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


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

Veerendra Kumar et al. Sci Rep.


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.


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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