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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--?-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.

SUBMITTER: Bowman GR

PROVIDER: S-EPMC3406870 | biostudies-other | 2012 Jul

REPOSITORIES: biostudies-other

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

Bowman Gregory R GR Geissler Phillip L PL

Proceedings of the National Academy of Sciences of the United States of America 20120702 29

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 model ...[more]

PMID: 22753506

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Project description:Cdt1 is a protein critical for DNA replication licensing and is well-established to be a binding partner of the minichromosome maintenance (MCM) complex. Cdt1 has also been demonstrated to have an emerging, "moonlighting" role at the kinetochore via direct binding to microtubules and to the Ndc80 complex. However, it is not known how the structure and conformations of Cdt1 could allow for these multiple, completely unique sets of protein complexes. And while there exist multiple robust methods to study entirely folded or entirely unfolded proteins, structure-function studies of combined, mixed folded/disordered proteins remain challenging. It this work, we employ multiple orthogonal biophysical and computational techniques to provide a detailed structural characterization of human Cdt1 92-546. DSF and DSCD show both folded winged helix (WH) domains of Cdt1 are relatively unstable. CD and NMR show the N-terminal and the linker regions are intrinsically disordered. Using DLS and SEC-MALS, we show that Cdt1 is polydisperse, monomeric at high concentrations, and without any apparent inter-molecular self-association. SEC-SAXS of the monomer in solution enabled computational modeling of the protein in silico. Using the program SASSIE, we performed rigid body Monte Carlo simulations to generate a conformational ensemble. Using experimental SAXS data, we filtered for conformations which did and did not fit our data. We observe that neither fully extended nor extremely compact Cdt1 conformations are consistent with our SAXS data. The best fit models have the N-terminal and linker regions extended into solution and the two folded domains close to each other in apparent "folded over" conformations. The best fit Cdt1 conformations are consistent with a function as a scaffold protein which may be sterically blocked without the presence of binding partners. Our studies also provide a template for combining experimental and computational biophysical techniques to study mixed-folded proteins.

Dataset Information

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

Publications

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

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