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• W3136191194 abstract "Rhodesain is the lysosomal cathepsin L-like cysteine protease of Trypanosoma brucei rhodesiense, the causative agent of Human African Trypanosomiasis. The enzyme is essential for the proliferation and pathogenicity of the parasite as well as its ability to overcome the blood–brain barrier of the host. Lysosomal cathepsins are expressed as zymogens with an inactivating prodomain that is cleaved under acidic conditions. A structure of the uncleaved maturation intermediate from a trypanosomal cathepsin L-like protease is currently not available. We thus established the heterologous expression of T. brucei rhodesiense pro-rhodesain in Escherichia coli and determined its crystal structure. The trypanosomal prodomain differs from nonparasitic pro-cathepsins by a unique, extended α-helix that blocks the active site and whose side-chain interactions resemble those of the antiprotozoal inhibitor K11777. Interdomain dynamics between pro- and core protease domain as observed by photoinduced electron transfer fluorescence correlation spectroscopy increase at low pH, where pro-rhodesain also undergoes autocleavage. Using the crystal structure, molecular dynamics simulations, and mutagenesis, we identify a conserved interdomain salt bridge that prevents premature intramolecular cleavage at higher pH values and may thus present a control switch for the observed pH sensitivity of proenzyme cleavage in (trypanosomal) CathL-like proteases. Rhodesain is the lysosomal cathepsin L-like cysteine protease of Trypanosoma brucei rhodesiense, the causative agent of Human African Trypanosomiasis. The enzyme is essential for the proliferation and pathogenicity of the parasite as well as its ability to overcome the blood–brain barrier of the host. Lysosomal cathepsins are expressed as zymogens with an inactivating prodomain that is cleaved under acidic conditions. A structure of the uncleaved maturation intermediate from a trypanosomal cathepsin L-like protease is currently not available. We thus established the heterologous expression of T. brucei rhodesiense pro-rhodesain in Escherichia coli and determined its crystal structure. The trypanosomal prodomain differs from nonparasitic pro-cathepsins by a unique, extended α-helix that blocks the active site and whose side-chain interactions resemble those of the antiprotozoal inhibitor K11777. Interdomain dynamics between pro- and core protease domain as observed by photoinduced electron transfer fluorescence correlation spectroscopy increase at low pH, where pro-rhodesain also undergoes autocleavage. Using the crystal structure, molecular dynamics simulations, and mutagenesis, we identify a conserved interdomain salt bridge that prevents premature intramolecular cleavage at higher pH values and may thus present a control switch for the observed pH sensitivity of proenzyme cleavage in (trypanosomal) CathL-like proteases. Human African Trypanosomiasis (HAT) or African Sleeping Sickness is a so-called neglected tropical disease (NTD) as classified by the World Health Organization (www.who.int). HAT is caused by two subspecies of African trypanosomes, Trypanosoma brucei gambiense and T. brucei rhodesiense (1Simarro P.P. Cecchi G. Franco J.R. Paone M. Diarra A. Ruiz-Postigo J.A. Fèvre E.M. Mattioli R.C. Jannin J.G. Estimating and mapping the population at risk of sleeping sickness.PLoS Negl. Trop. Dis. 2012; 6: 1-12Crossref Scopus (235) Google Scholar). These unicellular, protozoan parasites are transmitted to the host via the bite of a Tse-Tse fly. The disease progresses in two stages. In the early hemolymphatic stage, the parasites are present in the blood and in the lymphatic systems leading to unspecific symptoms such as fever and headache. In the later meningoencephalitic stage, the parasites cross the blood–brain barrier (BBB) and enter the central nervous system of the host. At this point, patients suffer from severe neurological symptoms, deregulation of sleep–wake cycles, coma, and ultimately death (2Kennedy P.G.E. Human African trypanosomiasis of the CNS: Current issues and challenges.J. Clin. Invest. 2004; 113: 496-504Crossref PubMed Scopus (222) Google Scholar). The few available treatment options often have severe side effects (3Pépin J. Milord F. The treatment of human African trypanosomiasis.Adv. Parasitol. 1994; 33: 1-47Crossref PubMed Scopus (273) Google Scholar). T. brucei spp. parasites express two main lysosomal cysteine proteases that belong to the papain family, the cathepsin B-like T. brucei cathepsin B (TbCathB) and the cathepsin L-like rhodesain (also called TbCathL, brucipain, or trypanopain). Rhodesain plays an important role in the progression into the second disease stage as it is involved in the crossing of parasites into the central nervous system via the BBB of the host (4Grab D.J. Garcia-Garcia J.C. Nikolskaia O.V. Kim Y.V. Brown A. Pardo C.A. Zhang Y. Becker K.G. Wilson B.A. De A Lima A.P.C. Scharfstein J. Dumler J.S. Protease activated receptor signaling is required for African trypanosome traversal of human brain microvascular endothelial cells.PLoS Negl. Trop. Dis. 2009; 3: 1-13Crossref Scopus (58) Google Scholar, 5Nikolskaia O.V. De A Lima A.P.C. Kim Y.V. Lonsdale-Eccles J.D. Fukuma T. Scharfstein J. Grab D.J. Blood-brain barrier traversal by African trypanosomes requires calcium signaling induced by parasite cysteine protease.J. Clin. Invest. 2008; 116: 2739-2747Crossref Google Scholar). The expression of TbCathB mRNA varies strongly during the life cycle of the parasite, while rhodesain is constitutively expressed (6Mackey Z.B. O’Brien T.C. Greenbaum D.C. Blank R.B. McKerrow J.H. A cathepsin B-like protease is required for host protein degradation in Trypanosoma brucei.J. Biol. Chem. 2004; 279: 48426-48433Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). In agreement with an important role in parasite survival, proliferation, and pathogenicity, RNAi-based rhodesain knockdown or inhibition of rhodesain but not TbCathB led to diminished parasitic growth and an increased sensibility to lytic factors in human serum (7Alsford S. Currier R.B. Guerra-Assunção J.A. Clark T.G. Horn D. Cathepsin-L can resist lysis by human serum in trypanosoma brucei brucei.PLoS Pathog. 2014; 10: 1-12Crossref Scopus (28) Google Scholar, 8Steverding D. Sexton D.W. Wang X. Gehrke S.S. Wagner G.K. Caffrey C.R. Trypanosoma brucei: Chemical evidence that cathepsin L is essential for survival and a relevant drug target.Int. J. Parasitol. 2012; 42: 481-488Crossref PubMed Scopus (59) Google Scholar). Rhodesain is thus generally assumed to be the main lysosomal cysteine protease in trypanosomes and presents a promising antitrypanosomal drug target. In general, proteases are attractive drug targets because the affinities of inhibitors toward active site residues can be tuned and potential inhibitors can easily be identified in high-throughput in vitro assay screenings (9Schirmeister T. Kesselring J. Jung S. Schneider T.H. Weickert A. Becker J. Lee W. Bamberger D. Wich P.R. Distler U. Tenzer S. Johé P. Hellmich U.A. Engels B. Quantum chemical-based protocol for the rational design of covalent inhibitors.J. Am. Chem. Soc. 2016; 138: 8332-8335Crossref PubMed Scopus (46) Google Scholar). Nevertheless, structural similarities to host proteins can lead to problems with off-target effects. Rhodesain shares 46% (60%) sequence identity (similarity) with human cathepsin L (hsCathL). The irreversible, vinylsulfone-based rhodesain inhibitor K11777 (N-methylpiperazine-urea-Phe-homophenylalanine-vinylsulfone-benzene), was shown to prevent T. brucei from overcoming a barrier formed by brain microvascular endothelial cells in an in vitro BBB model (4Grab D.J. Garcia-Garcia J.C. Nikolskaia O.V. Kim Y.V. Brown A. Pardo C.A. Zhang Y. Becker K.G. Wilson B.A. De A Lima A.P.C. Scharfstein J. Dumler J.S. Protease activated receptor signaling is required for African trypanosome traversal of human brain microvascular endothelial cells.PLoS Negl. Trop. Dis. 2009; 3: 1-13Crossref Scopus (58) Google Scholar, 5Nikolskaia O.V. De A Lima A.P.C. Kim Y.V. Lonsdale-Eccles J.D. Fukuma T. Scharfstein J. Grab D.J. Blood-brain barrier traversal by African trypanosomes requires calcium signaling induced by parasite cysteine protease.J. Clin. Invest. 2008; 116: 2739-2747Crossref Google Scholar). Encouragingly, while K11777 was indeed observed to not be biochemically selective, it was nontoxic to mammals, efficient against related protozoans, and could cure mice from a Trypanosoma cruzi infection (10McKerrow J.H. Update on drug development targeting parasite cysteine proteases.PLoS Negl. Trop. Dis. 2018; 12: 4-7Google Scholar, 11Doyle P.S. Zhou Y.M. Engel J.C. McKerrow J.H. A cysteine protease inhibitor cures Chagas’ disease in an immunodeficient-mouse model of infection.Antimicrob. Agents Chemother. 2007; 51: 3932-3939Crossref PubMed Scopus (155) Google Scholar, 12Clayton J. Chagas disease: Pushing through the pipeline.Nature. 2010; 465: 12-15Crossref PubMed Scopus (119) Google Scholar). In the cell, cathepsins are expressed as inactive zymogens where the ∼215 amino acid papain-fold core protease domain is preceded by a ∼20 amino acid signal peptide required for translation of the protein into the endoplasmatic reticulum, and a ∼100 amino acid prodomain required for correct folding and lysosomal targeting of the protease (13Huete-Pérez J.A. Engel J.C. Brinen L.S. Mottram J.C. McKerrow J.H. Protease trafficking in two primitive eukaryotes is mediated by a prodomain protein motif.J. Biol. Chem. 1999; 274: 16249-16256Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 14Tao K. Stearns A. Dong J. Wu Q. Sahagian G.G. The proregion of cathepsin L is required for proper folding, stability and ER exit.Arch. Biochem. Biophys. 1994; 311: 19-27Crossref PubMed Scopus (122) Google Scholar, 15Eakin A.E. Mills A.A. Harth G. McKerrow J.H. Craik C.S. The sequence, organization, and expression of the major cysteine protease (cruzain) from Trypanosoma cruzi.J. Biol. Chem. 1992; 267: 7411-7420Abstract Full Text PDF PubMed Google Scholar, 16Smith S.M. Gottesman M.M. Activity and deletion analysis of recombinant human cathepsin L expressed in Escherichia coli.J. Biol. Chem. 1989; 264: 20487-20495Abstract Full Text PDF PubMed Google Scholar, 17Lecaille F. Kaleta J. Brömme D. Human and parasitic papain-like cysteine proteases: Their role in physiology and pathology and recent developments in inhibitor design.Chem. Rev. 2002; 102: 4459-4488Crossref PubMed Scopus (434) Google Scholar). The prodomain interacts with the active site of the protease and keeps it in an autoinhibited state until it has been successfully trafficked to the acidic lysosome lumen. Here, the prodomain is removed by proteolytic cleavage, which may occur intra- or intermolecularly (18Nishimura Y. Kawabata T. Furuno K. Kato K. Evidence that aspartic proteinase is involved in the proteolytic processing event of procathepsin L in lysosomes.Arch. Biochem. Biophys. 1989; 271: 400-406Crossref PubMed Scopus (65) Google Scholar, 19Nishimura Y. Kawabata T. Kato K. Identification of latent procathepsins B and L in microsomal lumen: Characterization of enzymatic activation and proteolytic processing in vitro.Arch. Biochem. Biophys. 1988; 261: 64-71Crossref PubMed Scopus (154) Google Scholar, 20Wiederanders B. Kirschke H. The processing of a cathepsin L precursor in vitro.Arch. Biochem. Biophys. 1989; 272: 516-521Crossref PubMed Scopus (31) Google Scholar, 21Ménard R. Carmona E. Takebe S. Dufour É. Plouffe C. Mason P. Mort J.S. Autocatalytic processing of recombinant human procathepsin L: Contribution of both intermolecular and unimolecular events in the processing of procathepsin L in vitro.J. Biol. Chem. 1998; 273: 4478-4484Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). The cleavage site is located in the linker between the prodomain and the core protease domain (22Caffrey C.R. Hansell E. Lucas K.D. Brinen L.S. Alvarez Hernandez A. Cheng J. Gwaltney S.L. Roush W.R. Stierhof Y.D. Bogyo M. Steverding D. McKerrow J.H. Active site mapping, biochemical properties and subcellular localization of rhodesain, the major cysteine protease of Trypanosoma brucei rhodesiense.Mol. Biochem. Parasitol. 2001; 118: 61-73Crossref PubMed Scopus (142) Google Scholar, 23Kerr I.D. Wu P. Marion-Tsukamaki R. Mackey Z.B. Brinen L.S. Crystal structures of TbCatB and rhodesain, potential chemotherapeutic targets and major cysteine proteases of Trypanosoma brucei.PLoS Negl. Trop. Dis. 2010; 4: 1-9Crossref Scopus (67) Google Scholar). While the rhodesain core protease domain has been crystallized in complex with different inhibitors including K11777 (PDB: 2P7U) (23Kerr I.D. Wu P. Marion-Tsukamaki R. Mackey Z.B. Brinen L.S. Crystal structures of TbCatB and rhodesain, potential chemotherapeutic targets and major cysteine proteases of Trypanosoma brucei.PLoS Negl. Trop. Dis. 2010; 4: 1-9Crossref Scopus (67) Google Scholar, 24Giroud M. Dietzel U. Anselm L. Banner D. Kuglstatter A. Benz J. Blanc J.B. Gaufreteau D. Liu H. Lin X. Stich A. Kuhn B. Schuler F. Kaiser M. Brun R. et al.Repurposing a library of human cathepsin L ligands: Identification of macrocyclic lactams as potent rhodesain and trypanosoma brucei inhibitors.J. Med. Chem. 2018; 61: 3350-3369Crossref PubMed Scopus (17) Google Scholar, 25Kerr I.D. Lee J.H. Farady C.J. Marion R. Rickert M. Sajid M. Pandey K.C. Caffrey C.R. Legac J. Hansell E. Mckerrow J.H. Craik C.S. Rosenthal P.J. Brinen L.S. Vinyl sulfones as antiparasitic agents and a structural basis for drug design.J. Biol. Chem. 2009; 284: 25697-25703Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 26Berman H.M. Westbrook J. Feng Z. Gilliland G. Bhat T.N. Weissig H. Shindyalov I.N. Bourne P.E. The protein data bank.Acta Crystallogr. D Biol. Crystallogr. 2002; 58: 899-907Crossref PubMed Scopus (1772) Google Scholar), a structure of the zymogen as an important intermediate of the parasitic protease maturation process is currently not available and the molecular details of the pH-dependent cleavage process remain unclear. Here, we present the 2.8 Å crystal structure of the active site C150A mutant of the uncleaved T. brucei rhodesiense cysteine protease pro-rhodesain under acidic conditions. The rhodesain prodomain shares many structural features with other CathL prodomains, but is unique in the presence of an extended α-helix in its C-terminal end. Using photoinduced electron transfer–fluorescence correlation spectroscopy (PET-FCS) (27Sauer M. Neuweiler H. Probing rapid structural fluctuations of proteins and nucleic acids by single-molecule fluorescence quenching.Fluoresc. Spectrosc. Microsc. 2014; 1076: 371-417Google Scholar), we investigated the interdomain dynamics of the proenzyme. At low pH values, where the protease undergoes autocleavage, the “blocking peptide” region of the prodomain covering the protease active site shows increased dynamics. We further found evidence that pro-rhodesain can be processed both intra- and intermolecularly in a pH-dependent manner and identified a highly conserved interdomain salt bridge that can prevent premature intramolecular cleavage at high pH and may thus act as a “delay switch” for pro-cathepsin autocleavage. Cathepsin-like cysteine proteases are expressed as an inactive proform. pH-dependent cleavage of the prodomain from the globular core protease domain is an important step in the protease maturation process contingent on the correct localization of the protein to the lysosome. For a better understanding of pro-rhodesain inhibition and activation, we determined the crystal structure of T. brucei rhodesiense pro-rhodesain. While rhodesain expression in P. pastoris relies on the secretion of the folded protein (22Caffrey C.R. Hansell E. Lucas K.D. Brinen L.S. Alvarez Hernandez A. Cheng J. Gwaltney S.L. Roush W.R. Stierhof Y.D. Bogyo M. Steverding D. McKerrow J.H. Active site mapping, biochemical properties and subcellular localization of rhodesain, the major cysteine protease of Trypanosoma brucei rhodesiense.Mol. Biochem. Parasitol. 2001; 118: 61-73Crossref PubMed Scopus (142) Google Scholar, 25Kerr I.D. Lee J.H. Farady C.J. Marion R. Rickert M. Sajid M. Pandey K.C. Caffrey C.R. Legac J. Hansell E. Mckerrow J.H. Craik C.S. Rosenthal P.J. Brinen L.S. Vinyl sulfones as antiparasitic agents and a structural basis for drug design.J. Biol. Chem. 2009; 284: 25697-25703Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar), all currently available expression and purification protocols for rhodesain from Escherichia coli are based on its expression in inclusion bodies and subsequent protein refolding (28Pamer E.G. Davis C.E. So M. Expression and deletion analysis of the Trypanosoma brucei rhodesiense cysteine protease in Escherichia coli.Infect. Immun. 1991; 59: 1074-1078Crossref PubMed Google Scholar). To obtain the folded proenzyme for structural studies, we found both production routes to be ineffective. We thus optimized the purification of mature and pro-rhodesain from E. coli in a manner that circumvents secretion and refolding (see Figs. S1–S4 and Extended Materials and Methods for details). Purified mature rhodesain proteins from P. pastoris and E. coli have similar structural and functional properties as elucidated by size-exclusion chromatography (SEC), circular dichroism (CD) spectroscopy (Fig. 1, A and B), and a fluorescent peptide-cleavage assay using the fluorescent peptide Z-Phe-Arg-AMC (Z-phenylalanine-arginine-7-amido-4-methylcoumarin) to probe rhodesain activity (see Extended Materials and Methods) (9Schirmeister T. Kesselring J. Jung S. Schneider T.H. Weickert A. Becker J. Lee W. Bamberger D. Wich P.R. Distler U. Tenzer S. Johé P. Hellmich U.A. Engels B. Quantum chemical-based protocol for the rational design of covalent inhibitors.J. Am. Chem. Soc. 2016; 138: 8332-8335Crossref PubMed Scopus (46) Google Scholar). E. coli can thus be used as a reliable source for the production of active rhodesain for optimal flexibility in biophysical studies. To prevent autocleavage and to stabilize the proenzyme, we introduced a mutation into the active site of the protein, C150A. Pro-rhodesain C150A displays the expected increase in molecular weight during SEC compared with the mature protease as well as an additional small increase in α-helical content in CD spectroscopy measurements (Fig. 1, B and C). We obtained a 2.8 Å crystal structure for T. b. rhodesiense pro-rhodesain at acidic pH (Fig. 2, Table 1, Fig. S5). The proprotease structure contains two parts, the N-terminal prodomain (residues E38 to T123 according to uniprot numbering, ID.: Q95 PM0) and the C-terminal core protease domain (residues A126 to P342). The first 18 amino acids belonging to the prodomain, a short loop between the first two helices (residues 77/78) and residues G124/R125 at the C-terminus of the prodomain, are not resolved. High disorder, in agreement with the crystallographic B factors in the C-terminal prodomain region (Fig. 2, C and D), coincides with the expected intermolecular cleavage site of pro-rhodesain at position T123 or R125 (22Caffrey C.R. Hansell E. Lucas K.D. Brinen L.S. Alvarez Hernandez A. Cheng J. Gwaltney S.L. Roush W.R. Stierhof Y.D. Bogyo M. Steverding D. McKerrow J.H. Active site mapping, biochemical properties and subcellular localization of rhodesain, the major cysteine protease of Trypanosoma brucei rhodesiense.Mol. Biochem. Parasitol. 2001; 118: 61-73Crossref PubMed Scopus (142) Google Scholar, 23Kerr I.D. Wu P. Marion-Tsukamaki R. Mackey Z.B. Brinen L.S. Crystal structures of TbCatB and rhodesain, potential chemotherapeutic targets and major cysteine proteases of Trypanosoma brucei.PLoS Negl. Trop. Dis. 2010; 4: 1-9Crossref Scopus (67) Google Scholar). The entire C-terminus of the core protease up to residue P342 as well as the remaining residues of a TEV cleavage site (ENLYFQ), which was used to remove the C-terminal purification tag, could be reliably placed in the electron density. The remainder of the TEV cleavage site mediates crystal contacts to the neighboring protein’s prodomain and may have thus aided crystallization of the construct but does not interfere with the interaction of the prodomain with its corresponding catalytic domain.Table 1Crystallographic data collection and refinement statistics for T. brucei rhodesiense pro-rhodesain C150APDB entry7AVMData collection Wavelength1.54179 Space groupH3 Cell parametersa; b; c123.19 Å; 123.19 Å; 53.91 Åα; β; γ90.00°; 90.00°; 120°Resolution range [Å]37.92–2.8 (2.9–2.8)Unique reflections7512 (729)Multiplicity20.6 (19.8)Overall completeness [%]97.23 (92.73)<I/σ(I)>3.63 (at 2.81 Å)Refinement Rwork, Rfree0.255, 0.264 Rmerge0.611 Average B-Factor [Å2]41.2 Protein residues308 No. of solvent molecules38 Ligand atoms6 Total non-H atoms2379 RMSD from ideal geometryBond lengths [Å]0.010Bond angles [°]1.60 RamachandranFavored91.7%Allowed8.3%Outliers0%Numbers in parentheses characterize the highest-resolution shell. Open table in a new tab Numbers in parentheses characterize the highest-resolution shell. The catalytic core domain follows the typical two-lobed fold of the papain family of cysteine proteases with an α-helical L-domain and a β-sheet containing R-domain. The cleft between these domains harbors the active site triad residues C150 (mutated to alanine in our construct), H287 and N307 (Fig. 2B). In the same position as in papain (PDBs: 9PAP, 3TNX) (29Kamphuis I.G. Kalk K.H. Swarte M.B.A. Drenth J. Structure of papain refined at 1.65 Å resolution.J. Mol. Biol. 1984; 179: 233-256Crossref PubMed Scopus (460) Google Scholar, 30Roy S. Choudhury D. Aich P. Dattagupta J.K. Biswas S. The structure of a thermostable mutant of pro-papain reveals its activation mechanism.Acta Crystallogr. D Biol. Crystallogr. 2012; 68: 1591-1603Crossref PubMed Scopus (16) Google Scholar), pro-rhodesain contains three intramolecular disulfide bonds between residues C147-C188 and C181-C226 in the L-domain as well as between C280–C328 in the R-domain (Fig. S5). As apparent from the electron density, these residues all form disulfide bridges. The backbone RMSD between our structure of the pro-rhodesain core catalytic domain (PDB: 7AVM, residues 125–342) and the previously determined structure of mature rhodesain (PDB: 2P7U) (25Kerr I.D. Lee J.H. Farady C.J. Marion R. Rickert M. Sajid M. Pandey K.C. Caffrey C.R. Legac J. Hansell E. Mckerrow J.H. Craik C.S. Rosenthal P.J. Brinen L.S. Vinyl sulfones as antiparasitic agents and a structural basis for drug design.J. Biol. Chem. 2009; 284: 25697-25703Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar) is 0.39 Å, thus the presence of the prodomain does not affect the structure of the catalytic domain, including the relative side-chain orientations of the residues of the active site catalytic triad (Fig. 2B). The prodomain constitutes about one-third of the protein in immature cathepsin proteases and consists of three helices, H1–H3 (Fig. 2). In pro-rhodesain, the N-terminal helix 1 (H1, residues E38pro -K47pro) is shorter compared with H1 from other crystallized CathL zymogens. The lack of resolution for the first 18 amino acids indicates that this region may be highly flexible and potentially not structured in pro-rhodesain. H1 is connected to the orthogonal helix 2 (H2, residues A55pro-A76pro) via a long loop. H2 then leads into a long third helix (H3, residues E96pro-K113pro). Helix 3 covers the active site and constitutes the main difference between the prodomain of rhodesain and other CathL-like protease prodomains (see below). CathL and CathB proteases both belong to the CA clan and the C1 family of proteases (31Rawlings N.D. Barrett A.J. Bateman A. MEROPS: The database of proteolytic enzymes, their substrates and inhibitors.Nucleic Acids Res. 2012; 40: 343-350Crossref PubMed Scopus (701) Google Scholar) and thus share a papain-like fold. Nevertheless, their prodomains differ. For instance, the conserved ER(I/V)FNIN motif is only found in CathL-like precursors within helix 2 of the prodomain and is slightly modified in the trypanosomal CathL proteases (32Karrer K.M. Peiffer S.L. Ditomas M.E. Two distinct gene subfamilies within the family of cysteine protease genes.Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3063-3067Crossref PubMed Scopus (337) Google Scholar). Here, the ER(A/V)FNAA motif (consensus sequence Ex3Rx2(A/V)(F/W)x2Nx3Ax3A, with x = any amino acid) (Fig. 3, Fig. S6) is involved in the correct folding of the prodomain and mediates intradomain interactions (33Martínez J. Henriksson J. Rydåker M. JoséCazzulo J. Pettersson U. Genes for cysteine proteinases from Trypanosoma rangeli.FEMS Microbiol. Lett. 1995; 129: 135-141Crossref PubMed Scopus (11) Google Scholar, 34Mottram J.C. North M.J. Barry J.D. Coombs G.H. A cysteine proteinase cDNA from Trypanosoma brucei predicts an enzyme with an unusual C-terminal extension.FEBS Lett. 1989; 258: 211-215Crossref PubMed Scopus (121) Google Scholar). In most zymogens, H2 is longer than in our pro-rhodesain structure, and we do not observe density for the C-terminal end of this region (residues 77pro–78pro). Together with the elevated B-factor values for the C-terminal end of H2 indicate that this region may be flexible in the prodomain of pro-rhodesain as has been stated for human CathL (35Reif M.M. MacH L. Oostenbrink C. Molecular insight into propeptide-protein interactions in cathepsins L and O.Biochemistry. 2012; 51: 8636-8653Crossref PubMed Scopus (4) Google Scholar). H2 connects to Helix 3 (H3, residues 96pro–113pro) via a long linker. This linker partially forms a short antiparallel β-sheet (residues 82pro–84pro) with the propeptide binding loop (PBL, residues 271–275, Fig. 3C), a loop extending from the R subdomain of the catalytic core domain. Notably, both the short β-sheet in the H2/H3 linker of the prodomain and the interacting PBL in the protease domain display very low B-values indicating high rigidity (Fig. 2, C and D). In cathepsin prodomains, the β-strand in the H2/H3 linker is typically followed by a GNFD motif with the GxNxFxD (x = any amino acid) consensus sequence, which is required for proper protein folding and autoactivation (36Vernet T. Berti P.J. De Montigny C. Musil R. Tessier D.C. Menard R. Magny M.C. Storer A.C. Thomas D.Y. Processing of the papain precursor. The ionization state of a conserved amino acid motif within the pro region participates in the regulation of intramolecular processing.J. Biol. Chem. 1995; 270: 10838-10846Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). In pro-rhodesain, this sequence corresponds to the GNFD-like motif G85VT87PF89SD91. Residues in this motif form multiple contacts within both the prodomain and to the core protease domain (Fig. 3D). Our structure may thus provide some insights why the core protease domain’s PBL β-strand harbors a di-glycine motif (G274/G275) that is highly conserved among CathL proteases (Fig. S6) and why this region could be crucial for protease folding (37Coulombe R. Grochulski P. Sivaraman J. Ménard R. Mort J.S. Cygler M. et al.Structure of human procathepsin L reveals the molecular basis of inhibition by the prosegment.EMBO J. 1996; 15: 5492-5503Crossref PubMed Scopus (329) Google Scholar) as larger amino acid side chains may sterically interfere with the PBL interactions (Fig. 3, C and D). An interaction network involving mostly hydrophobic and aromatic residues positions the remainder of the prodomain H2/H3 linker whose P88pro and F89pro side chains dip into a hydrophobic basket formed by the side chains of F269, M270, W309, and W313 in the R-domain of the core protease (Fig. 3D). In addition, the backbone carbonyl of M270 forms a hydrogen bond with the side chain of T87pro while its side chain forms a hydrophobic interaction with F97pro in the first turn of H3, the long α-helix covering the entire length of the cleft between the L- and R-domains of the catalytic domain. In all available structures of other cathepsin proenzymes, this region is mainly unstructured (Fig. 4A). This α-helix therefore presents the most notable structural difference between pro-rhodesain and closely related nontrypanosomal proteases. The residues in H3 facing the catalytic domain are involved in numerous, mostly hydrophobic interactions with the catalytic domain, thereby efficiently blocking the rhodesain active site (Fig. 4A). In agreement with structures from multiple CathL proenzymes, the pro-rhodesain H3 helix starts out as a regular α-helix. However, and so far unique to pro-rhodesain, a bulge forms around residues N103pro and G104pro, whose backbone amides are not involved in hydrogen bonds. The hydrogen bond acceptor group for the amide group of G104pro in the context of a regular α-helix would be the oxygen of the backbone carbonyl of R100Pro. This group, however, is 5.1 Å away from the G104pro amide group, thus precluding formation of a hydrogen bond and instead locally distorting the α-helix in proximity to the active site of the core protease (Fig. 4B). Importantly, this bending of the α-helix enables the guanidino group of R100pro to interact with the side chain of Q144 in the core protease domain. Notably, Q144 is conserved across all CathL proteases while R100pro is conserved in the majority of homologous trypansomal CathL proproteases (Fig. S6). Below the bulge, H3 continues as a regular α-helix up to residue K113pro from where the prodomain leads into the R-domain of the catalytic core domain via a C-terminal 15 amino acid unstructured linker (Figs. 2 and 4B). The electron density in our X-ray structure allows tracing the backbone of the entire H3 α-helix unambiguously for all residues in H3 (E95pro–K113pro) (Fig. S5B). The B-factor values for the two N-terminal helical turns of H3 are among the high" @default.
• W3136191194 created "2021-03-29" @default.
• W3136191194 creator A5003295553 @default.
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• W3136191194 title "Structure, interdomain dynamics, and pH-dependent autoactivation of pro-rhodesain, the main lysosomal cysteine protease from African trypanosomes" @default.
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