Reassessing a sparse energetic network within a single protein domain

Abstract

Understanding the molecular principles that govern allosteric communication is an important goal in protein science. One way allostery could be transmitted is via sparse energetic networks of residues, and one such evolutionary conserved network was identified in the PDZ domain family of proteins by multiple sequence alignment [Lockless SW, Ranganathan R (1999) Science 286:295-299]. We have reassessed the energetic coupling of these residues by double mutant cycles together with ligand binding and stability experiments and found that coupling is not a special property of the coevolved network of residues in PDZ domains. The observed coupling for ligand binding is better explained by a distance relationship, where residues close in space are more likely to couple than distal residues. Our study demonstrates that statistical coupling from sequence analysis is not necessarily a reporter of energetic coupling and allostery.

Fig. 1.

The crystal structure of PSD95 PDZ3 bound to its peptide ligand (thick blue sticks) [PDB entry 1BE9 (Doyle)]. Residues mutated in the present study are shown as spheres. (A) Residues located close and making direct contact with H372. (B) Residues that are distal to H372 and not making direct contact. Note that residue 328 is an Ile in this crystal structure but that in our construct we have the wild-type residue, Val. This figure was drawn in PyMOL (47).

Fig. 2.

Equilibrium ligand binding experiments. Shown are equilibrium titrations of the wild-type PDZ3 (A), single mutants F340A (filled triangles) and K380A (✸) (B), H372Y mutant (C), and double mutants F340A/H372Y (✕) and K380A/H372Y (filled circles) (D). K380 is a network residue, whereas F340 is a non-network residue (7). (See Materials and Methods for experimental conditions.)

Fig. 3.

Plots of measured coupling energies. The coupling free energies were obtained by fitting K_D values to Eq. 3. (A) Binding coupling energies in kcal·mol⁻¹. Error bars are propagated standard errors determined from at least three independent measurements of each K_D (for V328A, A376G, and K380A) or propagated fitting errors and standard errors of independent measurements. Note that the error bars thus depend on the individual errors of each of the four K_D values used to calculate the coupling energy. (B) C_α–C_α distance (Å) as a function of binding coupling energy. (C and D) Binding coupling energy ΔΔΔG_C as a function of stability coupling energy ΔΔΔG_D-N (C) and statistical coupling energy ΔΔG_STAT (D). Statistical coupling energies were extracted from figure 2b of ref. . The units of ΔΔG_STAT are arbitrary. Note that the absolute values for the coupling energies are plotted in B and D.