Equilibrium fluctuations of a single folded protein reveal a multitude of potential cryptic allosteric sites

Abstract

Cryptic allosteric sites--transient pockets in a folded protein that are invisible to conventional experiments but can alter enzymatic activity via allosteric communication with the active site--are a promising opportunity for facilitating drug design by greatly expanding the repertoire of available drug targets. Unfortunately, identifying these sites is difficult, typically requiring resource-intensive screening of large libraries of small molecules. Here, we demonstrate that Markov state models built from extensive computer simulations (totaling hundreds of microseconds of dynamics) can identify prospective cryptic sites from the equilibrium fluctuations of three medically relevant proteins--β-lactamase, interleukin-2, and RNase H--even in the absence of any ligand. As in previous studies, our methods reveal a surprising variety of conformations--including bound-like configurations--that implies a role for conformational selection in ligand binding. Moreover, our analyses lead to a number of unique insights. First, direct comparison of simulations with and without the ligand reveals that there is still an important role for an induced fit during ligand binding to cryptic sites and suggests new conformations for docking. Second, correlations between amino acid sidechains can convey allosteric signals even in the absence of substantial backbone motions. Most importantly, our extensive sampling reveals a multitude of potential cryptic sites--consisting of transient pockets coupled to the active site--even in a single protein. Based on these observations, we propose that cryptic allosteric sites may be even more ubiquitous than previously thought and that our methods should be a valuable means of guiding the search for such sites.

Fig. 1.

Crystal structures of TEM-1 β-lactamase (A) in the absence of ligand and (B) in the presence of an allosteric inhibitor that reveals a cryptic binding site between helices 11 and 12. The backbone is colored from blue to red starting at the N terminus, a key active site residue (Ser70) is highlighted in green, and the allosteric inhibitor is shown in cyan (with helix 12 to its left and helix 11 to its right).

Fig. 2.

The three most frequently open pockets (yellow spheres), two of which coincide with the known allosteric ligand (cyan). Each sphere represents a pocket with a radius of up to 5 Å (SI Text). The spheres are overlaid on the apo crystal structure with the backbone colored from blue to red starting at the N terminus and a key active site residue (Ser70) highlighted in green.

Fig. 3.

The highest flux binding pathway, depicted as a series of configurations exemplifying intermediate Markov states. The crystallographically determined structure of helix 11 in the holo state is superimposed in yellow, emphasizing movement in this part of the protein during binding. One interesting feature of the pathway is that the two helices surrounding the cryptic site open 2–3 Å more widely than in the holo structure (particularly in states D and E) and then close around the ligand. The backbone is colored from blue to red starting at the N terminus, and the allosteric inhibitor is shown in cyan. The number associated with each structure quantifies progress along the binding pathway. Specifically, it indicates the probability that a trajectory initiated in the corresponding state reaches the bound state (F) before first reaching the unbound state (A).

Fig. 4.

A structure highlighting the community of coupled residues encompassing the known cryptic allosteric site. Side chains in this community are shown as sticks and are colored green if they are in the active site, cyan if they are in the allosteric site (i.e., within 3 Å of the cryptic ligand in the holo structure), and blue otherwise. The backbone is colored from blue to red starting at the N terminus.

Fig. 5.

A multitude of potential cryptic allosteric sites in (A) β-lactamase, (B) IL-2, and (C) RNase H. The 50 most frequently open pockets are shown as yellow spheres, each representing a pocket with a radius of up to 5 Å (SI Text). Sidechains within each coupled community are rendered in the same color. They are depicted as spheres if they are in the active site and as sticks otherwise. There are five communities for β-lactamase, six for IL-2, and four for RNase H. Almost every community contains at least one active site residue in each protein, so the vast majority of transient pockets could serve as cryptic sites. The light-blue, dashed circles encompass the known allosteric sites in β-lactamase and IL-2 and the backbone of each protein is colored from blue to red starting at the N terminus.