Structural Basis of Protein Kinase Cα Regulation by the C-Terminal Tail  Yuan Yang, Chang Shu, Pingwei Li, Tatyana I. Igumenova  Biophysical Journal  Volume 114, Issue 7, Pages 1590-1603 (April 2018) DOI: 10.1016/j.bpj.2017.12.030 Copyright © 2017 Biophysical Society Terms and Conditions

Figure 1 Host protein context and interactions between C2 and V5 domains. (a) Domain sub-structure of conventional PKC isoenzymes and schematic representation of the inactive form. The Ca2+-sensing C2 and C-terminal V5 domains are shown in gray and red, respectively; PS is a pseudo-substrate region. (b) Sequence alignment of C-terminal V5 domains from conventional PKCs. The boxes indicate three conserved motifs, two of which, the HM and the TM, are phosphorylated during enzyme maturation. (c) Expansions of 15N-1H HSQC spectra of Ca2+-bound C2 domain (C2⋅Can, n = 2,3), in the absence (black) and presence of four V5-derived peptides: pHM15, S657E, uHM, and SCB. The changes in crosspeak positions indicate the changes in electronic environment of the backbone N-H groups of C2 due to binding. To see this figure in color, go online. Biophysical Journal 2018 114, 1590-1603DOI: (10.1016/j.bpj.2017.12.030) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 2 The HM interacts with apo and Ca2+-bound C2. (a and b) Expansions of 15N-1H HSQC spectra of [U-15N] apo C2 (a) and C2⋅Can (b), showing the effect of increasing the pHM15 concentration, [pHM15]. The spectra are overlaid and color-coded according to [pHM15]. The N-H groups of C2 whose chemical shift changes as a result of binding are labeled. (c) CSPs, Δ, due to pHM15 binding for all spectrally resolved residues of C2⋅Can (top) and apo C2 (bottom). Residue-specific Δ values were calculated using spectra with zero and 1.5 mM [pHM15] for C2⋅Can (top) and apo C2 (bottom). Asterisks indicate residues that are either prolines or broadened/not spectrally resolved. The insets show CSPs for the indole side-chain amine groups of the four Trp residues. (d) Crystal structure of the C2 domain complexed to three Ca2+ ions, PDB: 3GPE. The CSPs of the Ca2+-complexed C2 domain due to pHM binding are color-coded and mapped onto the structure. Ca2+ ions are shown as green spheres. To see this figure in color, go online. Biophysical Journal 2018 114, 1590-1603DOI: (10.1016/j.bpj.2017.12.030) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 3 Synergistic action of Ca2+ and the HM. (a) Representative curves for pHM15 binding to apo C2 (open circles) and C2⋅Can (solid circles). (b) Representative curves for Ca2+ binding to apo C2 (open triangles) and C2⋅pHM15 (solid triangles). In both cases, the affinity of one ligand is enhanced in the presence of the other. To see this figure in color, go online. Biophysical Journal 2018 114, 1590-1603DOI: (10.1016/j.bpj.2017.12.030) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 4 (a) Backbone superposition of the C2⋅(Ca2+)2 NMR ensemble (PDB: 2NCE, gray) in the pHM complex with the crystal structures of the apo (PDB: 3RDJ, blue) and Ca2+-complexed C2 (PDB: 1DSY, orange). (b) Expansions of the 15N-1H HSQC spectra showing Ln3+-induced pseudocontact shifts of C2⋅Ln3+ in the absence and presence of a 15-fold molar excess of pHM. (c) The corresponding correlation plot of the PCS values. The inset shows the PCS correlation data for the LRC residues; the LRC is the interaction site of pHM with C2. The solid lines are x = y, whereas dashed lines are x = y ± 0.15; ±0.15 is a typical uncertainty of PCS measurements. To see this figure in color, go online. Biophysical Journal 2018 114, 1590-1603DOI: (10.1016/j.bpj.2017.12.030) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 5 The C2-pHM interface is stabilized by electrostatic and aromatic interactions. (a and b) Twenty top-ranking structures of the C2⋅(Ca2+)2⋅pHM complex, with pHM and LRC/CMBL3 highlighted in red and purple, respectively. Ca2+ ions are represented as green spheres. (c and d) The interface of the complex is stabilized by aromatic (c) and electrostatic (d) interactions. The charged/polar and aromatic side chains of C2 are shown in green and dark gray, respectively; all pHM side chains are colored gold. The interacting residues are labeled in red (pHM) and black (C2). To see this figure in color, go online. Biophysical Journal 2018 114, 1590-1603DOI: (10.1016/j.bpj.2017.12.030) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 6 Mutations at the C2-V5 interface affect the conformation of PKCα. (a) Identity of PKCα variants. (b) Fluorescence emission spectra of the wild-type PKCα and selected variants. The schematic diagram depicts the PKCα construct used for in vitro fluorescence measurements. (c) FRET ratios (mean ± SE) of all variants obtained from at least three independent measurements. The grouping is the same as in (a). To see this figure in color, go online. Biophysical Journal 2018 114, 1590-1603DOI: (10.1016/j.bpj.2017.12.030) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 7 Externally added pHM activates PKCα in the absence of PKC agonists and in the presence of only the C1 agonist, PDBu. The activity data are shown as the mean ± SD. The data obtained with a full complement of effectors, Ca2+, and LUVs comprising POPC/POPS/DAG 65:30:5 (mol %), are shown on the same plot for comparison. To see this figure in color, go online. Biophysical Journal 2018 114, 1590-1603DOI: (10.1016/j.bpj.2017.12.030) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 8 Proposed sensitizing and inhibitory roles of the C2-V5 interaction in PKCα. (a) Relative placement of the pHM and PtdIns(4,5)P2 ligands, obtained from the backbone superposition of the C2 complexes: C2⋅(Ca2+)2⋅pHM and PDB: 3GPE. The top-scoring C2⋅(Ca2+)2⋅pHM structure is colored gray; the backbone representation of PDB: 3GPE is omitted for clarity. The headgroup of PtdIns(4,5)P2 is shown in sphere representation. (b) Modulation of C2 electrostatic potential by Ca2+ and pHM. All electrostatic maps were calculated for apo C2, C2⋅pHM, and C2⋅(Ca2+)2⋅pHM using the structure of the complex obtained in this work. The color coding corresponds to the electrostatic potential ranging from −6kbT/e (red) to +6kbT/e (blue). Binding of pHM to the LRC decreases the electrostatic potential of C2 and thereby increases its affinity to Ca2+. (c) C2 domain crystal contacts in PKCβII (PDB: 3PFQ) that involve electrostatic interactions between C2 (K205) and V5 (E655 and D649). The relevance of this interaction for PKCβII auto-inhibition was established in a previous mutagenesis study (21). Other kinase residues that form an interface with the C2 domain are colored blue. (d) The proposed role of C2-V5 interactions in the first step of the PKCα activation process. To see this figure in color, go online. Biophysical Journal 2018 114, 1590-1603DOI: (10.1016/j.bpj.2017.12.030) Copyright © 2017 Biophysical Society Terms and Conditions