1. Volume 14, Issue 2, Pages 309-319 (February 2006)
Molecular Basis for Phosphorylation-Dependent, PEST-Mediated Protein Turnover 
Maria M. García-Alai, Mariana Gallo, Marcelo Salame, Diana E. Wetzler, Alison A. McBride, Maurizio Paci, Daniel O. Cicero, Gonzalo de Prat-Gay 
Structure 
Volume 14, Issue 2, Pages (February 2006)
DOI: /j.str
Copyright © 2006 Elsevier Ltd Terms and Conditions

2. Figure 1
Domain Arrangement of the BPV1 E2 Protein and Localization of the PEST Motif
Full-length E2 consists of two structural domains, the transactivation domain (left) and the DNA binding and dimerization domain (right) connected by a nonconserved region defined as the “hinge,” corresponding to residues 200–330. The peptide E2-PEST (283–311) contains the PEST switch region (underlined), poly-glutamic regions (bold), and serines 298 and 301 (∗) that can be phosphorylated by CKI and CKII.
Structure  , DOI: ( /j.str )
Copyright © 2006 Elsevier Ltd Terms and Conditions

3. Figure 2
Far-UV Circular Dichroism of the E2-PEST Peptide and the Phosphorylated Forms
(A) E2-PEST at different pHs. Inset: differential spectrum of E2-PEST between pH 8.0 and pH 3.0.
(B) E2-PEST at pH 7.5 at different temperatures; the spectrum at 20°C is shown as a dotted line. Inset: differential spectrum of E2-PEST between 2°C and 90°C.
(C) E2-PEST (full line), E2-PEST298p (broken line), E2-PEST301p (dashed line), and E2-PEST298p301p (dotted line) in 10 mM Tris-HCl (pH 7.5).
Structure  , DOI: ( /j.str )
Copyright © 2006 Elsevier Ltd Terms and Conditions

4. Figure 3
Presence of Regions Containing PII in E2-PEST Peptides; Effect of Phosphorylation
(A and B) Far-UV CD spectra of (A) E2-PEST and (B) E2-PEST301p in the presence of different concentrations of Gdm.HCl at pH 7.5. A positive band around 218 nm is stabilized as the concentration of the denaturant is increased for E2-PEST.
(C) Difference molar ellipticity at 222 nm of E2-PEST (black circle) and E2-PEST301p (white circle) as a function of [Gdm.Cl]. E2-PEST presents a cooperative transition, while no effect is observed for E2-PEST301p. The ellipticity change for PII was followed at 222 nm because of the inherent noise introduced by the high concentrations of denaturant around 215 nm.
Structure  , DOI: ( /j.str )
Copyright © 2006 Elsevier Ltd Terms and Conditions

5. Figure 4
Stabilization of α-Helical Populations in BPV1 E2-PEST Peptides by TFE
(A and B) Far-UV circular dichroism of (A) E2-PEST and (B) E2-PEST301p at different TFE concentrations ranging from 0% to 65% v/v at pH 4.5.
(C) Molar ellipticity at 222 nm E2-PEST (black circle) and E2-PEST301p (white circle) as a function of the percentage of TFE (v/v).
Structure  , DOI: ( /j.str )
Copyright © 2006 Elsevier Ltd Terms and Conditions

6. Figure 5 Structural Characterization of E2-PEST in Solution
(A) Backbone amide region of the 1H-NOESY spectrum of the E2-PEST peptide at 25°C (pH 4.5) in 1:1 TFE:H2O. The sequential NH-NH connectivity is shown.
(B) Plot of the chemical shift differences between the observed resonances and values found in a random coil conformation, Δδ = δ (observed) − δ (random coil), versus the position along the peptidic sequence for 1Hα, 13Cα, and 13Cβ on the top, medium, and lower panels, respectively. The tracts with defined elements of secondary structure are shaded on the plot.
(C) Summary of sequential and medium-range NOEs involving N1H and 1Hα protons. Strong, medium, and weak NOEs, classified according to the intensity of the crosspeak, are indicated by the different heights of the connecting boxes. The asterisks represent the NOEs that cannot be assigned due to the signal overlap. Bottom line: Hα(i)-Hδ(i+1) NOEs for proline residues, indicating that all of the prolines are in a trans conformation, are also shown.
Structure  , DOI: ( /j.str )
Copyright © 2006 Elsevier Ltd Terms and Conditions

7. Figure 6
Effect of Phosphorylation on the Structure of the E2-PEST Peptide
(A) Overlapping of the 13Cα-1Hα region of the [13C,1H]-HSQC spectra at natural abundance of E2-PEST (red) and E2-PEST301p (black) peptides.
(B) Plot of the differences between 13Cα chemical shifts observed between E2-PEST and E2-PEST301p peptides at 25°C, ΔδCα = δCα(E2-PEST) − δCα(E2-PEST301p), plotted against the position along the full-length E2 sequence. The value for S301 is out of scale. The tracts with defined elements of secondary structure are shaded on the plot.
Structure  , DOI: ( /j.str )
Copyright © 2006 Elsevier Ltd Terms and Conditions

8. Figure 7 Effect of Temperature, Gdm.Cl, and Phosphorylation on E2-PEST
(A–C) Plot of the differences between the 1JCαHα values for: (A) E2-PEST and E2-PEST301p, Δ1JCαHα = 1JCαHα (E2-PEST301p) − 1JCαHα (E2-PEST), at 25°C in a 1:1 TFE:H2O solution at pH 4.5; (B) E2-PEST at 25°C and at 10°C, Δ1JCαHα = 1JCαHα (E2-PEST at 10°C) − 1JCαHα (E2-PEST at 25°C), in a 1:1 TFE:H2O solution at pH 4.5; and (C) E2-PEST in 2 M Gdm.Cl and in a 1:1 TFE:H2O solution, Δ 1JCαHα = 1JCαHα (E2-PEST in Gdm.Cl) − 1JCαHα (E2-PEST in TFE:H2O), at 25°C and pH 4.5. (The constant values of residues E293 and E294, E295 and E296, P306 and P310, and P285 and P289 in the guanidinium solution may be exchangeable.) The tracts with defined elements of secondary structure are shaded on each plot and are depicted above the sequence.
Structure  , DOI: ( /j.str )
Copyright © 2006 Elsevier Ltd Terms and Conditions

9. Figure 8
Phosphorylation Triggers the Conformational Switch Required for Degradation
(A) Electrostatic potential surface representation of E2-PEST. Red, negative; blue, positive; white, neutral; green, serine 301.
(B) Model based on the structural features of the E2-PEST, showing the α helix, turn, and PII structures (left) that are disrupted after phosphorylation of S301 (right, S301 is depicted in backbone, and the phosphate added is marked in pink).
(C) Correlation between the half-lives of full-E2 proteins in vivo and the structure content in E2-PEST fragments. Data of percent α helix content (black) in water was obtained from Table 1. PII content (gray) was calculated from ΔG values of PII formation from Table 1 (see Experimental Procedures). Half-lives of full-length E2 proteins in vivo from Table 1 are shown (diagonal stripes). The half-life of unphosphorylated E2 cannot be calculated experimentally since it is not possible to discriminate between the phosphorylated and unphosphorylated species. The best estimate for this is the S290,S298,S301AAA triple mutant, since it cannot be phosphorylated by CKII (Penrose et al., 2004). Phosphorylation on S301 of the wild-type and S298A full-length E2 proteins is responsible for the accelerated turnover (Penrose et al., 2004), and therefore the half-life values for these species can be associated to the E2-PEST301p fragment.
Structure  , DOI: ( /j.str )
Copyright © 2006 Elsevier Ltd Terms and Conditions