan and with M13. ‘The interdomain [interlobe] interaction manifests itself only in the presence of such peptides: Tsuda et al. 1481 examined the binding of magnesium to TnC by [rH]-NMR and concluded: ‘Two Ca2’ binding sites of the N domain bind Mg2+ ions. Binding of Mg2’ induces a conformational change in the hydrophobic region of the C domain, but does not induce a change in the hydrophobic region of the N domain.’ Strynadka and James [491 presented ‘A model for the interaction of amphiphilic helices with

THELINKEROFCALMODULIN-TOHELEORNOTTOHELIX

troponin C and calmodulin’ based on the crystal packing of troponin C in which the first a-helix, which precedes helix El, ‘. . . of one molecule [packs] onto the exposed hydrophobic cleft of the C-terminal domain of a symmetry related molecule.’ They concluded that ‘. . . the increased length of melittin [relative to mastoparanl requires a significant bend in the central helix similar to that suggested recently for the MLCK cahnodulin binding peptide (Persechini & Kretsinger, 1988).’ Ma&all and Klee [SO] found that binding of calcium by apo-CaM stabilizes Arg-37 and Arg-106 (helices I-F and III-F) to trypsinolysis. In contrast Arg-74, Lys-75 and Lys-77 are cleaved 10 times faster when CaM is in the Ca2-form than when it is apo or in the Ca4-form They ‘. . . propose that at intermediate Ca2’ levels the flexibility of the central helix of calmodulin is greatly increased . . .’ Trewhella et al. [51] examined the interaction of CaM with two synthetic peptides - PhK5 (342-366) and PhK13 (301-326) - of the y subunit of skeletal muscle phosphorylase kinase, by both neutron diffraction and by SAXS. They found that the binding of PhK5 to CaM ‘. . . induces a dramatic contraction of cahnodulin.’ ‘In contrast, calmodulin remains extended upon binding PhK13.’ Further, in the presence of both PhK13 and PhK5 (or MLCK-1) CaM remains extended. They concluded ‘. . . that there is a fundamentally different type of calmodulin target enzyme interaction in the case of the catalytic subunit of phosphorylase kinase compared with that for myosin light chain kinase.’ Kataoka et al. [52] compared the SAXS of des81-84CaM and of des83-84CaM with that of wild type CaM in both uncomplexed and in melittin bound forms. All three proteins have bin&al vector length P(r), distributions consistent with their having dumbbell shapes. The lengths of des83-84 and des81-84 are shortened as expected for deleting two and four (6.0 A) residues respectively from the central helix. Unfortunately des84 was not investigated. All three molecules assume very similar spherical shapes upon binding melittin. Kataoka et al. [53] measured the SAXS of CaM bound to analogs of the 28 residue region of the plasma membrane Ca2+ pump of erytlnocytes. ‘C2OWcontains the 20 N-terminal amino acids of this domain, C24W the 24 C-terminal amino acids.

373

The complex between CaM and C2OWrevealed an interatomic length distribution function, P(r), similar to that of calmod& alone, indicating that the complex retains an extend@ dumbbell shaped structure. By contrast, the binding of C24W msulted in the formation of a globular structure similarto those observed with many other &M-binding proteins.’ lkura et al. [54] assigned nearly all ‘H and r3C resonances of Drosophila CaM in solution. They concluded that its stn~ctum *. . . is essentially identical to that of the X-ray crystal structure of mammalian CaM.’ However, ‘. . . NMR data indicate that residues Asp-78 to Ser-81 of this central-helix adopt a non-helical conformation with considerable flexibility.’ Even with this flexibility they did not observe ‘. . . any NOE interactions between the N-terminal and C-terminal domains.’ Ikura et al. [55] completed a ‘Multi-dimensional NMB study of Cahnodulin Complexed with the Binding Domain of Skeletal Muscle Myosin Light Chain Kinase’ and found numerous changes in resonances in both lobes of CaM. They judged their results consistent with the model of Persechini and Kretsinger [28, 291 and proposed that the bending involves changes in conformation of residues 75 through 81. Lime et al. [56] determined the calcium affinity of CaM and of its two tryptic fragments, l-77 and 78-148 over a range of salt concentrations using the chromophoric chelator 5,5’-BrzBAPTA (55’~dibromo-1,2-bis(2-aminophenoxy) ethane-N,N,N’,N’tetraacetic acid). ‘These measurements indicate that the separated globular domains retain the Ca2’ binding properties that they have in the intact molecule. The Ca2’ affinity is 6-fold higher for the C-tedal domain than for the N-tetinal domain.’ ‘Positive co-opcrativity of Ca2’ binding is observed within each globular domain at all ionic strengths. No interaction is observed between the globular domains.’ Campbell and Sykes [573 examined the interaction of the inhibitory dodecapeptide, Na-acetyl ~o~GKFKBPPLRBVR~IS, of troponin I with intact TnC by the 2-D NMB Nuclear Overhauser Effect Upon binding to TnC the peptide assumes a helical conformation with a bend in the middle to accommodate the two Pros thereby

314

forming a hydrophobic pocket consisting of the side chains of Val, Phe, Pro and Leu. They state that both ‘. . . hydrophobic and electrostatic forces...are involved’; and add that ‘One could envisage binding of the [amphiphilic] TnI peptide to the C-terminal domain of TnC-Ca(II)4 with a much weaker interaction of a second peptide with the N-terminal domain.’ They do not propose a bending of the central helix upon binding the inhibitory peptide. Rao et al. [58] showed by CD and by fluorescence spectroscopy that one equivalent of either &or al-purothionin (W’f) binds strongly to CaM. crPT contains two nearly antiparallel cl-he&s whose conformations are stabilized by (three) disulfide bonds; its conformation is assumed to be very similar when complexed with CaM as when in the crystal structum. Their model of the CaM*aPT complex is similar to that proposed by Persechini and Kretsinger [28,29].

Future research Since cahnodulin is essential to so many cell

functions involving calcium as a second messenger, it is appropriate that it is the topic of thousands of research papers. One should, though, appreciate that 29 distinct subfamilies of EI-hand homolog proteins have been identified (fable 1). Twenty-two of these contain four RF-hand domains. Nakayama et al. [59] proposed that eight - cahnodulin, troponin C, essential light chain of myosin, regulatory light chain of myosin, Call, squidulin, caltractia, and the four EF-hand part of cakiumdependent protein kinase - am congruent That is, certainly Cah& TnC, ELC and RLC and probably the other four, all evolved from a common four EF-hand domain precursor. The linkers of all eight either contain proline or are predicted to have a region with weak inclination to helix formation. One might anticipate similar functions and characteristics associated with their linkers. In contrast the other 14 four domain homologs am not congment Their linkers evolved by different gene splicing, tramlocation, and fusion events. As caution is appropriate when predicting characteristics of the linkers of the congruent eight, so abstinence is suggested for the other 14. Given the past two decades’ revelations about

CEILCALCIUM

calmodulin and troponin C, one should not accept the flexible tether model as explaining the very fundamental outstanding questions. To what extent, if any, does the binding of calcium or magnesium to domains 3 and 4 alter the structure of the linkr or of lobe 1,2 in either CaM or ThC? What is the structure or distribution of structures, assumed in solution by the 26 residue central ‘helix’ of CaM or the homologous 29 residue region of TnC, in solution? If the central helix and especially its linker region is more inclined to form an a-helix by its association with the remainder of the molecule, how is this stabilization imparted to the linker? If the linker does sometimes function as a flexible tether, how does the binding of a target shift the equilibrium toward the bent form; can the postulated change be described simply by mass law? Various research groups employing both different and similar techniques have reached contradictory conclusions. Do our existing paradigms provide au adequate power of interpretation, let alone prediction? Recently Raghunathan et al. [60] set out to confirm the predicted structure of desGlu84 calmodulin, that is a linker helix of seven residues with lobe 3,4 rotated loo” and translated 1.5 8, nearer lobe 1,2. The crystal structure reveals that the linker portion of the central helix is bent (Fig. 4). To paraphrase the motto of the Commonwealth of Virginia, Sic semper conjectoribus.

Acknowledgements RHK thanks NSF for grant DMB-8917285. He appreciates the criticisms and suggestions of numerous colleagues as well as access to matexials still in press. R. Macdonald’s literary talents and his preparation of figures were especially helpful.

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THE LINKER OF CALMODULIN-

TO HELIX OR NOT TO HELIX

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Please send reprint requests to : Dr Robert H. Kretsinger, Depaxtment of Biology, University of Virginia, Charlottesvilk, VA 22901, USA