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Shields, D., & Blobel, G. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 2059-2063. Wetmore, A. (1960) Smithson. M i x . Collect. 139, 1-37. White, T . J., Mross, G . A., Osserman, E. F., & Wilson, A. C. (1977) Biochemistry 16, 1430-1436.
H A I E C H ET A L .
White, T. J., Ibrahimi, I. M., & Wilson, A. C. (1978) Nature (London) 274, 92-94. Wilson, A. C., & Prager, E. M . (1974) in Lysozyme (Osserman, E. F., Canfield, R. E., & Beychok, S . , Eds.) pp 127-1 41, Academic Press, New York.
Magnesium and Calcium Binding to Parvalbumins: Evidence for Differences between Parvalbumins and an Explanation of Their Relaxing Function+ Jacques Haiech, Jean Derancourt, Jean-Francois PechEre, and Jacques G. Demaille*
ABSTRACT:
The calcium- and magnesium-binding properties of four parvalbumins, two from each genetic lineage a and 8, have been studied by equilibrium dialysis, flow dialysis, and ultraviolet differential spectroscopy. In the absence of calcium, a-parvalbumins from rabbit pZ = 5.55 and from frog p l = 4.88 and P-parvalbumin from frog p l = 4.50 bind two Mg2+ ions with high affinity (& = 16, 21, and 27 WM,respectively) at two equivalent and independent sites. Magnesium binding to the ion-free proteins induces conformational changes a t the level of the hydrophobic core and of the AB loop, which can be monitored by UV differential spectroscopy. Absorbance differences increase linearly with the molar ratio up to 2 mol of Mg2+/mol of parvalbumin. The affinity of parvalbumins for Ca2+ decreases in the presence of Mg2+ according to a simple competition for the same sites. In the absence of Mg2+, two Ca2+ions are bound at equivalent and independent sites with a calculated Kd = 6.6, 7.8, and 2.2 nM for rabbit, frog p l = 4.88, and frog p l = 4.50 parvalbumins, respectively. Substitution of Mg2+ by CaZ+ions induces structural changes that are especially visible at the level of the hydrophobic core and of the AB loop, but the overall structure is similar in proteins that bind either Ca2+or Mg2+. Therefore, these three parvalbumins exhibit two independent and equivalent highaffinity Ca2+-Mg2+ sites. In contrast, when studied by the
above techniques, P-parvalbumin from hake exhibits two nonequivalent high-affinity Ca2+-Mg2+sites. Since no more than 1.5 Mg2+- or Ca2+-binding sites could be found, the protein may have lost part of its Ca2+-binding capacity upon removal of divalent metals. Hake parvalbumin was shown to bind 2 Ca2+ ions at independent sites with Kd, = 3-5 n M and Kdz 2 17 nM, when conformational changes induced by addition of EGTA were followed by UV differential spectroscopy. Binding of EGTA to parvalbumin = 35 mM) was also demonstrated by this technique. @-ParValbumins appear to bind Ca2+ions more strongly than a-parvalbumins. The Ca2+-Mg2+ sites of parvalbumins exhibit either a lysyl residue (CD sites) or a glycyl residue (EF sites) between the Y and Z coordination sites. There is, therefore, no correlation between the nature of the residue in this position and the class of site. Parvalbumins bind 2 Mg2+ions in resting muscle. The delay in calcium binding resulting from the dissociation of bound Mg2+ explains why Ca2+ can trigger contraction. Relaxation occurs when Ca2+is displaced from the low-affinity sites of troponin C to the high-affinity Ca2+-Mg2+ sites of parvalbumins. More generally, low-affinity Ca2+-specificsites. which pick up Ca2+ with diffusion-limited kinetics, are "triggering sites", whereas high-affinity Ca2+-Mg2+ sites are "relaxing sites".
E r v a l b u m i n s are low M , (ca. 12000) acidic proteins present in abundance in the sarcoplasm of vertebrate fast skeletal muscles (PechEre et al., 1973; Blum et al., 1977; Pechbre, 1977). They belong to two evolutionarily distinct lineages a and /3, which were recognized in phylogenetic trees built by the maximum parsimony method (Goodman & PechEre, 1977) and confirmed by the absence of immunological cross-reactions between a and 8 proteins (Demaille et al., 1974). The knowledge of a number of primary structures (see Goodman & Pechbre, 1977, for a review) and of the tertiary structure (Moews & Kretsinger, I975a) of parvalbumins is in sharp contrast with the fact that their biological function is as yet
poorly understood. There is some evidence, however (PechEre, 1977), that this function is related to their calcium-binding properties. By using the Chelex partition technique, parvalbumins were shown (Benzonana et al., 1972) to bind 2 mol M in the presence of of Ca2+/mol with a Kd = 2 X physiological levels of Mg2+, Le., 2 mM. They are mostly found in fast muscle and nervous tissue (Baron et al., 1975), which both possess Ca2+ sequestration devices such as sarcoplasmic reticulum, allowing fast triggering and release (Hasselbach, 1978; Blaustein et al., 1978). It was thus recently proposed that parvalbumins play the role of a soluble relaxing factor, capable of removing Ca2+from the myofibrillar troponin C before being themselves deionized by the sarcoplasmic reticulum Ca2+-Mg2+-ATPase (PechEre et al., 1977; PechEre, 1977). This hypothesis has received experimental support from kinetic experiments conducted on myofibrils (Pechbre et al., 1977) as well as from the ability of sarcoplasmic reticulum vesicles to remove Ca2+ from parvalbumins (Blum et al., 1977; Gerday & Gillis, 1976). Parvalbumins must therefore exist in resting muscle in the Ca2+-free form. This raises the question as to how calcium ions can reach troponin C through the parvalbumin "barrier"
'From the Centre de Recherches de Biochimie Macromoldculaire, CNRS, BP 5051, 34033 Montpellier Cedex, France. Receiued December 20, 1978. This is Contribution No. 168. This work was supported in part by grants from CNRS, ATP Structure primaire et tridimensionnelle des protdines, and Fondation pour la Recherche Mddicale Francaise to J.-F.P. and by grants to J.G.D. from INSERM (CRL 78.4.086.1 and ATP 63.78.95 Biologie et pharmacologie moleculaire et cellulaire de la fibre cardiaque normale et pathologique), from DGRST (ACC Pharmacobiochimie des polypeptides and ACC Biologie et fonction du myocarde), and from NATO.
0006-2960/79/0418-2752$01 .OO/O
t 2 1979 American Chemical Society
MG(II) AND CA(II)
at the onset of contraction when Ca2+ is released from the sarcoplasmic reticulum. The concentration of parvalbumin is indeed very high in fast muscle fibers (Blum et al., 1977; Baron et al., 1975) and their affinity for Ca2+is ca. one order of magnitude higher than the one of troponin C (Potter & Gergely, 1975). As a first step toward the understanding of calcium fluxes between the calcium store (sarcoplasmic reticulum), the soluble parvalbumin and the myofibrillar troponin C, Ca2+-free parvalbumins have been shown to bind magnesium (Pechbre, 1977; Potter et al., 1977; Cox et al., 1977), the two metals competing for the same sites. This paper reports the detailed study of Ca2+ and Mg2+ binding to four different parvalbumins, two from a lineage, rabbit Pa p l = 5.55 and frog Pa p l = 4.88, and two from @ lineage, frog Pa PI= 4.50 and hake Pa p l = 4.36. Parvalbumins exhibit two independent Ca2+-MgZ+high-affinity sites, similar to sites I11 and IV of skeletal troponin C (Potter & Gergely, 1975; Leavis et al., 1978). a-and P-parvalbumins exhibit different affinities for Ca2+,and, among the proteins investigated, one of them (hake p l = 4.36) appeared to exhibit nonequivalent sites and to lose part of its Ca2+-bindingcapacity upon removal of divalent cations. Besides this first report of differences in ion-binding properties, a general scheme is presented which accounts for the relaxing properties of parvalbumins and provides a rationale for the existence of two classes (low and high affinity) of Ca2+-bindingsites in proteins. Materials and Methods Materials. All chemicals were reagent grade or the pdrest commercially available. 45CaC12(29.7 Ci/g) was obtained from CEA, Gif sur Yvette. Ultrapure water (Milli-Q instrument from Millipore Corp.) and acid-washed plastic ware were used throughout to minimize calcium contamination. Whenever glassware had to be used, it was washed with 50% nitric acid and extensively rinsed with ultrapure water (pCa > 7). Dialysis tubing was boiled in neutral EGTA' and rinsed before use. Parvalbumins from rabbit (Oryctolagus cuniculus), p I = 5.55, from hake (Merluccius merluccius), PI= 4.36, and from frog (Rana esculenta), p l = 4.88 and 4.50, were prepared as previously described (Pechbre et al., 1971a,b; Capony et al., 1975, 1976). Methods. The protein concentration of stock parvalbumin solutions was determined by amino acid analysis (Analyzer Beckman Multichrom Model 4255) after 24-H hydrolysis in 6 N HCl at 110 OC by using norleucine as internal standard (Moore & Stein, 1963). Dilutions from stock solutions were checked by ultraviolet spectrophotometry (Beckman Acta I11 instrument). Calcium and magnesium were measured by atomic absorption spectrophotometry by using a Varian Model 1150 apparatus. Parvalbumins used in this study were found to contain 2 f 0.2 mol of Ca2+/mol, except for the rabbit protein which, after desalting in the presence of 0.2 M acetic acid, contained only 0.15 mol of Ca2+/mol. Equilibrium-dialysis experiments were carried out on parvalbumins freed from Ca2+(
