X-Ray Solution Scattering of Squid Heavy Meromyosin: Strengthening the Evidence for an Ancient Compact off State

<div><p>The overall conformations of regulated myosins or heavy meromyosins from chicken/turkey, scallop, tarantula, limulus, and scorpion sources have been studied by a number of techniques, including electron microscopy, sedimentation, and pulsed electron paramagnetic resonance. These studies have indicated that the binding of regulatory ions changes the conformation of the molecule from a compact shape found in the “off” state of the muscle to extended relationships between the tail and independently mobile heads that predominate in the “on” state. Here we strengthen the argument for the generality of this conformational change by using small angle X-ray scattering on heavy meromyosin from squid. Small angle X-ray scattering allows the protein to be visualized in solution under mild and relatively physiological conditions, and squid differs from the other species studied by at least 500 million years of evolution. Analysis of the data indicates that upon addition of Ca²⁺ the radius of gyration increases. Differences in the squid “on” and “off” states are clearly distinguishable as bimodal and unimodal pair distance distribution functions respectively. These observations are consistent with a Ca²⁺-free squid heavy meromyosin that is compact, but which becomes extended when Ca²⁺ is bound. Further, the scattering profile derived from the current model of tarantula heavy meromyosin in the “off” state is in excellent agreement with the measured “off” state scattering profile for squid heavy meromyosin. The previous and current studies together provide significant evidence that regulated myosin's compact off-state conformation is an ancient trait, inherited from a common ancestor during divergent evolution.</p></div

Schematic figure of a myosin dimer showing location of domains.

<p>The structures of regions with variable shades of grey have been determined to atomic resolution.</p

Pair distance distribution function of squid HMM derived from small angle x-ray solution scattering data.

<p>The pair distance distribution function (denoted P(r)) function may be interpreted as the r²-weighted distribution of all possible electron pair distances within the protein. <b>A</b>) In the absence of calcium (EGTA+AMP.PNP), the protein assumes a unimodal P(r) distribution characteristic of a simple compact globular shape (solid line). In the presence of calcium (Ca²⁺+AMP.PNP) the protein gives a more extended bimodal P(r) distribution indicating the presence of two separated domains (dashed line). <b>B</b>) Similar P(r) functions are observed when ADP is used instead of AMP.PNP. For comparison, curves are shown together with the same maximum diameter cutoff (Dmax) of 300 Å.</p

Comparison of model-based and measured SAXS profiles.

<p>The predicted scattering profile is based on an electron microscopy derived off-state tarantula HMM model. Integrated scattering intensity (I in arbitrary units) is given as a function of momentum transfer (q in Å⁻¹, approximately proportional to the scattering angle). The model used is PDB i.d. 3DTP, with the deletion of the 50 N-terminal residues of the RLC unique to this tarantula myosin. The fit of the PDB computed profile to the squid HMM EGTA profile is quite good (slight aggregation is noted at smallest q), and is much better than the fit to the squid HMM Ca2+ data.</p

Probability that a feature is conserved as a function of sample size.

<p>The observation that squid HMM is relatively compact in the off state, together with this same observation in each of the other myosins previously examined (see text), increases the probability that this conformation is conserved in myosin-linked regulation. The formula for probability (P), shown on the y-axis, as a function of the size of a sample for which all members share the conformational feature, was developed by Ji Li (see Methods). Shaded region reflects the approximate number of independent species for which the compact conformation of off-state myosin has been observed (see text).</p

Myosin cleft closure determines the energetics of the actomyosin interaction

Formation of the strong binding interaction between actin and myosin is essential for force generation in muscle and in cytoskeletal motor systems. To clarify the role of the closure of myosin's actin-binding cleft in the actomyosin interaction, we performed rapid kinetic, spectroscopic, and calorimetric experiments and atomic-level energetic calculations on a variety of myosin isoforms for which atomic structures are available. Surprisingly, we found that the endothermic actin-binding profile of vertebrate skeletal muscle myosin subfragment-1 is unique among studied myosins. We show that the diverse propensity of myosins for cleft closure determines different energetic profiles as well as structural and kinetic pathways of actin binding. Depending on the type of myosin, strong actin binding may occur via induced-fit or conformational preselection mechanisms. However, cleft closure does not directly determine the kinetics and affinity of actin binding. We also show that cleft closure is enthalpically unfavorable, reflecting the development of an internal strain within myosin in order to adopt precise steric complementarity to the actin filament. We propose that cleft closure leads to an increase in the torsional strain of myosin's central β-sheet that has been proposed to serve as an allosteric energy-transducing spring during force generation.—Takács, B., O'Neall-Hennessey, E., Hetényi, C., Kardos, J., Szent-Györgyi, A. G., Kovács, M. Myosin cleft closure determines the energetics of the actomyosin interaction