Polycomb repressive complex 2 (PRC2) is a key chromatin modifier responsible for methylation of lysine 27 in histone H3. PRC2 has been shown to interact with thousands of RNA species in vivo, but understanding the physiological function of RNA binding has been hampered by the lack of separation-of-function mutants. Here, we use comprehensive mutagenesis and hydrogen deuterium exchange mass spectrometry (HDX-MS) to identify critical residues for RNA interaction in PRC2 core complexes from Homo sapiens and Chaetomium thermophilum, for which crystal structures are known. Preferential binding of G-quadruplex RNA is conserved, surprisingly using different protein elements. Key RNA-binding residues are spread out along the surface of EZH2, with other subunits including EED also contributing, and missense mutations of some of these residues have been found in cancer patients. The unusual nature of this protein-RNA interaction provides a paradigm for other epigenetic modifiers that bind RNA without canonical RNA-binding motifs.
Background: Recombinant tissue-type plasminogen activator (tPA) is a potent fibrinolytic agent used in clinics and is inactivated by endogenous PAI-1. Results: The crystal structure of the tPA⅐PAI-1 Michaelis complex was determined. Conclusion: Differences of inhibition of tPA and uPA by PAI-1 are revealed. Significance: This study offers important clues to design a newer generation of tPA thrombolytics with reduced PAI-1 inactivation.
Quercetin is a member of the flavonoids and was previously demonstrated to inhibit trypsin-like serine proteases at micromolar potencies. Different molecular models were proposed to explain such inhibition. However, controversies remain on the molecular details of inhibition. Here, we report the X-ray crystal structure of quercetin in a complex with the urokinase-type plasminogen activator (uPA), an archetypical serine protease. The structure showed that quercetin binds to the specific substrate binding pocket (S1 pocket) of uPA mainly through its two neighboring phenolic hydroxyl groups. Our study thus provides unambiguous evidence to support quercetin binding to serine proteases and defines the molecular basis of the interaction. Our results further establish that natural products with two adjacent phenolic hydroxyl groups (or catechol) are likely to inhibit other trypsin-like serine proteases, a new mechanism formerly under-recognized.
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