TIBS16-FEBRUARY1991

REVIF, WS INTRACW.lJlJ~JI~ CALCIUM LEVEI~ control a large number of biochemical processes including all cytoplasmic movements that are mediated by actomyosin interaction. In vertebrate striated muscle, calcium binding to troponin C (TN-C) overcomes the inhibitory action of troponin I (TN-I), allowing the muscle cell to contractL In some invertebrate muscles, where TN-C is absent, contraction is activated by direct calcium binding to a light chain present in the myosin headL In smooth muscles and non-muscle cells, calcium binding to calmodulin provokes the phosphorylation of a myosin light chain, filament assembly and contractionL These examples show that distinct regulatory pathways have evolved in different contractile systems. Although these pathways are apparently unrelated, there is a calcium-binding motif, known as an EF-hand, that is preserved in the polypeptides responsible for sensing and buffering intracellular calcium concentration.

Calcium billding induces confoFmational cha lges in muscle legulatoFy proteins

Calcium binding to proteins containing the 'EF-hand' structural motif regulates a variety of biochemical processes including muscle contraction. Techniques such as protein crystallography, site-directed mutagenesis and domain transplantation experiments are being used to unravel the conformational changes induced by calcium binding.

and specificity of some of these proteins being measured, a simple relationship between the amino acid sequence of the loops and their calcium-binding constants has not been established. Sequence analysis of more than 160 binding loops highlights their common features (Tabl~ I) and demonstrates that the loop seq:~ence only partially determines affinity a.nd specificity I°. Although there is a large ~egree of/:onEF-hands servation in the coordinating residues, The EF-hand motif was first there is considerable vari,tion in the described in the crystal structure of remaining positions throughout the parvalbuminz. This motif has now been loop. The relative contributions of each identified in approximately twenty sepposition can now be evaluated using arate groups of proteins~, including site-directed mutants. The results TN-C, calmodulin and the myosin light obtained so far indicate that modifichains. Each motif contains two cations of the conserved residues proa-helices oriented at approximately 90°, duce large changes in calcium linked by a loop with precisely spaced affinityTM. negatively charged residues and a cenExperiments in which the calcium tral glycine. The negatively charged affinities of single binding loops conresidues and a main-chain oxygen form taining variable lengths of the flanking a pentagonal bipyramid that coordia-helices were compared show that nates a calcium or magnesium ion (Fig. only part of the affinity is directly deterI). The structure resembles a right mined by the loop sequence 4'12. The hand with the open thumb and index analysis of fragments of TN-C containfinger representing the two a-helices ing one or more binding loops shows and the closed third finger representing that the context in which the loop is the loop3. The predictive value of the located can be responsible for some of definition of the EF-hand motif in both the properties of the metal-binding sequence and structural terms is shown site t3. These observations, and the fact by the following examples. (I) Synthetic that long range interactions in TN-C can calcium-binding proteins that mimic affect calcium affinity~4, suggest that there will be no simple rule relating Determinantsof affinity and specificity A. C. R. da Silva and E C. Relnaehare at the Despite the sequencing of a large loop sequence and affinity. It is now Departmentode Bioquimica,Institutode number of proteins containing one or clear that properties such as metal Quimica, Universidadede Sao PauloCP more EF-hands, and the calcium affinity affinity and specificity are probably 20.780, CEP01498 Sao PauloSP, Brazil. 53 © 1991,ElsevierSciencePublishersLtd,(UK) 0376-5067/91/$02.00 EF-hands bind calcium with low affinity4 and assume a similar structure in solution s. (2) A helix-loop-helix motif found in human lysozyme was converted into a calcium-binding site when properly spaced coordinating residues were introduced by site-directed mutagenesis 6. (3) A similar approach was used to reconstruct the inactive calcium-binding site found in cardiac TN-C (Ref. 7). Sequence comparisons suggested a high degree of structural similarities among proteins containing EF-hands3. This observation was confirmed by the determination of the crystallographic structures of other members of this family including TN-C and calmodulin s'9. These two proteins have a pair of globular domains linked by a long a-helix. Each globular domain contains two EFhands that share a common hydrophobic core. In some proteins these globular domains may have only one functional metal-binding site. The crystal structure~ of proteins containing a single functional site (such as the myosin light chains) have not been established, but sequence comparisons show that these proteins are probably derived from a two or four EF-hand protein where amino acid insertions, deletions and substitutions have destroyed one or more of the four metal-binding sites.

TIBS 16-FEBRUARY1991

Helix

Loop

... F D M F ... M K D S

Helix

- z ~

Ii i i i:-i I

/",, I~_

A

G

T

L...

K

N

F

F...

~ ---

TN-C (Site 1)

#I

~"~/~ X

X,'. .4

TN-C (Site 4) --Z

%

Figure 1 Segments of the TN-Csequence involved in coordinatingthe metal ion in sites 1 (site 1) and 4 (site 4). The coordinating residues present in the loop are flanked by two e~-helicesforming the EF-hand.The coordinating residues are marked (X, Y, Z, -Y, -X, -Z) and their three-dimensional position in the pentagonal bipyramid is indicated in the drawing. All coordinating groups are side-chains with the exception of position -Y where it is the main-chain oxygen. Both oxygens of the side-chain in position -Z are involved in coordinating the metal ion. (For a detailed discussion of the geometry see Ref. 3.)

determined by an interaction of se- cium-dependent fashion, regulating undergo different conformational quence determinants in the EF-hand with their activity. Troponin C interacts with changes upon metal binding. other secondary structure elements other components of the thin filament To describe the molecular physiolpresent in the protein. This view is in to allow muscle contraction while ogy of these calcium sensors it is necessagreement with the crystallographic calmodulin, on binding calcium, regu- ary to identify the structural changes structure of TN-C and calmodulin in lates a large number of enzymes. It is that occur on metal binding to the EFwhich extensive interactions between expected that these calcium sensors hands and to understand how these the two sites in each domain are are capable of undergoing confor- changes are used to relay the signal to observed s,9. By the use of site-directed mational changes responsible for the other components of the regulatory mutagenesis it may be possible to transmission of information to other system. Myosin regulatory light chains modulate these properties and devise proteins. A second group, which (RLC), calmodulins, TN-C and calgeneral procedures for increasing affin- includes parvalbumins and the 9 kDa bindins have been cloned and ity and specificity. intestinal Ca2÷-binding protein (ICBP or expressed in E. coil Site-directed mutacalbindin D9k) is probably responsible genesis and domain swapping experSensors and buffers for the maintenance of a buffered intra- iments are being used to elucidate In theory, there are at least two dis- cellular calcium concentration. These some of these problems. tinct roles for intracellular calcium- proteins were not shown to be involved binding proteins. Some are sensors of in regulation of metabolic processes. In Transplants of metal binding sites the intracellular calcium levels and contrast to the calcium sensors, these The myosin molecule is composed of include calmodulin and TN-C. These protein buffers may not have to relay two heavy chains and four light chains. interact with other proteins in a cal- signals to other proteins and could The C-terminal portions of the two

Table I. Aminoacids that occur In the calcium-bindingloop of EF-hands X 1

Position in the metal-binding loop -Y 6 7

2

y 3

4

Z 5

8

-X 9

10

11

-Z 12

Asp 98

Lys 28

Asp 73

Gly 51

Asp 56

Gly 89

Phe 16

lie 70

Asp 33

Phe 16

Glu 31

Glu 86

Asn 0.6

Ala 10

Asn 24

Lys 14

Ser 22

Asp 2

Tyr 15

Val 13

Ser 19

Val 12

Asp 27

Asp

Tyr 0.6

Thr 10

Glu 2.5

Ash 9

Asn 17

Asn 2

Lys 13

Leu 9

Glu 14

Ala 10

Ala 10

Val 2

Glu 0.6

Gin 9

Set 0.6

Arg 6

Gly 2

Lys 2

Thr 13

Met 2.5

Thr 11

Glu 10

Lys 8

Asn 1

-

Val 8

-

Ala 5

Gin 0.6

Arg 1

Glu 6

Cys 2

Gly 10

Leu 10

Gin 5

Gin 0.6

9

The five amino acids that occur with higher frequency in each of the 12 positions along the calcium-binding loop of 165 EF-hands.The numbers are percentages of the total amino acids found. X, Y, Z, -Y, -X and -Z indicate the positions that coordinate the metal ion. In position -Y the coordination involves the main-chain oxygen, which explains the higher variability found. A detailed analysis can be found in Ref. 10.

54

TIBS 16-FEBRUARY1991

heavy chains are (x-helical and form the coiled-coil tail of the molecule. The Nterminal half of each heavy chain combines with two light chains [one regulatory light chain (RLC) and one essential light chain (ELC)] to form a globular head where the Ca2+-/Mgz+-, actin- and ATP-binding sites are located. The myosin present in some invertebrates, the scallop myosin being the best characterized, is directly regulated by calcium. In the scallop myosin, the RLC has an inhibitory action on the ATPase activity. Upon calcium binding to the ELC the inhibitory action of the RLC is relieved ~s.In smooth muscle myosin the phosphorylation of the RLC is the primary event that triggers contractionL The function of the RLC in skeletal muscle myosin is not clear, although there is some evidence that it has an inhibitory function ~6. Skeletal muscle RLC presents a high affinity non-specific metal-binding site u, which is believed to be always occupied by metal. The scallop myosin system has been used to assay the regulatory properties of RLCs, since the endogenous RLC can be removed and replaced by an exogenous RLC (Ref. 17). Kendrick-Jones et al. ~8 have used this assay to demonstrate that smooth muscle RLC can regulate the scallop actin-activated myosin ATPase. In the same system, skeletal muscle RLC locks the myosin in the 'off' state, a situation that can only be reversed by the disruption of the RLC metal-binding site, which now locks the myosin in the 'on' statefL The ability of the different RLCs to confer specific regulatory properties to the myosin head was used to dissect the regions of the RLC responsible for these regulatory properties. Hybrid RLCs were constructed using cDNAs from different origins (Fig. 2). The N-terminal domains (containing the metalbinding sites) of the smooth muscle RLC and skeletal muscle RLCs were exchanged |9. The hybrid proteins expressed in E. coli were used to replace the endogenous scallop RLC. It was observed that the type of regulation obtained depends on the origin of the C-terminal domain, which does not contain the metal-binding site. Hybrids containing the smooth muscle C-terminal domain regulated scallop myosin in the same way as smooth muscle RLC, even if they contained a skeletal muscle-derived EF-handR The same is true for the reverse experiment: a metal-binding loop from smooth muscle cannot confer regulatory

Myosin ATPase Activity

:.-cO 0

'~

--Ca2+

,,

I

. . . .

.......

-i-Ca2+

Low

Low

Low

High

Low

High

Low

Low

High

High

...........

SMOOTH RLC

1 SKELETAL/SMOOTH HYBRID

...........

i

....

.....

~ ' ~

TN-C/SKELETAL HYBRID llfll/~f

ll/'~=llllLlllllll/f

lllllf]

Rgule 2 The structure of TN~, skeletal muscle RLC,smooth muscle RLC and the three hybrid RLCs (:Skeletal/Smooth; Smooth/Skeletal and TN-C/Skeletal) are indicated. These RLCs were used to replace the endogenous RLC in scallop myosin. The levels of the actin-activated myosin ATPasein the preserlce and absence of calcium are indicated. (Redrawnfrom Ref. 19 and Silva, A. C. R., Kendrick-Jones,J. and Reinach, F. C., submitted.)

properties to the skeletal C-terminal domain, indicating that the regulatory properties of the protein are determined by the C-terminal domainR To understand further the role of the C- and N-terminal domains in the regulation of the ATPase activity, a hybrid skeletal RLC with a calcium-specific site was constructed (b.~ C. R. Silva, J. Kendrick-Jones and E C. Reinach, unr, ublished). A low-affinity, calciumspecific metal-binding site from Tn-Cz° was transplanted into the skeletal RLC. The transplanted Tn-C site .retained only some of its characteristics. The affinity and specificity were reduced in the RLC context. In these hybrids, in contrast to the hybrids containing the smooth muscle metal-binding site linked to a skeletal muscle C-terminal domain, the C-terminal domain of the skeletal RLC lost its inhibitory action when tested in the scallop myosin system (A. C. R. Silva, J. Kendrick-Jones and E C. Reinach, unpublished). These transplantation experiments show that the properties related to regulatory characteristics of RLC are not

restricted to the sequences surrounding the metal-binding domain or the C-terminal domain, but are the result of specific interactions involving the whole protein. It is therefore expected that the metal-induced conformational change involves a sophisticated interplay between the EF-hand and the remainder of the structure. Similar conclusions were recently obtained when cardiac TN-C domains were transplanted into calmodulin. The resulting chimeras had specific activating properties when tested with different target enzymesZL MetaHnduced coMonnati0nal change Although four calcium-binding proteins have now been crystallized and their structures solved, there are no examples where both the calcium-free and the calcium-bound form of a single protein have been elucidated, in TN-C crystals the two H-terminal metal sites are empty while the two sites in the Cterminal domain have metal bound s. This is probably due to differences in calcium affinity between the two pairs 55

TIBS 1 6 - FEBRUARY1991

TN-C

f + C a 2+ r

-Ca2+

TN-I

C a 2+

C a 2+

Figure 3 The crystal structure of TN-C is shown at the lefts and the predicted model for the calcium bound form is shown at the right23. A cartoon of the TN.I molecule shows the proposed conformational change. On calcium binding there is a separation of the helices of TN-C and the movement of the inhibitory region of TN-I to cover the exposed hydrophobic patch formed in TN-C. The glutamic residues (position 57 and 88 of TN-C) described in the text are indicated. (Redrawn from Ref. 14.)

of sites. The two N-terminal sites, which are responsible for the regulation of muscle contraction, have a lower affinity and higher specificity when compared with the two C-terminal sites 22. Based on the observation that the structures of all EF-hands crystallized in the presence of metals are very similar, a model for the N-terminal sites in the presence of calcium was constructed 23. The basic assumption of this model is that the N-terminal domain assumes a conformation similar to the C-terminal domain when calcium is bound. The basic conformational change predicted by the model is a movement of a helix-linker-helix away from the core of the N-terminal domain and of the long interdomain helix (Fig. 3). This movement opens a cleft and exposes a small hydrophobic patch. Predictions based on this model were recently tested by two separate groups using site-directed mutants of TN-C~4,24. To test the hypothesis that the helix-linker-helix moves away from the 56

interdomain helix on calcium binding, we identified a pair of glutamic residues (E57 and E88) that form a carboxyl-carboxylate interaction at low pH in the crystal (Fig. 3). Mutating each of these residues separately into lysines, we predicted the formation of salt bridges (E57-K88 or K57-E88) between the two helices, which could stabilize the calcium-free state of the protein by increasing the energy necessary for the separation of the helices. This would decrease the calcium affinity of the regulatory sites if calcium binding and the conformational change were energetically coupled. This prediction was confirmed ~4.We have also shown that these mutant TN-Cs, when used to replace the endogenous TN-C of skinned fibers, were capable of regulating muscle contraction and increased the calcium levels necessary to obtain the same level of tension, providing experimental evidence for the helix separation hypothesis TM. The model was also tested by an ingenious experiment where two sep-

arate cysteines, capable of forming a disulfide bridge between a linker sequence and a position in the long helix, were introduced in TN-C by sitedirected mutagenesis in order to block the separation of the helices. This mutant TN-C, when oxidized, had a reduced affinity for calcium and was not capable of regulating acto-myosin activity. When the cysteines were reduced and blocked no change in calcium affinity was observed 24. A second prediction of the model is that a cluster of hydrophobic residues is exposed to the solvent in the calcium-bound state. This unfavorable energetic configuration can in theory be alleviated by replacing these residues with more hydrophilic residues, displacing the equilibrium of the molecule to the calcium-bound state and increasing the calcium affinity of the regulatory sites. Two of these mutants (V45 to T and M48 to A) were shown to have an increased calcium affinity. When incorporated into skinned fibers, they were

TIBS16-FEBRUARY1991 capable of regulating muscle contraction at much lower calcium concentrations, as predicted by the model (A. Herklotz, A. B. Araujo, M. Sorenson and E C. Reinach, unpublished). The longrange effects of these mutations clearly demonstrated that structures outside the EF-hand play an important role in determining the affinity of calciumbinding sites, as suggested by the trans. plantation experiments. These experiments show that the predictions of the model are correct and that this conformational change is responsible for the transmission of information to the other components of the thin filament. It is likely that the model is also applicable to calmodulin, since the sequences and the structures of TN-C and calmodulin are closely related. With calmodulin, the situation may be more complicated, since it recognizes a large number of different proteins 2s and there are indications that changes in the general shape of the molecule are also involved 26.

8 Herzberg, O. and James, M. N. G. (1988) proteins known to contain this motif is J. MoL BioL 203, 761-779 growing, as is the number of biochemi9 Babu, Y. S., Sack, J. S., Greenhough, T. J., cal processes known to be regulated by Bugg, C. E., Means, A. R. and Cook, W. J. (1985) Nature 315, 37-40 these proteins. How are the different B. J., Shaw, G. S. and Sykes, B. D. regulatory mechanisms built around 10 Marsden. (1990) Biochem. Cell Biol. 68, 587-601 the EF-hand motif? Do all EF-hands 11 Reinach, F. C., Nagai, K. and Kendrick-Jones, J. (1986) Nature 322, 80-83 transmit the signal with a similar conformational change? Have all proteins 12 Reid, R. E., Clare, D. M. and Hodges, R. S. (1980) J. Biol. Chem. 255, 3642-3646 that detect the EF-hand conformational 13 Leavis, P. C., Rosenfeld, S. S., Gergely, J., change converged during evolution? If Grabarek, Z. and Drabikowski, W. (1978) J. Biol. Chem. 253, 5452-5459 not, how are they related? The EF-hand K., Sorenson, M., Herzberg, 0., Moult, family of proteins and the associated 14 J.Fujimori, and Reinach, F. C. (1990) Nature 345, biochemical pathways provide a rich 182-184 source to study evolutionary solutions, 15 Kwon, H., Goodwin, E. B., Nyitray, L., Berliner, E., O'NealI-Hennessey, E., Melandri, F. D. and which can only expand over the next Szent-Gy6rgyi, A. G. (1990) Proc. Natl Acad. Sci. few years. USA 87, 4771-4775

Acknowledgements We would like to thank J. KendrickJones, R. H. Kretsinger and M. Whittle for their critical reading ot the manuscript. This work was supported by FAPESP, CNPq, The Muscular Dystrophy Association (USA) and The Rockerfeller Foundation. A.C.R.S. is a pro-doctoral fellow from FAPESP.

Transmittingthe signal If this model is correct, it will be necessary to understand how this conformational change is transmitted to the other components of the troponin complex. Closely associated with TN-Cis troponin I (TN-I), which has an inhibitory function. When TN-C is bound to TN-I the regulatory sites of TN-C have a tenfold increase in their calcium affinity22. This increase can be understood as a stabilization of the calcium-bound form, which is similar to the one found in mutants that lack the hydrophobic residues in the hydrophobic patch. Since it is unlikely that the hydrophobic patch in the troponin complex is exposed to the solvent, we propose that upon calcium binding a segment of TN-I, probably containing the inhibitory region 27, moves into the groove, covering the hydrophobic patch (Fig. 3). A second region of TN-I could also stabilize the calcium-bound state of the two C-terminal sites. The mapping of contact sites between TN-C and TN-I using cross-linkers supports this modeP .~9. This hypothesis explains why the TNC-TN-I complex has a higher affinity for calcium, and allows the formulation of predictions that can be tested with mutants of both TN-I and TN-C. Future pempeutlves Evolutionary aspects of the molecular switch designed around the EF-hand are largely unexplored. The number of

References 1 Adelstein, R. S. and Eisenberg, E. (1980) Annu. Rev. Biochem. 49, 921-956 2 Kretsinger, R. H. and Nockolds, C. E. (1973) J. Biol. Chem. 248, 3313-3326 3 Moncrief, N. D., Kretsinger, R. H. and Goodman, M. (1990) J. Mol. Evol. 30, 522-562 4 Reid, R. E., Gariepy, J., Saund, A. K. and Hedges, R. S. (1981) J. BioL Chem. 256, 2742-2751 5 Marsden, B. J., Hodges, R. S. and Sykes, B. D. (1989) Biochemistry 28, 8839-8847 6 Kuroki R., Taniyama, Y., Seko, C., Nakamura, H., Kikuchi, M. and Ikehara, M. (1989) Proc. Natl Acad. Sci. USA 86, 6903-6907 7 Putkey, J. A., Sweeney, H. L. and Campbell, S. T. (1989) J. BioL Chem. 264,12370-12378

16 Metzger, J. M., Greaser, M. L. and Moss, R. L. (1989) J. Gen. Physiol. 93, 855-883 17 Sellers, J. R., Chantler, P. D. and Szent~y6rgyi, A. G. (1980) J. MoL Biol. 144, 223-245 18 Kendrick-Jones, J., Jakes, R., Tooth, P., Craig, R. and Scho~ey,J. (1990) in Basic Biolot~yof Muscles: A Comparative Approach (Twarong, B. M., Levine, R. J. C. and Dewey, M. M., eds), pp. 255-272, Raven Press 19 Messer, N. G. and Kendrick-Jones, J. J. MoL Biol. (in press) 20 Reinach, F. C. and Karlsson, R. (1988) J. Biol. Chem. 263, 2371-2376 21 George, S. E., VanBerkum, M. F. A., Ono, T., Cook, R., Hanley, R. M., Putkey, J. A. and Means, A. R. (1990) J. BioL Chem. 265, 9228-9235 22 Potter, J. D. and Gergely, J. (1975) J. Biol. Chem. 250, 4628-4633 23 Herzberg, 0., Moult, J. and James, M. N. G. (1986) J. Biol. Chem. 261, 2638-2644 24 Grabarek, Z., Tan, R. Y., Wang, J., Tao, T. and Gergely, J. (1990) Nature 345,132-135 25 O'Neil, K. T. and DeGrado, W. F. (1990) Trends Biochem. Sci. 15, 59-64 26 Persechini, A. and Kretsinger, R. H. (1988) J. Biol. Chem. 263, 12175-12178 27 Eyk, J. E. V. and Hodges, R. S. (1988) J. Biol. Chem. 263, 1726-1732 28 Leszyk, J., Grabarek, Z., Gergely, J. and Collins, J. H. (1990) Biochemistry 29, 299-304 29 Wang, Z., Sarkar, S., Gergely, J. and Tao, T. (1990) J. BioL Chem. 265, 4953-4957

Contribution of articles to TIBS Articles for TIBSare generally invited by the Editors but ideas for Reviews and, in particular, Talking Point or Open Question features are welcome. Prospective authors should send a brief summary, citing key references, to the Staff Editor in Cambridge, or to a member of the Editorial Board, who will provide guidelines on manuscript preparation if the proposal is accepted. The submission of completed articles without prior consultation is strongly discouraged. Since the journal's content is planned in advance, such manuscripts may be rejected primarily because of lack of space. 57