Katti, Sachin S (2020-07). Deciphering The Molecular Determinants of Membrane Association By Phospholipid-Binding Domains. Doctoral Dissertation. Thesis uri icon

abstract

  • Assemblies of enzymes and adaptor complexes at the membranes relay the external signals to the cellular machineries in order to induce physiological responses. As a result, controlled and transient translocation of signaling complexes to the membranes is indispensable to signal transduction. Nature has designed a limited number of specialized phospholipid-binding domains to achieve such translocation. These domains are ubiquitous among modular protein complexes and respond to specific effectors and second messengers. Among these membrane-targeting modules, the diacylglycerol-binding C1 domains and Ca2+-sensitive C2 domains represent some of the most abundant classes in eukaryotes. These domains were first identified on protein kinase C (PKC), the membrane-activated kinase of AGC family that regulates cell proliferation, survival and apoptosis. The ligand/agonist preferences of most PKC-type C1 and C2 domains are known, yet their respective mechanisms of membrane association are not completely understood. Deciphering the molecular determinants that govern the membrane interactions of these domains is essential to fully realize how they modulate the host signaling complexes. That is the primary goal of the work presented here. Towards this goal, we systematically studied the tandem C1 domains (C1A and C1B) of PKCd isoform and tandem C2 domains (C2A and C2B) of synaptic Ca2+ sensor protein, Synaptotagmin 1 (Syt1). The novel structural features along with the agonist-binding studies reported here reveal that C1A domain has a weak dependence on diacylglycerol but sensitive towards tumor-promoting phorbol esters and PtdIns(4,5)P2. These findings combined with the existing knowledge provide a mechanistic model of diacylglycerol recognition by C1 domains. On the other hand, our work on C1B domain provides unprecedented atomic resolution insight into the agonist-induced membrane insertion of C1 domains. The determinants of membrane association for Syt1 C2 domains were studies using xenobiotic divalent cations, Cd2+ and Pb2+. These metal ions provided unique advantage over Ca2+ due to their high affinities. Using them, we were able to identify the changes in C2 domains necessary for effective membrane association. Together, the results presented here contribute significantly to our understanding of these conserved domains and their roles in signal transduction.

publication date

  • August 2020
  • August 2020
  • July 2020
  • August 2020
  • August 2020
  • August 2020
  • August 2020