Intrinsically Disordered PEP-19 Confers Unique Dynamic Properties to Apo and Calcium Calmodulin

Abstract

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...

Crystal Structure of Calmodulin Binding Domain of Orai1 in Complex with Ca2+•Calmodulin Displays a Unique Binding Mode

“…Therefore, CaM⅐Orai1-CMBD displays a unique binding mode of CaM to its target peptides, and again shows the flexibility of CaM to bind a large variety of target proteins. In addition to Orai1-CMBD and GAD-CMBD, IQ calmodulin binding peptides from Na V 1.5 (31), Na V 1.2 (32), and PEP-19 (33)(34)(35) bind selectively to the C-lobe of CaM, leaving the N-lobe unoccupied, and thus fall in this group 1-0.…”

Neurogranin Alters the Structure and Calcium Binding Properties of Calmodulin

“…In this case unconstrained tumbling of the tethered N-domain could propagate an increase in dynamics in the C-lobe. Increased CaM dynamics have previously been associated with rapid dissociation of Ca 2ϩ (33,(57)(58)(59). Alternatively the N-lobe of CaM may make contacts with Ng.…”

“…This leads to the hypothesis that Ng and PEP-19 may serve analogous roles in the postsynaptic cell but that they might possess unique properties (like crosstalk with the PKC pathway) that permits cell type-specific tuning of their impact on the Ca 2ϩ /CaM signaling pathway. We focus in this report on a quantitative understanding of the biochemical properties of Ng and its interactions with CaM, and we contrast these findings with published information on the related protein PEP-19 (14,33). Ng (ϳ7.8 kDa) and PEP-19 (ϳ6.8 kDa) are small intrinsically disordered proteins that both contain an IQ motif (34,35) preceded by an acidic region composed largely of Asp or Glu residues.…”

Relative Cosolute Size Influences the Kinetics of Protein-Protein Interactions

“…Wild-type mammalian calmodulin (CaM) (31)(32)(33), T34C/T110C CaM (17), K75C CaM (34), D2C CaM (35,36), and 15 N-isotope-labeled CaM (31,37,38) were expressed and purified as previously described in the cited works. The target peptide representing the CaM recognition motif for CaMKI (AKSKWKQAFNATAVVRHMRKLQ) was purchased from LifeTein (Somerset, NJ).…”