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[摘要]：PEP-19 (Purkinje cell protein 4) is an intrinsically disordered protein with an IQ calmodulin (CaM) binding motif. Expression of PEP-19 was recently shown to protect cells from apoptosis and cell death due to Ca2+ overload. Our initial studies showed that,PEP-19 causes novel and dramatic increases in the rates of association of Ca2+ with and dissociation of Ca2+ from the C-domain of CaM. The goal of this work was to study interactions between the C-domain of CaM (C-CaM) and PEP-19 by solution nuclear magnetic resonance (NMR) to identify mechanisms by which PEP-19 regulates binding of Ca2+ to CaM. Our results show that PEP-19 causes a greater structural change in apo C-CaM than in Ca2+-C-CaM, and that the first Ca2+ binds preferentially to site IV in the presence of PEP-19 with exchange characteristics that are consistent with a decrease in Ca2+ binding cooperativity. Relatively weak binding of PEP-19 has distinct effects on chemical and conformational exchange on the microsecond to millisecond time scale. In apo C-CaM, PEP-19 binding causes a redistribution of residues that experience conformational exchange, leading to an increase in the number of residues around Ca2+ binding site IV that undergo conformational exchange on the microsecond to millisecond time scale. This appears to be caused by an allosteric effect because these residues are not localized to the PEP-19 binding site. In contrast, PEP-19 increases the number of residues that exhibit conformational exchange in Ca2+-C-CaM. These residues are primarily localized to the PEP-19 binding site but also include Asp93 in site III. These results provide working models for the role of protein dynamics in the regulation of binding of Ca2+ to CaM by PEP-19. |
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