cMyBP-C phosphorylation modulates the time-dependent slowing of unloaded shortening in murine skinned myocardium

In myocardium, phosphorylation of cardiac myosin-binding protein-C (cMyBP-C) is thought to modulate the cooperative activation of the thin filament by binding to myosin and/or actin, thereby regulating the probability of cross-bridge binding to actin. At low levels of Ca2+ activation, unloaded shortening velocity (Vo) in permeabilized cardiac muscle is comprised of an initial high-velocity phase and a subsequent low-velocity phase. The velocities in these phases scale with the level of activation, culminating in a single high-velocity phase (Vmax) at saturating Ca2+. To test the idea that cMyBP-C phosphorylation contributes to the activation dependence of Vo, we measured Vo before and following treatment with protein kinase A (PKA) in skinned trabecula isolated from mice expressing either wild-type cMyBP-C (tWT), nonphosphorylatable cMyBP-C (t3SA), or phosphomimetic cMyBP-C (t3SD). During maximal Ca2+ activation, Vmax was monophasic and not significantly different between the three groups. Although biphasic shortening was observed in all three groups at half-maximal activation under control conditions, the high- and low-velocity phases were faster in the t3SD myocardium compared with values obtained in either tWT or t3SA myocardium. Treatment with PKA significantly accelerated both the high- and low-velocity phases in tWT myocardium but had no effect on Vo in either the t3SD or t3SA myocardium. These results can be explained in terms of a model in which the level of cMyBP-C phosphorylation modulates the extent and rate of cooperative spread of myosin binding to actin.

Conserved and divergent features of neuronal CaMKII holoenzyme structure, function, and high-order assembly

SUMMARYNeuronal CaMKII holoenzymes (α- and β-isoforms) enable molecular signal computation underlying learning and memory, but also mediate excitotoxic neuronal death. Here, we provide a comparative analysis of these signaling devices, using single particle EM in combination with biochemical and live-cell imaging studies. In the basal state, both isoforms assembled mainly as 12-mers (but also 14-mers, and even 16-mers for the β-isoform). CaMKIIα and β-isoforms adopted an ensemble of extended activatable states (with average radius of 12.6 versus 16.8 nm, respectively), characterized by multiple transient intra- and inter-holoenzyme interactions associated with distinct functional properties. The extended state of CaMKIIβ allowed EM analysis to directly resolve intra-holoenzyme kinase-domain dimers that could enable the cooperative activation mechanism by calmodulin, which was found for both isoforms. Surprisingly, high-order CaMKII clustering mediated by inter-holoenzyme kinase-domain dimerization was reduced for the β isoform for both basal and excitotoxicity-induced clusters, both in vitro and in neurons.

Dinuclear zinc-catalyzed asymmetric [3+2] cyclization reaction for direct assembly of chiral α-amino-γ-butyrolactones bearing three stereocenters

An efficient enantioselective [3+2] cyclization reaction of α-hydroxy-1-indanones and alkylidene azlactones has been developed with chiral dinuclear zinc catalysts via a Brønsted base and Lewis acid cooperative activation model. This...