Mapping Full Conformational Transition Dynamics of Intrinsically Disordered Proteins Using a Single-Molecule Nanocircuit.

Intrinsically disordered proteins (IDPs) are emerging therapeutic targets for human diseases. However, probing their transient conformations remains challenging because of conformational heterogeneity. To address this problem, we developed a biosensor using a point-functionalized silicon nanowire (SiNW) that allows for real-time sampling of single-molecule dynamics. A single IDP, N-terminal transactivation domain of tumor suppressor protein p53 (p53TAD1), was covalently conjugated to the SiNW through chemical engineering, and its conformational transition dynamics was characterized as current fluctuations. Furthermore, when a globular protein ligand in solution bound to the targeted p53TAD1, protein-protein interactions could be unambiguously distinguished from large-amplitude current signals. These proof-of-concept experiments enable semiquantitative, realistic characterization of the structural properties of IDPs and constitute the basis for developing a valuable tool for protein profiling and drug discovery in the future.

Integrative structural modeling of a multidomain polo-like kinase.

Polo-like kinase 1 (PLK1) is a key regulator and coordinator for mitotic signaling that contains two major functional units of a kinase domain (KD) and a polo-box domain (PBD). While individual domain structures of the KD and the PBD are known, how they interact and assemble into a functional complex remains an open question. The structural model from the KD-PBD-Map205PBM heterotrimeric crystal structure of zebrafish PLK1 represents a major step in understanding the KD and the PBD interactions. However, how these two domains interact when connected by a linker in the full length PLK1 needs further investigation. By integrating different sources of structural data from small-angle X-ray scattering, hydroxyl radical protein footprinting, and computational sampling, here we report an overall architecture for PLK1 multidomain assembly between the KD and the PBD. Our model revealed that the KD uses its C-lobe to interact with the PBD via the site near the phosphopeptide binding site in its auto-inhibitory state in solution. Disruption of this auto-inhibition via site-directed mutagenesis at the KD-PBD interface increases its kinase activity, supporting the functional role of KD-PBD interactions predicted for regulating the PLK1 kinase function. Our results indicate that the full length human PLK1 takes dynamic structures with a variety of domain-domain interfaces in solution.

Computational strategy for intrinsically disordered protein ligand design leads to the discovery of p53 transactivation domain I binding compounds that activate the p53 pathway.

Intrinsically disordered proteins or intrinsically disordered regions (IDPs) have gained much attention in recent years due to their vital roles in biology and prevalence in various human diseases. Although IDPs are perceived as attractive therapeutic targets, rational drug design targeting IDPs remains challenging because of their conformational heterogeneity. Here, we propose a hierarchical computational strategy for IDP drug virtual screening (IDPDVS) and applied it in the discovery of p53 transactivation domain I (TAD1) binding compounds. IDPDVS starts from conformation sampling of the IDP target, then it combines stepwise conformational clustering with druggability evaluation to identify potential ligand binding pockets, followed by multiple docking screening runs and selection of compounds that can bind multi-conformations. p53 is an important tumor suppressor and restoration of its function provides an opportunity to inhibit cancer cell growth. TAD1 locates at the N-terminus of p53 and plays key roles in regulating p53 function. No compounds that directly bind to TAD1 have been reported due to its highly disordered structure. We successfully used IDPDVS to identify two compounds that bind p53 TAD1 and restore wild-type p53 function in cancer cells. Our study demonstrates that IDPDVS is an efficient strategy for IDP drug discovery and p53 TAD1 can be directly targeted by small molecules.