High and low affinity Ca ++ binding sites in troponin-C studied by synchrotron radiation

Abstract

We have studied the structure of calcium binding-sites of Troponin-C using synchrotron radiation at the Frascati storage ring ‘Adone’. We have measured the X-ray absorption spectra of the protein. From the analysis of the X-ray Absorption Near Edge Structures (XANES) [1] we have found direct evidence of different local structures for the low and high affinity Ca ++ sites. Skeletal muscle Troponin-C (TNC) is one of the three subunits of Troponin which provides Ca ++ sensitivity to myosin ATPase: this occurs only when Ca ++, released from sarcoplasmic reticulum, is bound to TNC. TNC belongs to a family of homologous calcium-binding proteins, which includes, among others, parvalbumins and the ubiquitous calcium-dependent regulator [2]. A common structural feature of these proteins is the presence of a number of specific Ca ++-binding domains referred to as EF hands [2]. EF hands were observed first in the crystal structure of carp parralbumin and, thereafter, the presence of four EF hand structure has been predicted in TCN on the basis of the known amino-acid sequence [2]. The coordination of Ca ++ in every EF hand so far studied can be represented by an octahedron [3]. TNC has two classes of Ca ++-binding sites; the first class comprises two high affinity sites for Ca ++ (Kd = 2 × 10 −7 M) which are not fully specific since they also bind Mg ++, though with a much lower affinity (Kd = 10 −4 M); the second class comprises two sites specific for Ca ++ with a Kd of 2 × 10 −5 M [4, 5]. Two preparations were obtained by lyophilizing: first, 0.2 m M TNC in 0.38 m M CaCl 2; second, 0.2 m M TNC in 0.7 m M CaCl 2 and 2 m M MgCl 2. In the first preparation calcium is bound only at the high affinity sites, while in the second both the high and low affinity sites for Ca ++ should be occupied. Our results show that the XANES of the two high affinity sites of TNC in the absence of Mg ++ are very similar to those found in parvalbumin [2], suggesting an octahedral symmetry typical of the EF hand. The XANES of TNC shows different features under more physiological conditions, i.e. in presence of Mg ++ and with all the four Ca ++ sites occupied. The XANES structures are then broader probably because two different types of sites are occupied by Ca ++: the Mg-modified high affinity sites and the low affinity sites. The XANES, of the latter sites are clearly different both for a chemical shift of the transitions to t 1u final states of about 0.4 eV toward lower energies and for a different splitting of the structures. The different features of XANES should be associated with a different CaO charge transfer, a variation of the CaO distance and a distortion of octahedral symmetry.