Binding-induced folding of a natively unstructured transcription factor

Abstract

Transcription factors are central components of the intracellular regulatory networks that control gene expression. An increasingly recognized phenomenon among human transcription factors is the formation of structure upon target binding. Here, we study the folding and binding of the pKID domain of CREB to the KIX domain of the co-activator CBP. Our simulations of a topology-based Gō-type model predict a coupled folding and binding mechanism, and the existence of partially bound intermediates. From transition-path and Phi-value analyses, we find that the binding transition state resembles the unstructured state in solution, implying that CREB becomes structured only after committing to binding. A change of structure following binding is reminiscent of an induced-fit mechanism and contrasts with models in which binding occurs to pre-structured conformations that exist in the unbound state at equilibrium. Interestingly, increasing the amount of structure in the unbound pKID reduces the rate of binding, suggesting a "fly-casting"-like process. We find that the inclusion of attractive non-native interactions results in the formation of non-specific encounter complexes that enhance the on-rate of binding, but do not significantly change the binding mechanism. Our study helps explain how being unstructured can confer an advantage in protein target recognition. The simulations are in general agreement with the results of a recently reported nuclear magnetic resonance study, and aid in the interpretation of the experimental binding kinetics.

Figure 1. Structure of the pKID/KIX complex.

(A) Domain structure of CREB, consisting of DNA-binding domain basic region/leucine zipper (bZIP), the cAMP activated domain KID, and the glutamine-rich domains Q1 and Q2. The sequence of the structured part of pKID is shown with residues in helices α_A and α_B underlined. (B) Folding and binding of pKID (red: helix α_A; green: helix α_B) to the KIX domain of CBP (blue).

Figure 2. The fraction of native contacts for pKID, KIX, and the complex.

Fraction of native amino-acid contacts as a function of time for the intermolecular complex (Q_C; top), KIX (Q_KIX; center), and pKID (Q_KID; bottom). Data are shown for one out of thirteen simulations of the same length.

Figure 3. Free energy surface of the folding and binding process as function of Q_C.

Free energy as a function of the fraction of native intermolecular residue-residue contacts Q_C. Representative conformations along the reaction coordinate are shown (blue: KIX, red: pKID-α_A, and green: pKID-α_B). Inset: The probability p(TP|Q_C) of being on a transition path for a given value of Q_C. The TS region is marked in light gray.

Figure 4. 2D free energy surface for the binding-induced folding of pKID.

Potential of mean force for binding as a function of the fraction of intermolecular native contacts between helix α_A (Q_CA) and helix α_B (Q_CB) of pKID and KIX. The black line depicts one representative transition path from unbound to bound. Representative structures are shown for important regions of the free energy landscape.

Figure 5. Structural characteristics of the transition state ensemble.

(A) and (B) show the probability density function (pdf) of intramolecular native contacts of helix α_A (Q_αA) and helix α_B (Q_αB) of pKID for the unbound state, TS, intermediates I_A and I_B, and bound states. (C) and (D) show the probability density functions of native contact fractions Q_CA and Q_CB between KIX and helices α_A and α_B of pKID, respectively, in the bound and transition states.

Figure 6. Φ value analysis for the transition state.

Φ values for the folding/binding transition state for the residues of pKID forming native contacts with KIX. Red squares indicate amino acids belonging to helix α_A, green circles indicate amino acids belonging to helix α_B, and black triangles show Φ values for residues without secondary structure.

Figure 7. Non-specific encounter complexes.

Potential of mean force as function of the number of native and non-native contacts between pKID and KIX. Free energy surfaces are shown as a function of all non-native contacts (NNC) and native contacts (NC) (A–B) at equilibrium and (C–D) in transition paths. (E–F) The probability p(TP|NC,NNC) of being in the transition path as a function of NC and NNC. (A), (C), and (E) correspond to a potential with only native attractive interactions and (B), (D), and (F) correspond to the same potential with added non-native interactions at a strength of 40% of the native contacts.