Wildtype and Y220C L1 and L6 loops conformational landscape, with MSM-identified L6 states highlighted on the right.
The tumor suppressor p53 is the most frequently mutated gene in human cancer, and thus reactivation of mutated p53 is a promising avenue for cancer therapy. Analysis of wildtype p53 and the Y220C cancer mutant long-timescale molecular dynamics simulations with Markov state models and validation by NMR relaxation studies has uncovered the involvement of loop L6 in the slowest motions of the protein. Due to its distant location from the DNA-binding surface, the conformational dynamics of this loop has so far remained largely unexplored. We observe mutation-induced stabilization of alternate L6 conformations, distinct from all experimentally-determined structures, in which the loop is both extended and located further away from the DNA-interacting surface. Additionally, the effect of the L6-adjacent Y220C mutation on the conformational landscape of the functionally-important loop L1 suggests an allosteric role to this dynamic loop and the inactivation mechanism of the mutation. Finally, the simulations reveal a novel Y220C cryptic pocket that can be targeted for p53 rescue efforts. Our approach exemplifies the power of the MSM methodology for uncovering intrinsic dynamic and kinetic differences among distinct protein ensembles, such as for the investigation of mutation effects on protein function.
The E. coli cytidine repressor (CytR) is a member of the LacR family of bacterial repressors that regulates nine operons with distinct spacing and orientations of recognition sites. Understanding the structural features of the CytR DNA-binding domain (DBD) when bound to DNA is critical to understanding differential mechanisms of gene regulation. We previously reported the structure of the CytR DBD monomer bound specifically to half-site DNA and found that the DBD exists as a three-helix bundle containing a canonical helix-turn-helix motif, similar to other proteins that interact with DNA [Moody, et al (2011), Biochemistry 50:6622-32]. We also studied the free state of the monomer and found that since NMR spectra show it populates up to four distinct conformations, the free state exists as an intrinsically disordered protein (IDP). Here, we present further analysis of the DBD structure and dynamics in the context of full-site operator or nonspecific DNA. DBDs bound to full-site DNA show one set of NMR signals, consistent with fast exchange between the two binding sites. When bound to full-length DNA, we observed only slight changes in structure compared to the monomer structure and no folding of the hinge helix. Notably, the CytR DBD behaves quite differently when bound to nonspecific DNA compared to LacR. A dearth of NOEs and complete lack of protection from hydrogen exchange are consistent with the protein populating a flexible, molten state when associated with DNA nonspecifically, similar to fuzzy complexes. The CytR DBD structure is significantly more stable when bound specifically to the udp half-site substrate. For CytR, the transition from nonspecific association to specific recognition results in substantial changes in protein mobility that are coupled to structural rearrangements. These effects are more pronounced in the CytR DBD compared to other LacR family members.
Dystrophin is a 427 kDa rod-shaped protein encoded by one of the largest mammalian genes. The protein is formed by 27 domains, with 24 of them being single spectrin repeats interposed by two hinge regions. Single point and missing domain mutations within Dystrophin have been linked to Becker's Muscular Dystrophy (BMD), which is a degenerative muscle disease. This suggests that one role for Dystrophin is to reduce mechanical force between the sarcolemma membrane and the cytoskeleton. The mechanism for this function is unknown. The observation that single point mutations within Dystrophin are correlated with BMD suggests there is communication between each amino acid within each Dystrophin domain and that this communication extends to its neighboring domains. This led to the hypothesis that as force is transduced through the spectrin repeats, this results in the partial unfolding of the repeats which then dissipates force. If this partial unfolding of a given domain is accentuated when another repeat domain is in tandem, then this is consistent with negative coupling. Conversely, stabilization of one domain by another is positive coupling. Molecular dynamics (MD) forced unfolding experiments were consistent with negative coupling through non-additive force trajectories for tandem domains compared to individual domains. Therefore, we designed an experimental approach to test the computational results.Using monomeric spectrin 17, monomeric spectrin 18, and dimeric spectrin 17-18, we experimentally defined the thermodynamic and structural basis of energetic coupling between the spectrin domains through differential scanning calorimetry (DSC), fluorescence lifetime (FLT), and Overhauser dynamic nuclear polarization (ODNP). A combined approach utilizing these methods will provide insight into how coupling between spectrin repeats affects signal propagation and mechanical force dissipation. 1682-Pos Drug Resistance Induced by Local and Allosteric Conformational Changes in Oncogenic Tyrosine KinasesMitsugu Araki, Yasushi Okuno. Kyoto Univ, Kyoto, Japan. In non-small-cell lung cancer, the efficacies of tyrosine-kinase inhibitors (TKIs) are known to be decreased by mutations in target proteins such as ALK and RET, and in some cases there are no effective therapeutic strategies to overcome drug resistance. Here, we approach molecular mechanisms to acquire the resistance by combining molecular dynamics (MD) simulations on the nano-to micro-seconds timescales and our developed protein-ligand binding free energy computation method (Fujitani et al., Phys. Rev. E, (2009) 79, 021914 and Araki et al., J. Chem. Inf. Model., (2016) 56 (12), 2445-2456). Initial structures of mutants are modeled based on the crystal structures of the wild-type proteins, and their structural dynamics and binding affinities with TKIs are computed. In cases of drug resistance mutations in ALK, most of which occur near the drug binding site (< 10 angstroms), decreases in the efficacy tend to be successfully quantified by the binding free energy computatio...
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