1. 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 (April 2018)
DOI: /j.bpj
Copyright © 2017 Biophysical Society Terms and Conditions

2. 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  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

3. 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  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

4. 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  , DOI: ( /j.bpj )
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5. 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  , DOI: ( /j.bpj )
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6. 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  , DOI: ( /j.bpj )
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7. 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  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

8. 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  , DOI: ( /j.bpj )
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9. 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  , DOI: ( /j.bpj )
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