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Novel Structural Mechanism of Allosteric Regulation of Aspartic Peptidases Via an Evolutionarily Conserved Exosite

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Novel Structural Mechanism of Allosteric Regulation of Aspartic Peptidases Via an Evolutionarily Conserved Exosite Article Novel Structural Mechanism of Allosteric Regulation of Aspartic Peptidases via an Evolutionarily Conserved Exosite Graphical Abstract Authors Iva Ha´ nova´ ,Jirı´ Brynda, Radka Houstecka ´ , ..., Martin Horn, Ora Schueler-Furman, Michael Mares Correspondence [email protected] In Brief Aspartic peptidases (APs) are critically involved in numerous pathologies; however, little is known about their physiological regulation. Ha´ nova´ et al. discovered the first selective endogenous inhibitor of APs in higher organisms. It is generated by autoproteolysis and acts through a unique structural mechanism. Highlights d New inhibition mechanism revealed in aspartic peptidases (APs) d Oligopeptide inhibitor is proteolytically generated from the AP zymogen d This allosteric inhibitor binds to an exosite on the mature AP Ha´ nova´ et al., 2018, Cell Chemical Biology 25, 1–12 March 15, 2018 ª 2018 Elsevier Ltd. https://doi.org/10.1016/j.chembiol.2018.01.001 Please cite this article in press as: Ha´ nova´ et al., Novel Structural Mechanism of Allosteric Regulation of Aspartic Peptidases via an Evolutionarily Conserved Exosite, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.01.001 Cell Chemical Biology Article Novel Structural Mechanism of Allosteric Regulation of Aspartic Peptidases via an Evolutionarily Conserved Exosite Iva Ha´ nova´ ,1,2 Jirı´ Brynda,1 Radka Houstecka ´ ,1,3 Nawsad Alam,5 Daniel Sojka,4 Petr Kopa´ cek, 4 Lucie Maresova ´ ,1 Jirı´ Vondra´ sek, 1 Martin Horn,1 Ora Schueler-Furman,5 and Michael Mares1,6,* 1Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, 16610 Prague, Czech Republic 2Department of Biochemistry, Faculty of Science, Charles University, 12840 Prague, Czech Republic 3First Faculty of Medicine, Charles University, 12108 Prague, Czech Republic 4Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, 37005 Ceske Budejovice, Czech Republic 5Department of Microbiology and Molecular Genetics, Institute for Biomedical Research IMRIC, Hebrew University, Hadassah Medical School, 91120 Jerusalem, Israel 6Lead Contact *Correspondence: [email protected] https://doi.org/10.1016/j.chembiol.2018.01.001 SUMMARY a few proteinaceous compounds that might serve as endogenous inhibitors in lower organisms have been identified (Lenarcic and Pepsin-family aspartic peptidases are biosynthesized Turk, 1999; Li et al., 2000). as inactive zymogens in which the propeptide blocks Therefore, it is generally accepted that the major mechanism the active site until its proteolytic removal upon regulating the activity of pepsin-family peptidases is activation enzyme activation. Here, we describe a novel dual of the proenzyme. These peptidases are biosynthesized in the regulatory function for the propeptide using a set of form of inactive proenzymes (zymogens), in which the active crystal structures of the parasite cathepsin D IrCD1. site is blocked by a propeptide domain located at the N terminus of the molecule. The propeptide is cleaved off at acidic pH either In the IrCD1 zymogen, intramolecular autoinhibition by autocatalytic processing (via a monomolecular or bimolecular by the intact propeptide is mediated by an evolution- reaction) or processing assisted by another peptidase, leading to arily conserved exosite on the enzyme core. After active mature enzyme (for review, see Dunn, 2002; Khan et al., activation, the mature enzyme employs the same 1999; Richter et al., 1998). So far, several crystal structures of zy- exosite to rebind a small fragment derived from the mogens and their activation intermediates have helped increase cleaved propeptide. This fragment functions as understanding of propeptide interactions and the process of an effective natural inhibitor of mature IrCD1 that zymogen activation (James and Sielecki, 1986; Kervinen et al., operates in a pH-dependent manner through a unique 1999; Khan et al., 1997; Lee et al., 1998; Moore et al., 1995; allosteric inhibition mechanism. The study uncovers Morales et al., 2012; Ostermann et al., 2004). the propeptide-binding exosite as a target for In this work, we investigated the cathepsin D-like aspartic the regulation of pepsin-family aspartic peptidases peptidase IrCD1 from the hard tick Ixodes ricinus, a blood- feeding parasite that can transmit Lyme disease and tick-borne and defines the structural requirements for exosite encephalitis. IrCD1 plays a critical role in triggering the proteo- inhibition. lytic degradation pathway responsible for the digestion of host hemoglobin in tick gut cells (Horn et al., 2009). Previously, the IrCD1 propeptide was reported to be much shorter than propep- INTRODUCTION tides of other pepsin-family peptidases (Sojka et al., 2012). This prompted us to investigate its functional competence in Aspartic peptidases of the pepsin family (A1 family in the MEROPS inactivating the IrCD1 zymogen. Using structural analysis of database) are broadly distributed in eukaryotic organisms. Human the zymogen, we found that the IrCD1 propeptide retains full family members are the most well studied, including the gastric regulatory capability, which is associated with a small evolution- enzymes pepsin and chymosin; the blood pressure regulator arily conserved segment. Importantly, we identified an additional renin; cathepsins D and E, involved in several pathologies, function for this propeptide segment. After it is released by including cancer; and b-secretase (BACE), implicated in Alzheimer fragmentation of the propeptide during autoactivation, it is able disease (Rawlings and Barrett, 2013). Despite the importance of to rebind and effectively inhibit mature IrCD1. pepsin-family peptidases, little is known about the molecules Previously, several reports showed that propeptide-derived that function as physiological regulators of their activity. There products of the proteolytic activation event in pepsin-family are no known specific endogenous inhibitors of human pepsin- peptidases possess inhibitory activity (for references, see family peptidases, other than recently discovered sphingolipid Masa et al., 2006); however, the molecular mechanism of this inhibitors of human cathepsin D (Zebrakovska et al., 2011). Only phenomenon remained unknown. Here, we demonstrate for Cell Chemical Biology 25, 1–12, March 15, 2018 ª 2018 Elsevier Ltd. 1 Please cite this article in press as: Ha´ nova´ et al., Novel Structural Mechanism of Allosteric Regulation of Aspartic Peptidases via an Evolutionarily Conserved Exosite, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.01.001 Figure 1. Autoactivation of the IrCD1 Zymogen (A) Time course of the autoactivation. The IrCD1 zymogen was incubated at pH 4.0, and the reaction mixture was analyzed at different time points. Proteolytic activity generated during the autoactivation was determined in a kinetic assay with an FRET-peptide substrate. Mean values ±SE of triplicates are expressed relative to the maximum value. Rel., relative. (B) The autoprocessing mixture analyzed in (A) was resolved by SDS-PAGE and visualized by protein staining. The positions for the IrCD1 zymogen (pCD) and mature enzyme (mCD) are indicated. (C) pH profile of the autoactivation. The IrCD1 zymogen was incubated (for 0.5 or 3 hr) at various pH values, and the activity was assayed at pH 4.0 as described in (A). Mean values ±SE of triplicates are expressed relative to the maximum value for the indicated incubation time. the first time that a free propeptide fragment interacts with an sulfated polysaccharides, the presence of which greatly exosite located outside the active-site cleft, and we show at suppressed the autoactivation reaction in our experimental the structural level that this fragment inhibits the parental enzyme setup (Figures S1E–S1G). This suggests that interactions with through a novel allosteric mechanism. This work provides in- sulfated polysaccharides, which are widespread in all animal sights into a new class of specific endogenous regulators of tissues, regulate the autoactivation process in vivo. pepsin-family peptidases that are generated by autocatalytic During autoactivation of the IrCD1 zymogen, the 22-residue processing and have an oligopeptide structure. Furthermore, propeptide is proteolytically removed, generating the mature we propose the propeptide-binding exosite, which is evolution- N-terminal Ile23 (zymogen numbering) as determined by Edman arily conserved, as a novel target for inhibitors of various medi- sequencing. Mass spectrometry analysis of proteolytic products cally important pepsin-family peptidases. of the autoactivation reaction (Figure S1H) revealed that the released full-length propeptide is subsequently cleaved at RESULTS the Phe9-Lys10 bond located between secondary structure elements of the propeptide. Simulated fragmentation of the syn- IrCD1 Zymogen Is Autoactivated by Propeptide thetic propeptide by mature IrCD1 also resulted in processing Cleavage at the Phe9-Lys10 site (Figure S1H). We produced recombinant IrCD1 zymogen in a bacterial expres- sion system. Autocatalytic activation of the zymogen was Structure of Mature IrCD1 and Its Active Site investigated using two different assays: SDS-PAGE visualization We solved two crystal structures of mature IrCD1: the of the processed mature form and kinetic measurement of its empty enzyme and a complex with the substrate-mimicking proteolytic activity. Autoprocessing of the zymogen (47 kDa) inhibitor pepstatin. Crystallographic details are given in the was effective at acidic
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