1. Volume 44, Issue 5, Pages 734-744 (December 2011)
Structural Instability Tuning as a Regulatory Mechanism in Protein-Protein Interactions 
Li Chen, Vassilia Balabanidou, David P. Remeta, Conceição A.S.A. Minetti, Athina G. Portaliou, Anastassios Economou, Charalampos G. Kalodimos 
Molecular Cell 
Volume 44, Issue 5, Pages (December 2011)
DOI: /j.molcel
Copyright © 2011 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2011 44, 734-744DOI: (10.1016/j.molcel.2011.09.022)
Copyright © 2011 Elsevier Inc. Terms and Conditions

3. Figure 1 The CesAB Chaperone Has Molten Globule-like Properties
(A) Far-UV CD data of native CesAB (blue) and in the presence of 10% TFE (magenta). The crossover point at 201 nm is characteristic of coiled coils, whereas the 222:208 nm ratio (∼0.9) of native CesAB suggests a poorly packed coiled coil (see Experimental Procedures). Addition of 10% TFE significantly increases CesAB helicity.
(B and C) Overlaid 1H-15N HSQC (B) and 1H-13C HMQC (C) spectra of U-[2H,12C], Ala-, Leu-, Met-, Val-, and Ile-δ1-[13CH3] CesAB under native conditions (blue) and in 10% TFE (magenta). Only a fraction of the expected amide signals in native CesAB are present due to severe line broadening, indicating the presence of structural fluctuations on the milli- to microsecond (ms-μs) time scale between conformations with different chemical shifts. The methyl resonances show poor dispersion, indicating a relatively loose packing of the hydrophobic regions in the CesAB homodimer. All these features are the hallmark of a poorly packed protein with conformational heterogeneity and dynamic fluctuations among multiple conformational states. Various conditions (pH, temperature, salt) exerted very little effect on the spectra of CesAB, consistent with molten globule-like conformation of the protein (Receveur-Bréchot et al., 2006). The addition of TFE (10% volume/volume) shifts the equilibrium toward the folded conformation of CesAB (magenta). Many of the well-dispersed peaks present in 10% TFE are already present in the native spectrum (representative resonances are plotted at lower contour and shown in the rectangular boxes), although broad, and the addition of TFE decreases their line width.
(D) Far-UV CD thermal denaturation of CesAB features a long transition suggesting that the protein unfolds in a noncooperative manner.
See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
CesAB Forms a Partially Folded, Loosely Packed Four-Helical Bundle
(A) Solution structure of the CesAB homodimer, colored using a gradient scheme (blue to red, from the N to the C terminus) for each subunit. Figure S2A shows the conformational ensemble.
(B) The interface of the helical bundle (delineated region in dashed box in [A]) is dominated by hydrophobic residues; thus, CesAB dimerization is primarily mediated by nonpolar interactions. Hydrogen bonds are represented by dashed lines (black). The two subunits are colored blue and green.
(C) CesAB dimerization buries significant amounts of hydrophobic surface. One subunit is displayed as a solvent-accessible surface with hydrophobic residues colored green, whereas the other subunit is displayed as a blue ribbon.
See also Figure S2.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3 Folding and Dynamic Properties of the CesAB Homodimer
(A) CesAB is colored according to SSP value, using a gradient coloring scheme. Higher SSP values indicate higher propensity to form α helical structure.
(B) CesAB is colored according to RCI-S2 value, using a gradient coloring scheme. Higher RCI-S2 values indicate rigidity, whereas lower values indicate flexibility.
(C–E) Residue-specific free energy of unfolding (ΔG0U-F) of CesAB (C), CesAB-E20L (D), and CesAB-D14L/R18D (E), as determined from NMR-monitored residue-specific urea denaturation experiments, are mapped by continuous-scale color onto the structure of the CesAB homodimer. Higher ΔG0U-F values indicate regions with higher resistance to urea-induced denaturation and, thus, higher stability.
See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4
Coiled Coil Irregularities Result in Suboptimal Contacts within the CesAB Helical Bundle
(A) Close-up view of the CesAB intersubunit α1−α1′ coiled-coil interface. The residues of the heptad sequence that mediate CesAB dimerization are shown. Sequence irregularities at positions 20 and 30 are highlighted.
(B) Helical wheel representation of the intersubunit α1−α1′ and α3−α1′ coiled coils highlighting (in yellow) the irregularities at positions 14, 18, 20, and 30 that prevent optimal juxtaposition.
(C) Far-UV CD data of CesAB and variants show that amino acid substitutions increase helicity.
(D) Far-UV CD thermal denaturation data, monitored at 222 nm as a function of the temperature of CesAB and variants, showing that amino acid substitutions increase CesAB stability.
(E) Effect of amino acid substitutions on the melting temperature (Tm) of CesAB, given as the difference between the Tm of substituted CesAB and the Tm of wild-type CesAB (ΔTm). Positive values denote increased stability of the substituted CesAB.
See also Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5
CesAB Homodimer versus CesAB−EspA Heterodimer: Autoinhibition and Structural Mimicry
(A) EspA binding to the CesAB homodimer displaces one CesAB subunit to form a CesAB:EspA 1:1 heterodimeric complex in an apparent subunit exchange mechanism. The coloring scheme matches the coloring of the corresponding protein subunits on the other panels.
(B) Superposition of the CesAB homodimer (subunits are colored green and blue) and the CesAB−EspA heterodimer (CesAB is in orange and EspA in magenta).
(C) CesAB and CesAB−EspA are superimposed as in (B) but the second CesAB subunit in the homodimer and the EspA subunit in the heterodimer are not shown for clarity.
(D) EspA binding to CesAB stabilizes and induces folding of the chaperone in the heterodimeric complex by providing compensatory contacts to CesAB residues that form unfavorable contacts in the homodimer. Hydrogen bonds/salt bridges are represented by dashed lines (black).
(E) Superposition of the homodimer and the heterodimer (as in [B]) reveals that CesAB adopts an autoinhibitory conformation. The CesAB helices α1 and α3 in the homodimer overlap structurally with helices α4 and α3, respectively, of EspA in the heterodimer. Colors are as in (B). CesAB is displayed as a solvent-accessible surface.
See also Figure S5.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6
CesAB Transient Opening Mechanism and Effect of Its Suppression on EspA Binding and Secretion
(A) Mechanism for the relief of autoinhibition in CesAB and subsequent binding to EspA. CesAB is poised for binding to the α1 helix of EspA by transiently detaching and exposing the α3 helices, actions that are facilitated by poor packing at the α1-α3′ interface. The first docking event is followed by CesAB subunit dissociation, which is facilitated by poor packing of the α1-α1′ interface, and formation of an ultimately stable CesAB−EspA heterodimer (see also Figures S6A–S6D).
(B) Effect of CesAB and EspA amino acid substitutions on the relative stability between the CesAB homodimer and the CesAB-EspA heterodimer (see Figures S6I and S6J) as assessed by measuring the amount of CesAB bound to EspA. Higher values of EspA-bound CesAB indicate that the heterodimer is more stable than the homodimer. In the case of lower EspA-CesAB values, EspA cannot be prevented from forming filaments (Figure S6J).
(C) In vivo secretion of EspA from EPECΔcesAB strains that contained pASK-IBA7 plasmids expressing wild-type or mutated CesAB. The graph reports the total amount of EspA secreted in 120 min (Experimental Procedures).
(D) Infection of HeLa cells by bacterial EPECΔcesAB strains carrying plasmids that express CesAB or CesAB-E20l/E30L. Because the presence of CesAB-E20L/E30L severely compromises EspA secretion and filament formation, the actin polymerization seen with wild-type CesAB (white arrow) does not occur; b, bacterial cells; n, HeLa cell nuclei (Experimental Procedures).
See also Figure S6.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7
Model for Relief of Autoinhibition in CesAB and EspA Binding Facilitated by Finely Tuned Instability
Once EspA has been synthesized it should be rapidly captured by CesAB (reaction a); otherwise EspA tends to quickly self-oligomerize to form filaments (reaction b). CesAB homodimer exists predominantly in a closed, autoinhibited conformation (1) but it transiently populates an open state, stimulated by packing defects at the α1-α3′ interface, wherein helix α3 is accessible to EspA for binding. Because the transient opening of helix α3 is fast (it occurs on the sub-millisecond time scale) it can effectively capture EspA in its monomeric state. Optimization of the contacts at the α1-α3′ interface results in suppression of the transient opening of helix α3 (2) and, thus, abrogation of the binding of EspA by CesAB (reaction a′).
See also Figure S7.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2011 Elsevier Inc. Terms and Conditions