Exploring the structural origins of cryptic sites on proteins

Abstract

Molecular dynamics (MD) simulations of proteins reveal the existence of many transient surface pockets; however, the factors determining what small subset of these represent druggable or functionally relevant ligand binding sites, called "cryptic sites," are not understood. Here, we examine multiple X-ray structures for a set of proteins with validated cryptic sites, using the computational hot spot identification tool FTMap. The results show that cryptic sites in ligand-free structures generally have a strong binding energy hot spot very close by. As expected, regions around cryptic sites exhibit above-average flexibility, and close to 50% of the proteins studied here have unbound structures that could accommodate the ligand without clashes. Nevertheless, the strong hot spot neighboring each cryptic site is almost always exploited by the bound ligand, suggesting that binding may frequently involve an induced fit component. We additionally evaluated the structural basis for cryptic site formation, by comparing unbound to bound structures. Cryptic sites are most frequently occluded in the unbound structure by intrusion of loops (22.5%), side chains (19.4%), or in some cases entire helices (5.4%), but motions that create sites that are too open can also eliminate pockets (19.4%). The flexibility of cryptic sites frequently leads to missing side chains or loops (12%) that are particularly evident in low resolution crystal structures. An interesting observation is that cryptic sites formed solely by the movement of side chains, or of backbone segments with fewer than five residues, result only in low affinity binding sites with limited use for drug discovery.

Druggability Assessment in TRAPP using Machine Learning Approaches

“…A single hot spot at the ATP binding site was detected by mapping unbound structures in the DFG-in loop conformation whereas multiple hot spots, that cover both the ATP binding site and the connected sub-pocket, were identified upon mapping a bound structure in the DFGout conformation. 46 Besides, it has been suggested that cryptic binding sites have a strong binding hot spot in the vicinity and exhibit above-average flexibility around the incipient pockets, 46 which coincides with the behavior of the cryptic sub-pocket of p38 MAP kinase.…”

Discovery of a hidden transient state in all bromodomain families

“…4b,c). These structures are the ones of ZMYND11 and PB1 (6), BDs that present substantial sequence variation -particularly in the ZA-loop region-compared to the general trend.…”

“…The other crystallographic evidence is a structure of PB1 (6), which is also an atypical BD having an unusually short ZA-loop. 20 In comparison with SMARCA2, a member of the same family, it shows a very similar opening despite having a low sequence identity (Figure 4c).…”

“…[1][2][3] Transitions between the different conformations of a protein may directly connect to changes in several properties, such as protein stability, interactions with binding-partners, or the appearance of "cryptic" pockets that could be druggable but are hidden in native structures. [4][5][6] Characterizing and determining such conformations is therefore essential to understanding the intricate relationship between structure, dynamics and function of proteins, with a direct impact on drug design and protein engineering. 7,8 Unfortunately, most of the conformations that a protein can adopt have low populations, and are consequently difficult to characterize with conventional biophysical techniques.…”

A cryptic pocket in Ebola VP35 allosterically controls RNA binding

“…Retrospective analysis of other validated drug targets suggests cryptic sites created by the movement of secondary structure elements, such as the displacement of helix 7, are often druggable. 40 The potential druggability of this cryptic site is also supported by application of the FTMap algorithm,41,42 which predicts a number of hotspots within the pocket where small molecules could form a variety of energetically-favorable interactions ( Supplementary Fig. 1).…”