Biochemistry 1994,33, 12800-12806
12800
A Model Structure of the Muscle Protein Complex 4Ca2+.Troponin COTroponin I Derived from Small-Angle Scattering Data: Implications for Regulation? Glenn A. Olah and Jill Trewhella’ Chemical Science and Technology Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545 Received June 14, 1994; Revised Manuscript Received August 17, 1994’
ABSTRACT: We report here a model structure for 4Ca2+.troponin Cdroponin I derived from small-angle X-ray and neutron scattering data using a Monte Carlo modeling method. In this model, troponin I appears as a spiral structure that wraps around 4Ca2+.troponin C which adopts an extended dumbbell conformation similar to that observed in the crystal structures of troponin C. The troponin I spiral has the approximate dimensions of an a-helix and winds through the hydrophobic “cups” in each globular domain of troponin C. The model is consistent with a body of previously published biochemical data on the interactions between troponin C and troponin I, and suggests the molecular mechanism for the Ca2+-sensitiveswitch that regulates the muscle contraction/relaxation cycle involves a signal transmitted via the central spiral region of troponin
I In the sliding filament model of muscle contraction, interdigitating thick and thin filaments move past each other, resulting in contraction and relaxation. The thick filaments are composed of myosin, while the thin filaments are made from a helical assembly of actin monomers with tropomyosin polymerized head to tail in the grooves of the actin helix, and each tropomyosin bound to one troponin. The contractile force is thought to be generated when the myosin heads, S1, cyclically attach and detach from specific sites on the actin monomers, whereby a power stroke, driven by a c t i n 4 1myosin ATPase activity, occurs sometime during the attachment phase of the cycle. Troponin and tropomyosin form a Ca2+-sensitiveswitch which regulates the interactions between myosin and actin [reviewed by Leavis and Gergely (1 984) and Zot and Potter (1987)l. Troponin has three subunits: troponin C (TnC)’ which binds Ca2+,troponin I (TnI) which inhibits the actin-S l-myosin ATPase activity, and troponin T (TnT) which binds troponin to tropomyosin. The X-ray crystal structures of TnC (Herzberg & James, 1985; Sundaralingam et al., 1985) show a dumbbell-shaped molecule, with C- and N-terminal globular domains connected by an extended 8-9-turn solvent-exposed a-helix. The C-terminal domain contains two high-affinity Ca2+/Mg2+binding sites (sites I11 and IV), thought to always be occupied in muscle, while the N-terminal domain contains two low-affinity Ca2+-specific sites (sites I and 11) (Potter & Gergely, 1975), which are not occupied in the crystal structure. Comparison of the structurally similar N- and C-terminal domains indicates the effect 7 This work was performed under the auspices of the Department of Energy (Contract W-7405-ENG-36) and supported in part by an appointment of G.A.O. to the Alexander Hollaender Distinguished Postdoctoral Fellowship Program sponsored by DOE/Office of Health and Environmental Research, and administered by the Oak Ridge Institute for Science and Education. It was also supported by National Institutes of Health Grant GM40528 (J.T.) and DOE/OHER Project KP-04-0100-0(J.T.). The neutron data used for this study were obtained using facilities at the Manuel Lujan Jr. Neutron Scattering Center, a national user facility funded by DOE/Office of Basic Energy Sciences,and facilities at the National Institute of Standards Technology, U.S. Department of Commerce, supported by the National Science Foundation under Agreement DMR-9 122444. Abstract published in Aduance ACS Abstracts, October 1, 1994. Abbrevations: d,,,, maximum linear dimension; MLCK, myosin light chain kinase; R,, radius of gyration; R,, radius of gyration of cross section; TnC, troponin C; TnI, troponin I; TnT, troponin T.
0006-2960/94/0433- 12800$04.50/0
of Ca2+binding is to open the “cup”-shaped domain, exposing hydrophobic residues lining the inner surface of the cup (Herzberg et al., 1986). Interactions with these hydrophobic cup regions are thought to be important in the Ca2+-dependent regulatory function. It is Ca2+binding to the two low-affinity sites in the N-terminal domain of TnC which regulates the contractile event by altering the interaction of TnI with actin and releasing the inhibition of myosin attachment to actin. The TnI sequence segment 96-115 is implicated in the regulatory function and is believed to alternately bind to actin or to 4Ca2+.TnC (Syska et al., 1976;Talbot & Hodges, 1981). Recently we completed small-angle X-ray and neutron scattering experiments on 4Ca2+.TnC.TnI and obtained new structural information on the complex in solution. These experiments are reported elsewherein detail (Olah et al., 1994), but briefly the neutron experiment capitalized on the different neutron scattering properties of hydrogen and deuterium by measuring the scattering from a complex of deuterated 4Ca2+-TnCand nondeuterated TnI dissolved in solvents with different Dz0:HzO ratios. We extracted from this “contrast series” the small-angle scattering profiles for each component of the complex. These profiles, combined with structural parameters also determined from the scattering data such as the radius of gyration, R,,the maximum linear dimension, d,,,, the separation of the centers-of-mass of the components, the symmetry of the components, and the relative dispositions of symmetry axes, provide a set of constraints that severely limit the possible models for the complex. While the onedimensional data obtained from small-angle solutionscattering experiments are in general not sufficiently constraining to uniquely define a three-dimensional structure, in the case of the 4Ca2+.TnC-TnI complex the combination of constraints from the scattering data, the previously determined crystal structure of TnC, and known molecular volumes allowed a low-resolution structure to be derived. The nucleosome core particle had a similar set of structural constraints which facilitated the successful modeling of its solution structure using small-angle scattering data, thus revealing the nucleosome protein core surrounded by double-helical DNA in a helical superstructure (Pardon et al., 1975, 1977; Hjelm et al., 1977). This structure was subsequently confirmed by X-ray crystallography experiments (Finch et al., 1977). 0 1994 American Chemical Society
Troponin C/Troponin I Structure To model the structure of 4Ca2+.TnC.TnI, we developed a Monte Carlo method that allowed us to rapidly evaluate possible models for the complex against the scattering data. This method has allowed an exhaustive search of possible models consistent with the starting model constraints. We present here a description of the modeling approach, the results of the model search and refinement, a comparison of the final model with published biochemical data, and a brief discussion of the implications of the model for regulation. Since highresolution structural data on the 4Ca2+.TnC-TnI complex are not currently available, the low-resolution model derived from the scattering data will serve as an important guide for experiments aimed at further delineating the structure/ function relationship for this key Ca*+-sensitive molecular switch.
MATERIALS AND METHODS Starting Model Parameters. Table 1 summarizes the structural parameters derived from the X-ray/neutron scattering experiments, while Figure 1 shows thederived scattering profiles [Z(Q) vs Q; Q is the scattering vector defined as (4a sin @/A, where 0 is half the scattering angle and X is the wavelength of the incident radiation] for the overall complex and its individual components (Olah et al., 1994). The inverse Fourier transform of the experimental scattering profiles (Moore, 1980) gives the respective vector length or pair distribution functions, P ( r ) (Figure 2). P ( r ) is the frequency of interatomic vector lengths within the molecular boundary and goes to zero at the maximum linear dimension, dmax. The second moment of P ( r ) gives the radius of gyration, R,, for the scattering particle. The P(r) profiles for each component and for the complex indicate highly asymmetric, elongated structures. A comparison of the P(r) profiles for TnC calculated from the crystal structure and from the neutron experiment shows them to be very similar (Figure 2B). Both profiles show the characteristic peak at =18 A corresponding to the most probable vector length within the individual globular domains, a secondary peak at =45 A corresponding to the most probable interdomain vector length, and a dmax of 72 A. We therefore concluded the structure of 4Ca2+.TnC in the complex is similar to the crystal structure in that the helix connecting the two globular domains fully extended. The P ( r ) profile for TnI indicates an even more asymmetric structure than TnC. The multiple peaks (at 43 and 86 A) and shoulder (at 10-20 A) preclude a simple uniform rod shape (which would give a single peak due to the diameter of the rod and steadily decrease with increasing vector length), but rather indicate a structure with repeating features or domains. Guinier analysis of the very low-Q scattering data gives average radius of gyration of cross section, R,, values for each component and the complex (Table 1) that indicate their long axes are all approximately coincident (Olah et al., 1994). The variation of R, with neutron scattering for the complex indicates the separations of their centers-of-mass are also approximately coincident ( < l o A) and the higher scattering density component (deuterated 4Ca2+.TnC) is more toward the inside of the complex than the lower scattering density component (TnI) (Olah et al., 1994). Additionally, the TnI component is significantly more extended than the TnC, with a d,,, equal to that of the entire complex. The scattering data thus constrain the possible models for the complex to structures in which the TnC component is in an extended configuration, similar to the crystal structure, with TnI encompassing and extending beyond TnC, approximately symmetrically, at both ends of the long axis of
Biochemistry, Vol. 33, No. 43, 1994
12801 ~
Table 1: Comparison of Structural Parameters Derived from Scattering Data’ and the Final “Best Fit” Model 4CaZ+.TnC TnI 4Ca2+.TnC.TnI
experiment model experiment model experiment model
R,@) 23.9 f 0.5 24.1 41.2 f 2.0 40.1 33.0 f 0.5 33.4
Rc(4 10.7 f 1.0 10.3 20.5 f 2.0 20.5 16.2 f 1.5 15.7
4l“A) 72 f 3 73 118 f 4 114 115 f 4 117
Separation (A) of the Centers-of-Mass of 4CaZ+.TnC and TnI experiment
