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 Ca 2+ overload. Our initial studies showed that PEP-19 causes novel and dramatic increases in the rates of Ca 2+ association and dissociation from the C-domain of CaM. The goal of this work was to study interactions between C-domain of CaM (C-CaM) and PEP-19 by solution NMR to identify mechanisms by which PEP-19 regulates Ca 2+ binding to CaM. Our results show that PEP-19 causes a greater structural change in apo C-CaM relative to Ca 2+ -CCaM, and that the 1 st Ca 2+ binds preferentially to site IV in the presence of PEP-19 with exchange characteristics that are consistent with a decrease in Ca 2+ binding cooperativity. Relatively weak binding of PEP-19 has distinct effects on chemical/conformational exchange on the μs-ms timescale. In apo C-CaM, PEP-19 binding causes a redistribution of residues that experience conformational exchange, leading to an increase in residues around Ca 2+ binding site IV that undergo conformational exchange on μs-ms timescale. This appears to be due to an allosteric effect since 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 Ca 2+ -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 regulation of Ca 2+ binding to CaM by PEP-19 (Purkinje cell protein 4) is a small 6.7 kDa polypeptide that was initially identified in the central nervous system (CNS), but is now known to be present in a variety of other tissues (for review see (1-5)). PEP-19 has no known intrinsic activity other than binding to calmodulin (CaM) in the presence or absence of Ca 2+ via an IQ CaM binding motif. This gives PEP-19 the potential to exert broad cellular effects as a regulator of CaM signaling. This idea is consistent with a general cytoprotective role for PEP-19 put forth based on the fact that Purkinje cells of the cerebellum and granule-cell neurons in the dentate gyrus, which have high levels of PEP-19, are largely spared from the effects of Alzheimer's Disease, while PEP-19 negative cells are severely affected (6). Conversely, cell types that are most affected by Huntington's disease exhibit a significant loss of expression of PEP-19 (7). Experimental studies also support a cytoprotective role for PEP-19 since expression of PEP-19 greatly inhibits cell death due to apoptosis (8,9), or glutamate-induced Ca 2+ cytotoxicity (9). Interestingly, PEP-19 is greatly increased in human leiomyoma versus matched myometrium (10), and high levels of PEP-19 are found in 58 of 60 NCI-60 tumor cell lines (11), even cells derived from tissues that express low or no PEP-19. This may be related to increased survival potential of transformed cells due to antiapoptotic effects of...

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