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Structural Basis for the Interaction of Unstructured Neuron Specific Substrates Neuromodulin and Neurogranin With Calmodulin

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Structural Basis for the Interaction of Unstructured Neuron Specific Substrates Neuromodulin and Neurogranin With Calmodulin

Veerendra Kumar et al. Sci Rep.

Abstract

Neuromodulin (Nm) and neurogranin (Ng) are neuron-specific substrates of protein kinase C (PKC). Their interactions with Calmodulin (CaM) are crucial for learning and memory formation in neurons. Here, we report the structure of IQ peptides (24aa) of Nm/Ng complexed with CaM and their functional studies with full-length proteins. Nm/Ng and their respective IQ peptides are intrinsically unstructured; however, upon binding with CaM, IQ motifs adopt a helical conformation. Ser41 (Ser36) of Nm (Ng) is located in a negatively charged pocket in the apo CaM and, when phosphorylated, it will repel Nm/Ng from CaM. These observations explain the mechanism by which PKC-induced Ser phosphorylation blocks the association of Nm/Ng with CaM and interrupts several learning- and memory-associated functions. Moreover, the present study identified Arg as a key CaM interacting residue from Nm/Ng. This residue is crucial for CaM-mediated function, as evidenced by the inability of the Ng mutant (Arg-to-Ala) to potentiate synaptic transmission in CA1 hippocampal neurons.

Figures

Figure 1
Figure 1. Structure-based sequence alignment of CaM binding peptides from different proteins.
Sequence analysis shows a critical aromatic amino acid (green) that anchors the peptide into CaM; a positively charged amino acid (blue) that determines the orientation of the peptide between two lobes of CaM; and hydrophobic amino acids (red) that are involved in binding. The pdb codes are given in parentheses.
Figure 2
Figure 2. Nm/Ng proteins and peptides interacting with CaM.
ITC experiments corresponding to (A) Nm (B) Ng (C) NgIQ and (D) NgR38A titrated against apoCaM. Upper panels show the raw thermogram data; lower panels show the heat release for each injection (binding isotherm). The data have been fitted using a single-site binding model.
Figure 3
Figure 3. Cartoon representations of the structure of (A) apo CaM-(Gly)5-NmIQ and (B) apo CaM-(Gly)5-NgIQ complexes, with CaM (orange), NmIQ (cyan) and NgIQ (magenta).
N and C-termini are labeled. 2Fo-Fc electron density maps of (C) NmIQ and (D) NgIQ peptides. Maps are contoured at a level of 1σ.
Figure 4
Figure 4. Interactions of (A) NmIQ and (B) NgIQ peptides with the C-lobe of CaM.
CaM is shown in surface representation and key side chains of IQ peptides involved in interactions are shown as sticks. The electrostatic surface potential of C-lobes of CaM are shown (negative charge, red; positive charge, blue).
Figure 5
Figure 5. Superposition of the structures of NmIQ (cyan) and NgIQ (magenta) bound to C-lobe of apo CaM (orange).
Side chains of Arg43 of Nm and Arg38 of Ng are shown as sticks.
Figure 6
Figure 6. NgR38A does not enhance AMPAR-mediated synaptic transmission.
Inset, sample traces of evoked AMPAR (left)- and NMDAR(right)-mediated synaptic responses recorded at −60 mV (the peak amplitude) and +40 mV (the amplitude at 60 ms latency, when AMPAR responses are decayed), respectively. Scale bars, 20 pA, 40 ms. Left graph, comparisons of evoked AMPAR-mediated responses from NgR38A-infected and control neurons. Right graph, simultaneous recordings of evoked NMDAR-mediated responses from NgR38A-infected and control neurons.

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