| Popis: |
Multidomain proteins reside at the heart of cellular signaling, where they can act as multivalent scaffolds for the assembly of protein complexes and as ligand-dependent effectors of signal transduction. Despite their prevalence, it is unclear how multidomain proteins simultaneously interact with multiple clients, and how each domain contributes to the overall activity of the protein. Challenges, including the large size and low solubility have largely limited studies to smaller truncated forms of these proteins. Although more tractable, the relevance of studies on truncated proteins to our understanding of the full-length protein is questionable. Otoferlin, which consists of 6 consecutive C2 domains, serves an example. Expressed almost exclusively in vertebrate sensory hair cells, otoferlin is a 240 kDa transmembrane protein hypothesized to act as the calcium sensor for neurotransmitter release at hair cell ribbon synapses during the encoding of sound. While studies have suggested that some C2 domains of otoferlin may associate with SNARE proteins and the voltage gated calcium channel CaV1.3, no study has quantitatively characterized multivalent and concomitant binding properties of multi-domain or full-length otoferlin owing to its size and solubility. Thus, it remains unclear whether otoferlin can bind these proteins simultaneously and more generally. To address this limitation, we have developed a generally applicable real-time single-molecule colocalization binding titration assay which in combination with single-molecule photobleaching can resolve the binding multivalencies, stoichiometries, and binding affinities of large full-length multidomain proteins enriched from mammalian cell culture. Using this methodology, we conclude that one otoferlin molecule may bind both up to 4 Cav1.3 and 4 SNARE proteins simultaneously, and that physiologically relevant calcium concentrations alter the stoichiometry and affinity. Our results motivate a model in which otoferlin acts as a scaffolding/priming protein that localizes SNARE proteins and the calcium channel in close proximity to synchronize calcium influx and exocytosis. |
