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The C-terminal domains of proteases play crucial roles in hydrolysis, substrate adsorption and targeted binding. Identifying and characterizing interactions between C-terminal domains and biomacromolecules can help to examine the diversity as well as the substrate-binding ability of C-terminal domains and to explore novel functions. The bacterial pre-peptidase C-terminal (PPC) domain is a typical C-terminal domain normally found at the C-terminus of bacterial secreted proteases. In this work, we successfully demonstrated that 8-anilinonaphthalene-1-sulfonic acid (ANS) could be used to rapidly determine the interactions between this C-terminal domain and biomacromolecules. The time-resolved ANS fluorescence of PPC and collagen interaction could be used for quantitative analysis of the collagen-binding capability based on the slope of the time-scanning curve. Using this method, we found that PPC domains had an obvious affinity to fibrillar proteins but had little or no capacity to bind polysaccharides or linear DNAs. Docking studies proved that collagen bound to the same hydrophobic site of PPC as the ANS probe, causing a decrease in the emission intensity. This method is simple and cost effective and provides an effective detection technique to analyze the interaction between this C-terminal domain and biomolecules.

Identification of a ligand binding site represents the starting point for a structure-based drug development program. Lack of a binding site hampers the development of improved ligands that modulate the protein of interest. In this letter, we describe the development of chemical tools that will allow for elucidation of the Hsp90 C-terminal ligand binding site. Our strategy is based on the preparation of paramagnetic analogs of KU-596, an investigational new drug that is currently undergoing clinical trials for the treatment of neuropathy and interacts with the Hsp90 C-terminal domain. In particular, we report the design and synthesis of three novel paramagnetic analogs of KU-596, which will be used to obtain long range distances for NMR structural studies of Hsp90 in complex with C-terminal ligands.

Heat shock protein 90 (Hsp90) is a molecular chaperone (90 kDa) that functions as a dimer. This protein facilitates the folding, assembly, and stabilization of more than 400 proteins that are responsible for cancer development and progression. Inhibiting Hsp90’s function will shut down multiple cancer-driven pathways simultaneously because oncogenic clients rely heavily on Hsp90, which makes this chaperone a promising anticancer target. Classical inhibitors that block the binding of adenine triphosphate (ATP) to the N-terminus of Hsp90 are highly toxic to cells and trigger a resistance mechanism within cells. This resistance mechanism comprises a large increase in prosurvival proteins, namely, heat shock protein 70 (Hsp70), heat shock protein 27 (Hsp27), and heat shock factor 1 (HSF-1). Molecules that modulate the C-terminus of Hsp90 are effective at inducing cancer-cell death without activating the resistance mechanism. Herein, we describe the design, synthesis, and biological binding affinity for a series of dimerized C-terminal Hsp90 modulators. We show that dimers of these C-terminal modulators synergistically inhibit Hsp90 relative to monomers.

Corrigendum: Lysophosphatidic acid directly activates TRPV1 through a C-terminal binding site