Characterization of Structural Determinants of Granzyme B Reveals Potent Mediators of Extended Substrate Specificity

Granzymes are trypsin-like serine proteases mediating apoptotic cell death that are composed of two genetically distinct subfamilies: granzyme A-like proteases resemble trypsin in their active site architecture, while granzyme B-like proteases are quite distinct. Granzyme B prefers substrates containing P4 to P1 amino acids Ile/Val, Glu/Met/Gln, Pro/Xaa, and aspartic acid N-terminal to the proteolytic cleavage. By investigating the narrow extended specificity of the granzyme B-like proteases the mediators of their unique specificity are being defined. The foci of this study were the structural determinants Ile99, Tyr174, Arg192, and Asn218. Even modest mutations of these residues resulted in unique extended specificity profiles as determined using combinatorial substrate libraries and individual fluorogenic substrates. As with other serine proteases, Ile99 completely defines and predicts P2 specificity, primarily through the binding constant Km. Asn218 variants have minor effects alone but in combination with mutations at Arg192 and Ile99 alter P2 through P4 extended specificity. For each variant, the activity on its cognate substrate was equal to that of granzyme B for the same substrate. Thus, mutations at these determinants change extended selectivity preferentially over catalytic power. Additionally Asn218 variants result in increased activity on the wild type substrate, while the N218A/I99A variant disrupts the additivity between P2 and P4 specificity. This defines Asn218 not only as a determinant of specificity but also as a structural component required for P2 and P4 independence. This study confirms four determinants of granzyme B extended substrate specificity that constitute a canon applicable to the study of the remaining family members. Granzymes are trypsin-like serine proteases mediating apoptotic cell death that are composed of two genetically distinct subfamilies: granzyme A-like proteases resemble trypsin in their active site architecture, while granzyme B-like proteases are quite distinct. Granzyme B prefers substrates containing P4 to P1 amino acids Ile/Val, Glu/Met/Gln, Pro/Xaa, and aspartic acid N-terminal to the proteolytic cleavage. By investigating the narrow extended specificity of the granzyme B-like proteases the mediators of their unique specificity are being defined. The foci of this study were the structural determinants Ile99, Tyr174, Arg192, and Asn218. Even modest mutations of these residues resulted in unique extended specificity profiles as determined using combinatorial substrate libraries and individual fluorogenic substrates. As with other serine proteases, Ile99 completely defines and predicts P2 specificity, primarily through the binding constant Km. Asn218 variants have minor effects alone but in combination with mutations at Arg192 and Ile99 alter P2 through P4 extended specificity. For each variant, the activity on its cognate substrate was equal to that of granzyme B for the same substrate. Thus, mutations at these determinants change extended selectivity preferentially over catalytic power. Additionally Asn218 variants result in increased activity on the wild type substrate, while the N218A/I99A variant disrupts the additivity between P2 and P4 specificity. This defines Asn218 not only as a determinant of specificity but also as a structural component required for P2 and P4 independence. This study confirms four determinants of granzyme B extended substrate specificity that constitute a canon applicable to the study of the remaining family members. Cytotoxic lymphocytes serve as the major immune system defense against virally and tumor-infected cells (1Hudig D. Gregg N.J. Kam C.M. Powers J.C. Biochem. Biophys. Res. Commun. 1987; 149: 882-888Crossref PubMed Scopus (33) Google Scholar). They efficiently initiate apoptosis in the target cell by the directional release of cytotoxic granules containing the granzyme family of serine proteases, the pore-forming protein perforin, and proteoglycans (2Trapani J.A. Genome Biol. 2001; 2Crossref PubMed Google Scholar). The granzymes are trypsin-like serine proteases expressed as preproproteases. The pre region targets them to the granules where activation occurs through the proteolytic processing of the pro region by dipeptidyl protease 1 (3Pham C.T. Ley T.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8627-8632Crossref PubMed Scopus (349) Google Scholar). Of particular interest is granzyme B because of its unique specificity for aspartic acid-containing substrates (4Poe M. Blake J.T. Boulton D.A. Gammon M. Sigal N.H. Wu J.K. Zweerink H.J. J. Biol. Chem. 1991; 266: 98-103Abstract Full Text PDF PubMed Google Scholar). Granzyme B initiates the apoptotic cascade by activating caspases 3 and 7 (5Van de Craen M. Van den Brande I. Declercq W. Irmler M. Beyaert R. Tschopp J. Fiers W. Vandenabeele P. Eur. J. Immunol. 1997; 27: 1296-1299Crossref PubMed Scopus (46) Google Scholar) and proteolyzing a specific set of intercellular substrates such a DNA fragmentation factor 45 (6Thomas D.A. Du C. Xu M. Wang X. Ley T.J. Immunity. 2000; 12: 621-632Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 7Sharif-Askari E. Alam A. Rheaume E. Beresford P.J. Scotto C. Sharma K. Lee D. DeWolf W.E. Nuttall M.E. Lieberman J. Sekaly R.P. EMBO J. 2001; 20: 3101-3113Crossref PubMed Scopus (115) Google Scholar). In addition to the strict requirement by granzyme B for an aspartic acid residue at the primary, or P1, 1Serine protease substrate recognition sites are labeled according to the method of Schecter and Berger (8Schechter I. Berger A. Biochem. Biophys. Res. Commun. 1968; 32: 898-902Crossref PubMed Scopus (340) Google Scholar). Labels increase in number from P1, P2,... Pn for the substrate amino acids N-terminal to the scissile bond and P1′, P2′,... Pn′ for the substrate amino acids C-terminal to the scissile bond. The corresponding substrate recognition pockets on the enzyme are labeled Sn... S2, S1, S1′, S2′... Sn′. position the substrate preferentially contains an Ile or Val at the P4 position; Glu, Gln, or Met at P3; any amino acid at P2; non-charged amino acids at P1′; and Ser, Ala, or Gly at P2′ for efficient hydrolysis (9Thornberry N.A. Rano T.A. Peterson E.P. Rasper D.M. Timkey T. Garcia-Calvo M. Houtzager V.M. Nordstrom P.A. Roy S. Vaillancourt J.P. Chapman K.T. Nicholson D.W. J. Biol. Chem. 1997; 272: 17907-17911Abstract Full Text Full Text PDF PubMed Scopus (1852) Google Scholar, 10Harris J.L. Peterson E.P. Hudig D. Thornberry N.A. Craik C.S. J. Biol. Chem. 1998; 273: 27364-27373Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Granzyme B homologues have been located on rat, mouse, and human chromosomes 14 in a tightly linked gene cluster with chymase, cathepsin G, rat mast cell protease, and granzymes C through H (11Heusel J.W. Hanson R.D. Silverman G.A Ley T.J. J. Biol. Chem. 1991; 266: 6152-6158Abstract Full Text PDF PubMed Google Scholar, 12Hanson R.D. Hohn P.A. Popescu N.C. Ley T.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 960-963Crossref PubMed Scopus (47) Google Scholar). Proteases in this gene cluster share >46% identity and >56% sequence similarity with granzyme B. Despite the homology, these proteases have five identified or predicted primary specificities and unique substrates (13Kam C.M. Hudig D. Powers J.C. Biochim. Biophys. Acta. 2000; 1477: 307-323Crossref PubMed Scopus (132) Google Scholar). The specific interactions that determine these narrow extended substrate specificities may occur through a constellation of amino acids located on the highly variable loops surrounding the active site (14Hof P. Mayr I. Huber R. Korzus E. Potempa J. Travis J. Powers J.C. Bode W. EMBO J. 1996; 15: 5481-5491Crossref PubMed Scopus Google Scholar, Craik C.S. 1997; PubMed Scopus Google Scholar, M.E. T. 1997; PubMed Scopus Google Scholar, R. P. E. M. R. D. R. Huber R. Bode W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The amino acids and acids are according to their with the serine protease D.M. Chem. Res. Scopus Google Scholar). and the loops containing them have been for their in P1 specificity in trypsin-like proteases PubMed Scopus Google Scholar, A. Powers J.C. Hudig D. Biol. PubMed Scopus Google Scholar, PubMed Scopus Google and their for specific recognition of to two amino acids been with the granzyme B-like serine proteases are the at and have a containing a at amino acid and have the primary specificity determinant to amino acid This amino acid is in granzyme and a with the of the substrate J.L. R. Craik C.S. Biol. 2000; PubMed Scopus Google Scholar). to in a protease with P1 specificity against substrates A. J.C. Powers J.C. 1999; PubMed Scopus Google Scholar). the to alter trypsin to specificity required active site loops and PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). the primary binding site in the granzyme B proteases and the P1 specificity is In this to the for amino acids and M.E. T. 1997; PubMed Scopus Google Scholar, P.J. Wang Huber R. Bode W. S. J. Biol. 1999; PubMed Scopus Google the cathepsin specificity for and amino acids (14Hof P. Mayr I. Huber R. Korzus E. Potempa J. Travis J. Powers J.C. Bode W. EMBO J. 1996; 15: 5481-5491Crossref PubMed Scopus Google and the granzyme B for aspartic The sequence identity of the granzyme B-like the number of structural determinants and that in substrate specificity. The of of granzyme B J.L. R. Craik C.S. Biol. 2000; PubMed Scopus Google Scholar, J. Garcia-Calvo M. Thornberry N.A. J.W. Chem. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, E. P. D. M. R. K. Huber R. Bode W. Biol. Chem. 2000; PubMed Scopus Google M.E. T. 1997; PubMed Scopus Google rat mast cell protease 27: PubMed Scopus Google and cathepsin (14Hof P. Mayr I. Huber R. Korzus E. Potempa J. Travis J. Powers J.C. Bode W. EMBO J. 1996; 15: 5481-5491Crossref PubMed Scopus Google Scholar) and combinatorial for substrate specificity J.L. S. J.A. Craik C.S. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar) a for extended substrate specificity using This study to through the of amino acids identified by the of rat granzyme B in the of its specificity. The of amino acids were with a of variants the active site binding and combination variants were with at Tyr174, Ile99, Arg192, and Asn218. P1 to P4 combinatorial libraries combinatorial of substrates to each variant for in specificity and individual substrates to activity through The the of the specificity and are to the of each on the selectivity of granzyme B. and were from and were from The was from and according to the and the system were from The was a from the of of The was a from Thornberry The substrate was a of of Granzyme B granzyme B amino acid sequence of rat granzyme B is the number or number was as a to granzyme and proteases from the for of of using J. W. Res. 1997; PubMed Scopus Google Scholar) and C. R. 1991; PubMed Scopus Google Scholar). The were using The of rat granzyme B in a with was to determine amino acids with of the substrate amino acid acids were also that in the human granzyme chymase, cathepsin G, and rat mast cell protease of the are from the for the rat granzyme human granzyme and chymase, and cathepsin G, and and rat mast cell protease of Granzyme wild and rat granzyme B were as J.L. Peterson E.P. Hudig D. Thornberry N.A. Craik C.S. J. Biol. Chem. 1998; 273: 27364-27373Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). variants of wild type rat granzyme B were using the The to were to mutations the are was by with to the and The was cells and with granzyme B variants were expressed and to by the wild type J.L. Peterson E.P. Hudig D. Thornberry N.A. Craik C.S. J. Biol. Chem. 1998; 273: 27364-27373Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar) with between and 3 was in the using 1 each variant was a containing and by at with wild type or J.L. Peterson E.P. Hudig D. Thornberry N.A. Craik C.S. J. Biol. Chem. 1998; 273: 27364-27373Abstract Full Text Full Text PDF PubMed Scopus (166) Google and at the completely and were and to a of the substrates in each are at a the of the variant protease, and the are to the specificity constant The granzyme B variants were for activity against and in granzyme activity to between and 1 that activity equal to wild type granzyme B. each variant was in the P1 of a in of of fluorogenic substrates where the P1 amino acid is was and was while the and P4 amino acids of amino acid increase the to and the number of the to caspases and human (9Thornberry N.A. Rano T.A. Peterson E.P. Rasper D.M. Timkey T. Garcia-Calvo M. Houtzager V.M. Nordstrom P.A. Roy S. Vaillancourt J.P. Chapman K.T. Nicholson D.W. J. Biol. Chem. 1997; 272: 17907-17911Abstract Full Text Full Text PDF PubMed Scopus (1852) Google Scholar) and rat granzyme B J.L. Peterson E.P. Hudig D. Thornberry N.A. Craik C.S. J. Biol. Chem. 1998; 273: 27364-27373Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar) was to extended through of containing each for the amino acids and are and and are activity in the was for 1 at on a and the of substrate hydrolysis was with the and were at and of was to and by acid C. a Scholar). The substrates were to by on a with a of acid and The of each substrate was by The of each substrate was by hydrolysis of the or using of wild type granzyme B. a of each substrate was to of and the was over of the substrate was were using a protease was to between and 1 in substrates were in to between and while substrates were to between and activity was at and of and for a of of substrate hydrolysis were determined using the and to the using the for a substrate were only was The substrates were for hydrolysis by granzyme B and variants at in for the of rat granzyme B with the J.L. R. Craik C.S. Biol. 2000; PubMed Scopus Google Scholar, J. Garcia-Calvo M. Thornberry N.A. J.W. Chem. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, E. P. D. M. R. K. Huber R. Bode W. Biol. Chem. 2000; PubMed Scopus Google amino acid of of the binding site were determined They and in the Ile99 and in the Arg192 and Asn218 in the and and in the acid been identified as the primary determinant of P1 specificity, and the determinants and between two or amino acids in the or are required for catalytic activity PubMed Scopus Google Scholar, A. PubMed Scopus Google Scholar). determinants of specificity from from because of structural of extended specificity. activity in the that granzyme B variants and substrates containing P1 aspartic acid not This result is with the strict specificity of granzyme B for aspartic acid J.L. S. J.A. Craik C.S. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). The is of substrates where P1 is aspartic acid in of By the P1 amino acid and with a the number of substrates was to the to the of the was to in substrate specificity, the activity in each of the is as a of the at the substrate This the specificity but the activity of each was to the specificity for each was to the of each amino acid to the specificity of granzyme B. The variant a on the specificity profiles at the P2 The increased the hydrolysis of or substrates and the hydrolysis of the or P4 profiles for the P2 amino acid was the activity for in the and extended to substrates where the specificity for and were and for granzyme B variants specificity for and substrate the of specificity for the of the wild type and the variant in for activity in the libraries and between individual substrates in a amino acids specificity at Asn218 was with to the of the bond between the amino of Asn218 and the of the in the J.L. R. Craik C.S. Biol. 2000; PubMed Scopus Google Scholar). granzyme B not change the P2 or P4 specificity profiles but the for substrates with at the the number of amino acids to and Ala, and increased the for Met and of the amino acid that is from the wild type by granzyme B was increased with wild type activity from to hydrolysis of was from to the variant in the the and sequence the specificity constant for granzyme B was the wild type the specificity not change the of the the not a in the of the P4 amino The mutations in substrate specificity at the P4 position where Ile and Val to a of on was The and variants amino acids in other of the granzyme occurs in the human of rat granzyme while is in granzymes and C The variant the wild type granzyme B for by the but is an increase in the specificity of substrates The for substrates is to for granzyme B where the specificity are and In the variant a at P2 but is that in the and substrates are with specificity and Asn218 was to the of the bond between and the of in were to this and in combination with mutations of The variant for the substrates containing at the a number of amino acids and Ala, and increased for Met and The not change the P2 or P4 the hydrolysis of was by the variant by the wild type was the amino while the specificity constant for amino acids such as the for The variant change at P2 and an increased for and at and a for over This variant also activity to the J.L. Peterson E.P. Hudig D. Thornberry N.A. Craik C.S. J. Biol. Chem. 1998; 273: 27364-27373Abstract Full Text Full Text PDF PubMed Scopus (166) Google with the variant, the completely the specificity from to the activity of was to by The a as The specificity for for and are and This a for the amino for wild type granzyme B against a of substrates for individual substrates the of specificity where amino acids from the wild type of the wild type and the variant in for activity in the libraries and between individual substrates not in a The of of in the to an substrate is where the enzyme preferentially to the of the the activation between the enzyme and the and proteases a catalytic in the substrate and enzyme to the The of the of the or scissile bond an and the by hydrolysis of the to the active protease, the and a This is in the substrate is at an bond as in and the is the and the to and The catalytic for granzyme B on a of substrates in its selectivity the substrate sequence at the P2 position of the substrate in and but not and was for the activity of the protease substrates of For was that extended substrates increase by and of binding to A. PubMed Scopus Google Scholar). The substrates with and P4 amino acids not this For and the and were wild The increased at from to and while the from to and substrate at and P4 have for the protease and are not to efficiently the binding catalytic for variants to wild type The for residues at P2 for the and N218A/I99A variants the increase in for the substrate. the for amino acids is with the variant, but the activity is not that of wild the and variants residues with and in Km. not in a The variant proteases in a the Ile99 variant proteases have that are the wild type activity on the same substrate, its as a highly substrate. The for to the wild type protease of the and The increase in that while the been in the and the increase by the of a residue is not to the binding constant to the For the Asn218 the result is The substrate is over by granzyme B but a to the variants and The binding constant constant for of the variants through while the result in the activity Despite the completely for by the variant, the activity is granzyme B on primarily to a The of in the activity of the variant from the of the variant to the binding with Asn218 a for and variants have a of amino acids and increased with wild type granzyme B. This that Asn218 is to substrates with residues to the The N218A/I99A a on extended specificity by the P2 through P4 specificity to the sequence the and the P2 specificity is to amino but in addition the and P4 specificity been to and amino The specificity constant of the N218A/I99A variant is to that of the variant against the substrate The substrate with the specificity constant is the This that in substrate is by the combinatorial of the and change in of wild type and the N218A/I99A variant on a of substrates the in activity as the substrates and the in and The change in of the N218A/I99A variant is also in to wild type granzyme B. not in a the between the P2 and P4 substrate amino acids and its to a of substrates and were for wild type and N218A/I99A granzyme B The of or between the extended is determined by the between the Biol. Google Scholar) using the in specificity between a substrate and the wild type The in is the wild type substrate, is a substrate the substrate. is between extended the for a amino acid not equal the for the same in the of an In wild type granzyme the for is and P4 is Ile or The same result is for the P4 amino acid change for Ile to and This the wild type granzyme B requirement of an extended substrate for efficient The identity of the P4 amino acid to the and is to the identity of the P2 amino a amino acid or amino acid at is in the substrate, the activity is of the identity of the P2 amino acid The N218A/I99A variant the specificity The for is the P4 amino acid is Ile and the P4 amino acid is is also the P4 amino acid is from Ile to and for and The of binding to the from an or P2 amino acid is on the identity of the P4 amino acid in the N218A/I99A The granzyme B-like of serine proteases an in immune They are on 14 of the rat, and and have been to have processing proteolytic is that processing the granzymes catalytic for in catalytic in for activity on a distinct extended substrate. The granzyme B an active site that is distinct trypsin-like This active the through the catalytic and the and the binding to the granzyme B in a of serine proteases D.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). this the amino acids that between are to have in specificity and For granzyme four of these structural determinants have been and The variants in this study a of mutations to extended specificity, and the a method for the in their specificity. Even mutations result in in the profiles of the a the specificity profiles by are in the individual of substrates. The profiles specificity by individual substrate and individual substrates. The of P2 specificity on the identity of the amino acid at position been by but the and variants the of the and of position as a determinant of P2 specificity. change in P2 specificity Ile99 was to and and in combination with the that residues and preferentially the by the number of For of the binding of to to because the binding of substrates at P2 are to each In a that the result for the variant, for the human and of granzyme P2 specificity J.K. S. J.L. Craik C.S. Biol. PubMed Scopus Google Scholar) where position is with proteases the specificity also the of the in the of and to the P2 by A. Bode W. Huber R. W. K. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). P2 substrate binding in A. A. K. E. Huber R. Bode W. 1999; Full Text Full Text PDF Scopus Google and of protein C is to an increased activity against substrates with a residue J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). The between interactions in the and substrate specificity that amino acids at structural to Ile99 of granzyme B in other trypsin-like serine proteases may result in selectivity for amino The other determinant with effects on specificity was Asn218. is unique to the granzyme B of serine proteases in granzyme as a bond for the amino acid at Asn218 resulted in variants with increased and specificity. This a in granzyme B specificity, the of substrates containing or amino acids and the of the P2 the amino acid at Arg192 is as the primary determinant as in of granzyme B J.L. Peterson E.P. Hudig D. Thornberry N.A. Craik C.S. J. Biol. Chem. 1998; 273: 27364-27373Abstract Full Text Full Text PDF PubMed Scopus (166) Google protein C C.T. J. Biol. Chem. Full Text PDF PubMed Google and C.T. Eur. J. Biochem. 1996; PubMed Scopus Google Scholar). in granzyme specificity is determined by amino acids and Arg192 against and substrate while the Asn218 as a to amino and amino As with Ile99, the in activity for substrates is primarily in that the is to in of the The of the that amino acids in with the extended substrate amino acids by to interactions to the extended specificity of the of the structural determinants the specificity profiles and the binding constant and the catalytic the hydrolysis of individual substrates by the variant proteases that the are primarily in selectivity catalytic power. Thus, the to specificity from the of substrates increased of substrates or an of the and between the extended substrate sites was also for granzyme B and its In granzyme an Ile or Val at the P4 position is required for efficient the that the catalytic on the identity of the P2 and P4 amino acids are completely with P2 amino acids have of the increase in activity of and on extended substrates. is that extended substrates increase by and of binding to A. PubMed Scopus Google Scholar). Thus, for granzyme B an catalytic where and structural effects to efficient of substrates by the of the Asn218 a in substrate specificity not to to an in the of a substrate for efficient the not alter the P4 specificity, the addition of to a on the N218A/I99A effects in substrate recognition by the between the P2 and P4 This also the of the identity of the P2 and P4 amino acids with wild the of may increase the number of of of the substrate This amino acids with to by the while efficient the between P2 and P4 amino acids that the at may have the of binding for The of Asn218 in granzyme B is to the substrate amino acid and to the catalytic activity of the By the amino acids that the active site of a granzyme B-like protease, of extended specificity been as a determinant of P2 specificity, and the combination of and defines specificity. the while and alter the P4 specificity. The remaining of the granzyme B have a of amino acids at the structural determinants in this study to alter and the to these remaining granzymes that granzymes C and have a P2 and or while granzyme H P2 amino extended specificity the specificities of the granzymes as unique distinct at extended that in the apoptotic and at of for the Thornberry and at for the and and for and