Summary Adenosine-to-inosine RNA editing is crucial for generating molecular diversity, and serves to regulate protein function through recoding of genomic information. Here, we discover editing within CaV1.3 Ca2+ channels, renown for low-voltage Ca2+-influx and neuronal pacemaking. Significantly, editing occurs within the channel’s IQ domain, a calmodulin-binding site mediating inhibitory Ca2+-feedback (CDI) on channels. The editing turns out to require RNA adenosine deaminase ADAR2, whose variable activity could underlie a spatially diverse pattern of CaV1.3 editing seen across the brain. Edited CaV1.3 protein is detected both in brain tissue and within the surface membrane of primary neurons. Functionally, edited CaV1.3 channels exhibit strong reduction of CDI; in particular, neurons within the suprachiasmatic nucleus show diminished CDI, with higher frequencies of repetitive action-potential and calcium-spike activity, in wildtype versus ADAR2 knockout mice. Our study reveals a mechanism for fine-tuning CaV1.3 channel properties in CNS, which likely impacts a broad spectrum of neurobiological functions.

Nonribosomal peptide synthetases (NRPSs) are microbial enzymes that produce a wealth of important natural products by condensing substrates in an assembly line manner. The proper sequence of substrates is obtained by tethering them to phosphopantetheinyl arms of holo carrier proteins (CPs) via a thioester bond. CPs in holo and substrate-loaded forms visit NRPS catalytic domains in a series of transient interactions. A lack of structural information on substrate-loaded carrier proteins has hindered our understanding of NRPS synthesis. Here, we present the first structure of an NRPS aryl carrier protein loaded with its substrate via a native thioester bond, together with the structure of its holo form. We also present the first quantification of NRPS CP backbone dynamics. Our results indicate that prosthetic moieties in both holo and loaded forms are in contact with the protein core, but they also sample states in which they are disordered and extend in solution. We observe that substrate loading induces a large conformational change in the phosphopantetheinyl arm, thereby modulating surfaces accessible for binding to other domains. Our results are discussed in the context of NRPS domain interactions.

Nuclear magnetic resonance (NMR) studies of larger proteins are hampered by difficulties in assigning NMR resonances. Human intervention is typically required to identify NMR signals in 3D spectra, and subsequent procedures depend on the accuracy of this so-called peak picking. We present a method that provides sequential connectivities through correlation maps constructed with covariance NMR, bypassing the need for preliminary peak picking. We introduce two novel techniques to minimize false correlations and merge the information from all original 3D spectra. First, we take spectral derivatives prior to performing covariance to emphasize coincident peak maxima. Second, we multiply covariance maps calculated with different 3D spectra to destroy erroneous sequential correlations. The maps are easy to use and can readily be generated from conventional triple-resonance experiments. Advantages of the method are demonstrated on a 37 kDa nonribosomal peptide synthetase domain subject to spectral overlap.