Person:

Kalocsay, Marian

Increased cardiac contractility during fight-or-flight response is caused by b-adrenergic augmentation of CaV1.2 channels. In transgenic murine hearts expressing fully PKA phosphorylation-site-deficient mutant CaV1.2 a1C and b subunits, this regulation persists, implying involvement of extra-channel factors. Here, we identify the mechanism by which b-adrenergic agonists stimulate voltage-gated Ca2+ channels. We expressed ascorbate-peroxidase-conjugated-a1C or -b2B subunits in mouse hearts and used multiplexed, quantitative proteomics to track hundreds of proteins in proximity of CaV1.2. We observed that the Ca2+ channel inhibitor Rad, a monomeric G-protein, is enriched in the CaV1.2 micro-environment but is depleted during b-adrenergic stimulation. PKA-catalyzed phosphorylation of specific Ser residues on Rad decreases its affinity for auxiliary b-subunits and relieves constitutive inhibition of CaV1.2 observed as an increase in channel open probability. Expression of Rad or Rem, a homolog, also imparted PKA-induced stimulation of CaV1.3 and CaV2.2, revealing an evolutionarily conserved mechanism that confers adrenergic-modulation upon voltage-gated Ca2+ channels.

Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) cause severe respiratory diseases in infants and elder adults. Neither a vaccine nor an effective antiviral therapy exists to control RSV or HMPV infections. During viral genome replication and transcription, the tetrameric phosphoprotein P serves as a crucial adaptor between the nucleoprotein-RNA (N-RNA) template and the L protein, which has RNA-dependent RNA polymerase (RdRp), GDP polyribonucleotidyltransferase (PRNTase) and cap-specific methyltransferases (MTases) activities. How P interacts with L and mediates association with the free form of N and with the ribonucleoprotein (RNP) is not clear for HMPV or other prominent human pathogens including measles, Ebola and rabies viruses. Here, we report a cryo-EM reconstruction showing the ring-shaped structure of the polymerase and capping domains of HMPV L, bound with a tetramer of P. The connector and MTase domains are mobile with respect to the core. The putative priming loop important for initiation of RNA synthesis is fully retracted, leaving space in the active-site cavity for RNA elongation. P interacts extensively with the N-terminal region of L, burying more than 4,016 Å2 of molecular surface area in the interface. Two of the four helices forming the coiled-coil tetramerization domain of P, and long C-terminal extensions projecting from these two helices, wrap around the L protein like tentacles. The structural versatility of the four P protomers, which are largely disordered in their free state, demonstrates an example of a “folding-upon-partner-binding” mechanism for carrying-out P adaptor functions. The structure shows that P has the potential to modulate multiple functions of L and should accelerate the design of specific antiviral drugs.