A novel bivalent interaction mode underlies a non-catalytic mechanism for Pin1-mediated protein kinase C regulation
- Department of Biochemistry & Biophysics, Texas A&M University, United States
- Department of Cell Biology & Genetics, Texas A&M University, United States
Figures
Figure 1 with 5 supplements
Pin1 binds the turn motifs of α and βII PKC isoenzymes through its WW domain.
(A) Multi-modular architecture of conventional PKC isoenzymes, shown along with the amino acid sequence alignment of the C-terminal V5 domains. Turn and hydrophobic motifs are highlighted in purple …
Figure 1—figure supplement 1
NMR-detected binding of the PKCβII turn motif (pTMβII) to full-length Pin1.
The protein concentration was 100 μM, and the pTMβII concentration varied from 0 mM to 1 mM. Most of the affected N-HN resonances are in fast exchange on the NMR chemical shift timescale (Supplementa…
Figure 1—figure supplement 2
NMR-detected binding of the PKCβII turn motif (pTMβII) to the isolated WW domain.
The protein concentration was 100 μM, and the pTMβII concentration varied from 0 mM to 1 mM. Most of the affected N-HN resonances are in fast exchange on the NMR chemical shift timescale (Supplementa…
Figure 1—figure supplement 3
NMR-detected binding of the PKCα turn motif (pTMα) to full-length Pin1.
The protein concentration was 100 μM, and the pTMα concentration varied from 0 mM to 1.8 mM. Most of the affected N-HN resonances are in fast exchange on the NMR chemical shift timescale (Supplementa…
Figure 1—figure supplement 4
NMR-detected binding of the PKCα turn motif (pTMα) to the isolated WW domain.
The protein concentration was 100 μM, and the pTMα concentration varied from 0 mM to 1.8 mM. Most of the affected N-HN resonances are in fast exchange on the NMR chemical shift timescale (Supplementa…
Figure 1—figure supplement 5
Mass spectrometry data for the synthesized peptides derived from the C-terminal V5 regions of PKC isozymes.
(A–I) Mass spectra of the phosphorylated peptides used in this work. Expected molecular masses are: 1328.4 (pTMα); 1826.8 (pHMα); 3421.5 (pV5α); 2689.8 (V5βII-pTM-HM); 1288.3 (pTMβII); 1540.5 …
Figure 2
Pin1 does not appreciably catalyze isomerization of the turn motif in α and βII PKC isoenzymes due to presence of proline at the +1 position.
No exchange cross-peaks characteristic of Pin1-catalyzed pThr-Pro cis-trans isomerization are present in the spectra of turn motif (TM) regions from the PKC βII (A) and α (B) isoforms. This is …
Figure 3 with 2 supplements
Hydrophobic motif interacts with Pin1 via two independent sites.
Comparison of the chemical shift perturbation (CSP) plots obtained at maximum concentrations of pHMβII versus ligand-free proteins for (A) full-length Pin1 and (B) isolated WW and PPIase domains. …
Figure 3—figure supplement 1
NMR-detected binding of hydrophobic motifs from PKCβII (A, pHMβII) and α (B, pHMα) to full-length Pin1.
The protein concentration was 100 μM, and the pHM concentration varied from 0 mM to 1.35 mM (pHMβII), and from 0 mM to 1.8 mM (pHMα). Most of the affected N-HN resonances are in fast exchange on the …
Figure 3—figure supplement 2
Pin1 binds hydrophobic motif of PKCα (pHMα) via two independent sites.
Comparison of the chemical shift perturbation (CSP) plots obtained at maximum concentrations of pHMα used in binding experiments versus ligand-free proteins for (A) full-length Pin1 and (B) isolated …
Figure 4 with 5 supplements
Unidirectional bivalent binding mode of the C-terminal PKCβII region to Pin1.
(A, B) Comparison of the chemical shift perturbation (CSP) plots of Pin1 obtained at maximum concentrations of pV5βII (A) and those of isolated domains, WWiso and PPIaseiso, at maximum …
Figure 4—figure supplement 1
NMR-detected binding of the fully phosphorylated C-term regions pV5βII (A) and pV5α (B) to full-length Pin1.
The protein concentration was 100 μM, and the pV5 concentration varied from 0 μM to 375 μM in case of pV5βII, and from 0 μM to 1530 μM in case of pV5α. The binding kinetics is in the …
Figure 4—figure supplement 2
Unidirectional bivalent binding mode of the C-terminal PKCα region to Pin1.
Comparison of the chemical shift perturbation (CSP) plots of Pin1 obtained at maximum concentrations of pV5α (A) and those of isolated domains, WWiso and PPIaseiso, at maximum concentrations of pTMα …
Figure 4—figure supplement 3
The C-term region of PKCβII binds to the catalytically deficient C113S Pin1 variant.
(A) The C113S Pin1 spectrum (red) shows minimum chemical shift perturbations compared to that of the wild-type (WT) Pin1 (black). (B) The C-term region pV5βII binds to C113S Pin1, evidenced by the …
Figure 4—figure supplement 4
Unidirectional bivalent binding mode of the C-terminal PKCβII region to C113S Pin1.
The chemical shift perturbation (CSP) plot was constructed using the chemical shifts of the apo and pV5βII-bound C113S Pin1. The similarity of CSP patterns between the pV5βII-complexed wild-type (Fig…
Figure 4—figure supplement 5
Thermodynamic benefits of bivalent of Pin1-C-term PKCα interactions.
Kd values for the monovalent interactions of the hydrophobic and turn motifs with isolated Pin1 domains and full-length Pin1 are contrasted with the Kd value for the bivalent Pin1-pV5α interactions. …
Figure 5 with 2 supplements
Phosphorylation state of the conserved C-term motifs defines the C-term interaction mode with Pin1.
(A) Chemical shift perturbation (CSP) pattern of full-length Pin1 due to interactions with the monophosphorylated V5βII-pTM-HM region. The concentrations of Pin1 and V5βII-pTM-HM are 100 μM and 0.46 …
Figure 5—figure supplement 1
Pin1 chemical shift perturbation (CSP) plots of Pin1 obtained at maximum concentrations of V5βII-TM-HM (A), V5α-pTM-HM (B), V5α-TM-pHM (C), and V5α-TM-HM (D).
The protein concentration was 100 μM. Other details are given in Supplementary file 2. TM, turn motif; HM, hydrophobic motif.
Figure 5—figure supplement 2
Representative inter-molecular 1H-1H NOEs between the pTM-WWPin1 (A) and pHM-PPIasePin1 (B).
The assignment labels are color-coded according to the Pin1 domain/C-terminal PKCβII region. (C) NMR ensemble of the Pin1::pV5βII complex (PDB ID 8SG2) reveals a novel Pin1 substrate-binding mode. …
Figure 6 with 5 supplements
Structure of the Pin1::pV5βII complex reveals the bivalent recognition mode.
(A) The lowest-energy NMR structure showing the pV5βII backbone (tan) forms an extensive binding interface with the WW (green) and PPIase (blue) domains of the full-length Pin1. pV5βII is broken …
Figure 6—figure supplement 1
2D LigPlot+ diagram of representative Pin1 interactions with residues 639–650 (‘pTM anchor’ and ‘turn’) of pV5βII.
The lowest-energy structure of the Pin1::pV5βII complex was used to generate the diagram. The contact cutoff for hydrophobic contacts is 4.0 Å. The turn motif is highlighted in yellow.
Figure 6—figure supplement 2
2D LigPlot+ diagram of representative Pin1 interactions with residues 651–661 (‘groove’ and ‘pHM anchor’ segments) of pV5βII.
The lowest-energy structure of the Pin1::pV5βII complex was used to generate the diagram. The contact cutoff for hydrophobic contacts is 4.0 Å. The hydrophobic motif is highlighted in gray.
Figure 6—figure supplement 3
The C-terminal tail in the structure of the PKCβII catalytic domain (PDB ID 2I0E).
(A) The C-terminal V5 domain (cyan) has elevated B-factors and peripherally interacts with the N-lobe of the catalytic domain (gray). (B) The intra-V5 R649-D646 salt bridge and the Q653(N-HN)-(O=C)I6…
Figure 6—figure supplement 4
The C-terminal part of pV5βII is threaded through the PPIase groove.
Space-filling representation showing the threading of pV5βII through the PPIase domain and its anchoring by the phosphate group of pS660. The ‘turn’ segment is shown in licorice representation. The …
Figure 6—figure supplement 5
Comparison of the binding poses between the D-peptide, a potent unnatural peptide inhibitor of Pin1, and the ‘pHM anchor’ segment of pV5βII.
Crystal structure of the monovalent Pin1::D-peptide complex (A) and the lowest-energy NMR structure of the bivalent Pin1::pV5βII complex (B). The phosphate group interacting with the catalytic loop …
Figure 7 with 1 supplement
Regulation of PKCα homeostasis by Pin1 in HEK293T cells.
(A) PKCα protein levels at steady-state are inversely proportional to Pin1 levels. HEK293T cells were transfected with CRISPR/Cas9 plasmids encoding Pin1 guide RNAs and clonal lines were generated. …
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Figure 7—source data 1
Original uncropped immunoblots for data in Figure 7A.
- https://cdn.elifesciences.org/articles/92884/elife-92884-fig7-data1-v1.zip
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Figure 7—source data 2
Original uncropped immunoblots for data in Figure 7C.
- https://cdn.elifesciences.org/articles/92884/elife-92884-fig7-data2-v1.zip
Figure 7—figure supplement 1
PKCα levels in cells with reduced Pin1 function.
(A) HEK293T cells were transfected with mock or Pin1 siRNA as indicated at top, incubated for 72 hr in serum-replete medium, and cell lysates were prepared and analyzed by immunoblotting. Immunoblot …
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Figure 7—figure supplement 1—source data 1
Original uncropped immunoblots for data in Figure 7—figure supplement 1A.
- https://cdn.elifesciences.org/articles/92884/elife-92884-fig7-figsupp1-data1-v1.zip
Figure 8
Non-catalytic role for Pin1.
(A) Non-isomerizable pSer/Thr-Pro-Pro turn motifs separated by <25 residues from the hydrophobic motifs are present in the C-term tails of other AGC kinases, such as AKT and PKN. (B) A possible …
Videos
Video 1
Visualization of the lowest energy structure of the Pin1::pV5βII NMR ensemble.
Additional files
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Supplementary file 1
Properties of the 18 PKC C-terminal-derived peptides used in this study.
All peptides have acetylated N-termini and amidated C-termini. TM and HM stand for the turn and hydrophobic motifs, respectively. The phosphorylated Thr of the TM and phosphorylated Ser of the HM are shown in red. Peptides having ‘V5’ in their name contain both TM and HM. Peptides starting with ‘p’ indicate that the peptide is phosphorylated at either one or both motifs, the latter only for the ‘V5’ peptides.
- https://cdn.elifesciences.org/articles/92884/elife-92884-supp1-v1.docx
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Supplementary file 2
List of binding experiments carried out in this study, with the corresponding values of the dissociation constants Kd obtained from the chemical shift binding curves and/or lineshape analysis.
- https://cdn.elifesciences.org/articles/92884/elife-92884-supp2-v1.docx
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Supplementary file 3
List of the NMR samples and experiments for the structure determination of the complex.
Sample 2* was prepared in the buffer containing 100% D2O.
- https://cdn.elifesciences.org/articles/92884/elife-92884-supp3-v1.docx
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Supplementary file 4
NMR restraints statistics for the CYANA structure calculation.
- https://cdn.elifesciences.org/articles/92884/elife-92884-supp4-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/92884/elife-92884-mdarchecklist1-v1.pdf
