Protein topology determines binding mechanism

Abstract

Protein recognition and binding, which result in either transient or long-lived complexes, play a fundamental role in many biological functions, but sometimes also result in pathologic aggregates. We use a simplified simulation model to survey a range of systems where two highly flexible protein chains form a homodimer. In all cases, this model, which corresponds to a perfectly funneled energy landscape for folding and binding, reproduces the macroscopic experimental observations on whether folding and binding are coupled in one step or whether intermediates occur. Owing to the minimal frustration principle, we find that, as in the case of protein folding, the native topology is the major factor that governs the choice of binding mechanism. Even when the monomer is stable on its own, binding sometimes occurs fastest through unfolded intermediates, thus showing the speedup envisioned in the fly-casting scenario for molecular recognition.

Fig. 1.

A phase diagram that correlates the association mechanism of the homodimers with their structural properties. The two- and three-state homodimers are structurally classified based on the number of interfacial and intramonomeric native contacts as well as the hydrophobicity of the interface (the 11 selected homodimers for simulation are designated by a star). The interface hydrophobicity was calculated based on the normalized occurrence of each amino acid in interfacial contacts multiplied by its hydrophobicity factor (50). The classification as two-state, three-state, and existence of dimeric intermediate is based on experimental data. In general, a two-state dimer is characterized by higher ratio of interfacial native contacts to monomeric native contacts, and a more hydrophobic interface, in comparison to a three-state dimer. The two-state homodimers include 1cta (Troponin C site III) (40), 1arr (Arc repressor) (33, 41), 2gvb (gene V protein) (34, 42), 1f36 (factor for inversion stimulation) (36), 1bet (β nerve growth factor) (43). The three-state homodimers include: 1lmb (λ repressor) (44), 1cop (λ Cro repressor) (45), 1lfb (LFB1 transcription factor) (46), 3ssi (S. subtilisn inhibitor) (47), 1xso (superoxide dismutase) (48). The class of three-state homodimers with dimeric intermediate is represented by 2wrp (Trp repressor) (49) and denoted by the same color as the two-state dimers because its dimerization does not involve a preexisting folded monomer. For references on other dimers in the phase diagram, see supporting information.

Fig. 2.

Free energy surfaces of folding and binding of obligatory (two-state) dimers. Free energy surfaces of the simulated homodimers are plotted as a function of the intrasubunit native contacts (Q_A and Q_B), intersubunit native contacts, (Q_interface), the total number of native contacts (Q_Total), and the separation distance between the two chains [Rcm(A)–Rcm(B)]. The simulations reproduce the experimentally inferred mechanisms regarding the coupling between folding and binding: the monomers constitute the dimers fold concurrently with their binding. The free energy surfaces are calculated at their transition temperatures (the folded and unfolded states have identical free energy values) defined by the peak of the specific heat profile as a function of temperature: 0.94 ε, 1.08 ε, 1.06 ε, 1.13 ε, and 1.21 ε for Troponin C site III, Arc repressor, Factor for inversion stimulation, gene V protein, and β nerve growth factor, respectively. The free energy is in units of ε. We note that an unfolded monomer is not entirely unfolded but is partially structured, containing ≈20–40% of the native contacts.

Fig. 3.

Typical trajectories of folding and association of representative homodimers presented in Figs. 2 and 4. The time evolution of the potential energy, the separation distance, as well as Q_A (green), Q_B (blue), and Q_Interface (red), illustrate the coupling between folding and binding [Troponin C site II (a), Arc repressor (b), and Trp repressor (c)], binding of two already folded monomers [λ repressor (d)], and the cases where recognition occurs by an unfolded subunit [λ Cro repressor (e) and LFB1 transcription factor (f)]. All of the trajectories are at the same temperatures as the corresponding free energy surfaces (Figs. 2 and 4).

Fig. 4.

Free energy surfaces of folding and binding of nonobligatory (three-state) dimers. An unbound folded monomer exist for the three-state dimers (a–e) [except for Trp repressor (f) with a dimeric intermediate]. The formation of some three-state dimers [λ Cro repressor (c), LFB1 transcription factor (d), and S. subtilisin inhibitor (e)] preferentially occurs by binding between folded and unfolded monomers and not by binding two already folded chains as found for λ repressor (a) and Cu/Zn superoxide dismutase (b). For Trp repressor, a coupling between folding and binding with a dimeric intermediate is observed. For three-state homodimers (may have more than a single peak in the specific heat curve), the free energy surfaces were plotted at temperatures in which the unfolded and folded states have the same free energy: 0.99 ε, 1.27 ε, 0.99 ε, 1.20 ε, 0.96 ε, and 1.04 ε for λ repressor, Cu/Zn superoxide dismutase, λ Cro repressor, LFB1 transcription factor, S. subtilisin inhibitor, and Trp repressor, respectively. The free energy is in units of ε.

Fig. 5.

Free energy profiles (units of ε) as a function of the separation distance between the two chains. Note that for each dimer the separation distance was shifted by subtracting the separation distance in the native dimer. A gradual decrease of the free energy indicates a weak interaction between at least a single unfolded chain and its target, showing that binding occurs by the fly-casting mechanism [for λ Cro repressor, the profiles are shown for linkers of 12 (filled circles), 20 (triangle), and 30 (open circles) glycine residues and without a linker (solid line), indicating similar effect starting at separation distance of 20 Å]. On the other hand, a flat free energy profile indicates a collision between two folded chains. For each dimer, the snapshots illustrate conformations with a shifted separation distance of 30, 20, 10, 5, and 0 Å (for clearer representation, the backbone was added to each Cα conformation).