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, 106 (4), 865-74

p15PAF Is an Intrinsically Disordered Protein With Nonrandom Structural Preferences at Sites of Interaction With Other Proteins

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p15PAF Is an Intrinsically Disordered Protein With Nonrandom Structural Preferences at Sites of Interaction With Other Proteins

Alfredo De Biasio et al. Biophys J.

Abstract

We present to our knowledge the first structural characterization of the proliferating-cell-nuclear-antigen-associated factor p15(PAF), showing that it is monomeric and intrinsically disordered in solution but has nonrandom conformational preferences at sites of protein-protein interactions. p15(PAF) is a 12 kDa nuclear protein that acts as a regulator of DNA repair during DNA replication. The p15(PAF) gene is overexpressed in several types of human cancer. The nearly complete NMR backbone assignment of p15(PAF) allowed us to measure 86 N-H(N) residual dipolar couplings. Our residual dipolar coupling analysis reveals nonrandom conformational preferences in distinct regions, including the proliferating-cell-nuclear-antigen-interacting protein motif (PIP-box) and the KEN-box (recognized by the ubiquitin ligase that targets p15(PAF) for degradation). In accordance with these findings, analysis of the (15)N R2 relaxation rates shows a relatively reduced mobility for the residues in these regions. The agreement between the experimental small angle x-ray scattering curve of p15(PAF) and that computed from a statistical coil ensemble corrected for the presence of local secondary structural elements further validates our structural model for p15(PAF). The coincidence of these transiently structured regions with protein-protein interaction and posttranslational modification sites suggests a possible role for these structures as molecular recognition elements for p15(PAF).

Figures

Figure 1
Figure 1
Amino acid sequence features and backbone dynamics of p15PAF. Sites of ubiquitylation (Ub) or phosphorylation (Ph) are indicated by arrows. Residues encompassing the conserved D-box, PIP-box, and KEN-box motifs are underlined. The solid line shows the disorder prediction by the metaPrDOS server (left axis) and dotted lines indicate the protein interaction regions predicted by ANCHOR (using a threshold of 0.5). Bars indicate the backbone amide 15N T2 relaxation times at 25°C and 23.4 T (right axis).
Figure 2
Figure 2
p15PAF is monomeric and has little structure in solution. (A) Size-exclusion chromatogram of p15PAF in PBS, pH 7.0, at room temperature (dotted line and left axis), and molar mass derived from MALLS (solid line and right axis). (B) CD spectrum of p15PAF in PBS, pH 7.0, at 25°C. (Inset) Thermal denaturation as measured by changes in the signal at 222 nm.
Figure 3
Figure 3
Residue-specific assignment of the 1H-15N HSQC NMR spectrum of p15PAF at pH 6.3 and 25°C. The pairs of signals from the side-chain amides of asparagine and glutamine residues are arbitrarily labeled ND1/ND2 and NE1/NE2, respectively, without implying a stereospecific assignment. (Inset) Signal from the W61 indole group.
Figure 4
Figure 4
Local nonrandom structures in the conformational ensemble of p15PAF. Comparison of the experimental RDCs (black circles) with those simulated using a random-coil ensemble without any structural bias (blue line) and those calculated including the conformational preferences described in the text (red line). The estimated error in the measured RDCs is ±0.4 Hz (indicated by the error bars). Calculated RDCs are plotted only if the corresponding experimental value was available. Those regions where nonrandom structures were detected are shaded in blue (extended structure), red (helical structure), or green (inverse γ-turn and KEN-box structures). To see this figure in color, go online.
Figure 5
Figure 5
SAXS data and analysis of p15PAF in PBS, pH 7.0, at 25°C. (A) Semilogarithmic representation of SAXS intensity versus momentum transfer (gray circles), and the back-calculated curve derived from the conformational ensemble that best explains the experimental RDCs (solid black line). The residuals of the fitting are shown below (dashed line and gray circles). (B) Kratky plot of the data shown in A.

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