Limits...
The C-terminal V5 domain of Protein Kinase Cα is intrinsically disordered, with propensity to associate with a membrane mimetic.

Yang Y, Igumenova TI - PLoS ONE (2013)

Bottom Line: Phosphorylation-mimicking mutations do not alter the overall conformation of the polypeptide backbone, as evidenced by the local nature of chemical shift perturbations and the secondary structure propensity scores.Upon micelle binding, V5α acquires a higher propensity to form helical structures at the conserved "NFD" motif and the entire C-terminal third of the domain.The ability of V5α to partition into the hydrophobic micellar environment suggests that it may serve as a membrane anchor during the PKC maturation process.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, Texas A & M University, College Station, Texas, United States of America.

ABSTRACT
The C-terminal V5 domain is one of the most variable domains in Protein Kinase C isoforms (PKCs). V5 confers isoform specificity on its parent enzyme through interactions with isoform-specific adaptor proteins and possibly through specific intra-molecular interactions with other PKC domains. The structural information about V5 domains in solution is sparse. The objective of this work was to determine the conformational preferences of the V5 domain from the α isoform of PKC (V5α) and evaluate its ability to associate with membrane mimetics. We show that V5α and its phosphorylation-mimicking variant, dmV5α, are intrinsically disordered protein domains. Phosphorylation-mimicking mutations do not alter the overall conformation of the polypeptide backbone, as evidenced by the local nature of chemical shift perturbations and the secondary structure propensity scores. However, the population of the "cis-trans" conformer of the Thr(638)-Pro(639)-Pro(640) turn motif, which has been implicated in the down-regulation of PKCα via peptidyl-prolyl isomerase Pin1, increases in dmV5α, along with the conformational flexibility of the region between the turn and hydrophobic motifs. Both wild type and dmV5α associate with micelles made of a zwitterionic detergent, n-dodecylphosphocholine. Upon micelle binding, V5α acquires a higher propensity to form helical structures at the conserved "NFD" motif and the entire C-terminal third of the domain. The ability of V5α to partition into the hydrophobic micellar environment suggests that it may serve as a membrane anchor during the PKC maturation process.

Show MeSH

Related in: MedlinePlus

Conformational preferences and sub-nanosecond dynamics of wtV5α and dmV5α.(A) Overlay of the 15N-1H HSQC spectra of wtV5α (black) and dmV5α (green). The asterisks indicate the mutation sites, Thr638 (TM) and Ser657 (HM). HS673 stands for homoserine lactone, which is the C-terminal residue generated upon CNBr cleavage of the (His)6-tag from V5α. (B) Chemical shift perturbation analysis of the dmV5α-wtV5α pair. Purple vertical lines mark the mutation sites; the NFD motif is shaded. (C) SSP scores plotted as a function of the primary structure. The secondary structure elements of the V5 domain in the structure of the catalytic domain from PKCα (PDB ID 3IW4) are shown for comparison. (D) Comparison of the hetero-nuclear {1H}-15N NOE values obtained for wtV5α (black) and dmV5α (green). The NOE values and their difference are plotted against the V5 primary structure in the top and bottom panels, respectively.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3675085&req=5

pone-0065699-g002: Conformational preferences and sub-nanosecond dynamics of wtV5α and dmV5α.(A) Overlay of the 15N-1H HSQC spectra of wtV5α (black) and dmV5α (green). The asterisks indicate the mutation sites, Thr638 (TM) and Ser657 (HM). HS673 stands for homoserine lactone, which is the C-terminal residue generated upon CNBr cleavage of the (His)6-tag from V5α. (B) Chemical shift perturbation analysis of the dmV5α-wtV5α pair. Purple vertical lines mark the mutation sites; the NFD motif is shaded. (C) SSP scores plotted as a function of the primary structure. The secondary structure elements of the V5 domain in the structure of the catalytic domain from PKCα (PDB ID 3IW4) are shown for comparison. (D) Comparison of the hetero-nuclear {1H}-15N NOE values obtained for wtV5α (black) and dmV5α (green). The NOE values and their difference are plotted against the V5 primary structure in the top and bottom panels, respectively.

Mentions: The 15N-1H hetero-nuclear single-quantum coherence (HSQC) spectra of the uniformly [15N-enriched] V5α variants are superimposed in Figure 2(A). The cross-peaks in the spectra correspond to the amide 15N-1H groups of the protein backbone. The assignment of cross-peaks to the specific residues was carried out using the triple-resonance NMR experiments on the V5α samples uniformly enriched with 15N and 13C. The HSQC spectra of Figure 2(A) are characterized by small chemical shift dispersion in the amide 1H region, which is a spectroscopic signature of intrinsically disordered proteins [48]. To evaluate the influence of phosphorylation-mimicking mutations, we carried out a chemical shift perturbation (CSP) analysis for the dmV5α-wtV5α pair using the 1HN, 15N, 13CO, 13Cα, and 13Cβ chemical shifts. Significant perturbations are observed at the mutation sites, Ser657 and Thr638, due to the change in amino acid identities. In addition, the residues adjacent to the phosphorylation-mimicking mutations experienced significant chemical shift changes. We attribute those to the changes in the local electrostatic environment caused by the introduction of two negative charges at the TM and HM.


The C-terminal V5 domain of Protein Kinase Cα is intrinsically disordered, with propensity to associate with a membrane mimetic.

Yang Y, Igumenova TI - PLoS ONE (2013)

Conformational preferences and sub-nanosecond dynamics of wtV5α and dmV5α.(A) Overlay of the 15N-1H HSQC spectra of wtV5α (black) and dmV5α (green). The asterisks indicate the mutation sites, Thr638 (TM) and Ser657 (HM). HS673 stands for homoserine lactone, which is the C-terminal residue generated upon CNBr cleavage of the (His)6-tag from V5α. (B) Chemical shift perturbation analysis of the dmV5α-wtV5α pair. Purple vertical lines mark the mutation sites; the NFD motif is shaded. (C) SSP scores plotted as a function of the primary structure. The secondary structure elements of the V5 domain in the structure of the catalytic domain from PKCα (PDB ID 3IW4) are shown for comparison. (D) Comparison of the hetero-nuclear {1H}-15N NOE values obtained for wtV5α (black) and dmV5α (green). The NOE values and their difference are plotted against the V5 primary structure in the top and bottom panels, respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3675085&req=5

pone-0065699-g002: Conformational preferences and sub-nanosecond dynamics of wtV5α and dmV5α.(A) Overlay of the 15N-1H HSQC spectra of wtV5α (black) and dmV5α (green). The asterisks indicate the mutation sites, Thr638 (TM) and Ser657 (HM). HS673 stands for homoserine lactone, which is the C-terminal residue generated upon CNBr cleavage of the (His)6-tag from V5α. (B) Chemical shift perturbation analysis of the dmV5α-wtV5α pair. Purple vertical lines mark the mutation sites; the NFD motif is shaded. (C) SSP scores plotted as a function of the primary structure. The secondary structure elements of the V5 domain in the structure of the catalytic domain from PKCα (PDB ID 3IW4) are shown for comparison. (D) Comparison of the hetero-nuclear {1H}-15N NOE values obtained for wtV5α (black) and dmV5α (green). The NOE values and their difference are plotted against the V5 primary structure in the top and bottom panels, respectively.
Mentions: The 15N-1H hetero-nuclear single-quantum coherence (HSQC) spectra of the uniformly [15N-enriched] V5α variants are superimposed in Figure 2(A). The cross-peaks in the spectra correspond to the amide 15N-1H groups of the protein backbone. The assignment of cross-peaks to the specific residues was carried out using the triple-resonance NMR experiments on the V5α samples uniformly enriched with 15N and 13C. The HSQC spectra of Figure 2(A) are characterized by small chemical shift dispersion in the amide 1H region, which is a spectroscopic signature of intrinsically disordered proteins [48]. To evaluate the influence of phosphorylation-mimicking mutations, we carried out a chemical shift perturbation (CSP) analysis for the dmV5α-wtV5α pair using the 1HN, 15N, 13CO, 13Cα, and 13Cβ chemical shifts. Significant perturbations are observed at the mutation sites, Ser657 and Thr638, due to the change in amino acid identities. In addition, the residues adjacent to the phosphorylation-mimicking mutations experienced significant chemical shift changes. We attribute those to the changes in the local electrostatic environment caused by the introduction of two negative charges at the TM and HM.

Bottom Line: Phosphorylation-mimicking mutations do not alter the overall conformation of the polypeptide backbone, as evidenced by the local nature of chemical shift perturbations and the secondary structure propensity scores.Upon micelle binding, V5α acquires a higher propensity to form helical structures at the conserved "NFD" motif and the entire C-terminal third of the domain.The ability of V5α to partition into the hydrophobic micellar environment suggests that it may serve as a membrane anchor during the PKC maturation process.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, Texas A & M University, College Station, Texas, United States of America.

ABSTRACT
The C-terminal V5 domain is one of the most variable domains in Protein Kinase C isoforms (PKCs). V5 confers isoform specificity on its parent enzyme through interactions with isoform-specific adaptor proteins and possibly through specific intra-molecular interactions with other PKC domains. The structural information about V5 domains in solution is sparse. The objective of this work was to determine the conformational preferences of the V5 domain from the α isoform of PKC (V5α) and evaluate its ability to associate with membrane mimetics. We show that V5α and its phosphorylation-mimicking variant, dmV5α, are intrinsically disordered protein domains. Phosphorylation-mimicking mutations do not alter the overall conformation of the polypeptide backbone, as evidenced by the local nature of chemical shift perturbations and the secondary structure propensity scores. However, the population of the "cis-trans" conformer of the Thr(638)-Pro(639)-Pro(640) turn motif, which has been implicated in the down-regulation of PKCα via peptidyl-prolyl isomerase Pin1, increases in dmV5α, along with the conformational flexibility of the region between the turn and hydrophobic motifs. Both wild type and dmV5α associate with micelles made of a zwitterionic detergent, n-dodecylphosphocholine. Upon micelle binding, V5α acquires a higher propensity to form helical structures at the conserved "NFD" motif and the entire C-terminal third of the domain. The ability of V5α to partition into the hydrophobic micellar environment suggests that it may serve as a membrane anchor during the PKC maturation process.

Show MeSH
Related in: MedlinePlus