Biomolecular Condensation of Trypsin Prevents Autolysis and Promotes Ca2+-Mediated Activation of Esterase Activity

Abstract

AbstractThe presence of Ca2+ions is known to facilitates the biocatalytic activity of trypsin-like serine proteases via structural stabilization against thermal denaturation and autolysis. Herein, we report a new and hidden regulatory role of Ca2+in the catalytic pathways of trypsin and α-chymotrypsin under physiological conditions. We discovered that macromolecular crowding promotes spontaneous homotypic condensation of native trypsin via liquid-liquid phase separation to yield membraneless condensates/droplets in a broad range of concentrations, pH, and temperature. These condensates are stabilized by multivalent hydrophobic interactions between short patches of hydrophobic residues. Importantly, no liquid-to-solid-like phase transition has been observed over a period of 14 days, indicating the structural intrigrity of phase-separated trypsin within the droplets. Structural insights revealed minimal conformational perturbation of trypsin upon phase separation. Interestingly, we found that Ca2+binding in the calcium binding loop reversibly regulates the biomolecular condensation of trypsin and α-chymotrypsin. While Ca2+-bound trypsin are ineffective to undergo LLPS to form condensate, its removal facilitates condensation under similar experimental conditions. More importantly, we show that biomolecular condensation effectively prevents autolysis of trypsin at physiological conditions and preserve its native-like esterase activity over a period of 14 days, whereas free trypsin loses 86% of its initial activity. In addition, it has been found that phase-separated trypsin responds to Ca2+-dependent activation of its esterase activity even after 14 days of storage while free trypsin failed to do so. Our findings indicate that biomolecular condensates of trypsin and trypsin-like serine proteases act as storage media to prevent autolysis and premature activation, and at the same time preserve their native-like active conformations. The present study highlights an important physiological aspect of biomolecular condensates of trypsin-like serine proteases by which cells can spatio-temporally regulate their biocatalytic efficacy via Ca2+-signalling.