Abstract
Numerous biomolecular interactions involve unstructured protein regions, but how to exploit such interactions to enhance the affinity of a lead molecule in the context of rational drug design remains uncertain. Here clarification was sought for cases where interactions of different ligands with the same disordered protein region yield qualitatively different results. Specifically, conformational ensembles for the disordered lid region of the N-terminal domain of the oncoprotein MDM2 in the presence of different ligands were computed by means of a novel combination of accelerated molecular dynamics, umbrella sampling, and variational free energy profile methodologies. The resulting conformational ensembles for MDM2, free and bound to p53 TAD (17-29) peptide identify lid states compatible with previous NMR measurements. Remarkably, the MDM2 lid region is shown to adopt distinct conformational states in the presence of different small-molecule ligands. Detailed analyses of small-molecule bound ensembles reveal that the ca. 25-fold affinity improvement of the piperidinone family of inhibitors for MDM2 constructs that include the full lid correlates with interactions between ligand hydrophobic groups and the C-terminal lid region that is already partially ordered in apo MDM2. By contrast, Nutlin or benzodiazepinedione inhibitors, that bind with similar affinity to full lid and lid-truncated MDM2 constructs, interact additionally through their solubilizing groups with N-terminal lid residues that are more disordered in apo MDM2.
Highlights
A large fraction of proteins contain substantial regions that are unstructured in native conditions [1,2]
In the simulations of small molecule bound complexes, complete lid opening was not observed with conventional MD (cMD) or accelerated molecular dynamics (aMD) protocols, but enhanced conformational fluctuations were observed with the latter protocol (S1 Fig)
While this conformation would hinder binding of the p53 transactivation domain (TAD) as a result of steric clashes with the lid, the Phe19-Trp23-Leu26 cleft was still accessible to small molecules such as Nutlins
Summary
A large fraction of proteins contain substantial regions that are unstructured in native conditions [1,2]. Protein disorder plays a key role in biomolecular function, enabling proteins to tune binding affinity and specificity to diverse partners [3]. In particular protein-complexes that involve interactions with disordered protein regions often involve disorder-to-order transitions (and vice versa) in one or both partner [4]. Much successful medicinal chemistry has arisen from efforts to mimic biomolecular recognition mechanisms, prominent examples include GPCR-(ant)agonists or transition state analogue enzyme inhibitors. There is evidence that small-molecules can productively target disordered protein regions [5]. For instance the Metallo lab has reported several small-molecule ligands that interact with disordered regions of the transcription factor c-Myc [6], though concerns about binding specificity have been raised [7]. How to anticipate productive interactions in the context of rational drug design with experimental or computational methods remains uncertain [10], and detailed investigations are necessary to progress our understanding of this molecular recognition mechanism
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