Crystal Structure of Human Cystatin D, a Cysteine Peptidase Inhibitor with Restricted Inhibition Profile

Cystatins are natural inhibitors of papain-like (family C1) and legumain-related (family C13) cysteine peptidases. Cystatin D is a type 2 cystatin, a secreted inhibitor found in human saliva and tear fluid. Compared with its homologues, cystatin D presents an unusual inhibition profile with a preferential inhibition cathepsin S > cathepsin H > cathepsin L and no inhibition of cathepsin B or pig legumain. To elucidate the structural reasons for this specificity, we have crystallized recombinant human Arg26-cystatin D and solved its structures at room temperature and at cryo conditions to 2.5- and 1.8-Å resolution, respectively. Human cystatin D presents the typical cystatin fold, with a five-stranded anti-parallel β-sheet wrapped around a five-turn α-helix. The structures reveal differences in the peptidase-interacting regions when compared with other cystatins, providing plausible explanations for the restricted inhibitory specificity of cystatin D for some papain-like peptidases and its lack of reactivity toward legumain-related enzymes. Cystatins are natural inhibitors of papain-like (family C1) and legumain-related (family C13) cysteine peptidases. Cystatin D is a type 2 cystatin, a secreted inhibitor found in human saliva and tear fluid. Compared with its homologues, cystatin D presents an unusual inhibition profile with a preferential inhibition cathepsin S > cathepsin H > cathepsin L and no inhibition of cathepsin B or pig legumain. To elucidate the structural reasons for this specificity, we have crystallized recombinant human Arg26-cystatin D and solved its structures at room temperature and at cryo conditions to 2.5- and 1.8-Å resolution, respectively. Human cystatin D presents the typical cystatin fold, with a five-stranded anti-parallel β-sheet wrapped around a five-turn α-helix. The structures reveal differences in the peptidase-interacting regions when compared with other cystatins, providing plausible explanations for the restricted inhibitory specificity of cystatin D for some papain-like peptidases and its lack of reactivity toward legumain-related enzymes. Cystatins are natural inhibitors of family C1 (papain-like) cysteine peptidases. In mammals, cystatins inhibit peptidases such as cathepsins B, H, K, L, and S both intra- and extracellularly following a reversible, tight binding mechanism (1Abrahamson M. Alvarez-Fernandez M. Nathanson C.M. Biochem. Soc. Symp. 2003; 70: 179-199Crossref PubMed Scopus (350) Google Scholar). The family C1 enzymes are involved in the normal lysosomal turnover of proteins, but they are also implicated in many disease processes, such as tumor invasion and connective tissue destruction on inflammation (2Sloane B.F. Moin K. Krepela E. Rozhin J. Cancer Metastasis Rev. 1990; 9: 333-352Crossref PubMed Scopus (188) Google Scholar, 3Mort J.S. Recklies A.D. Poole A.R. Arthritis Rheum. 1984; 27: 509-515Crossref PubMed Scopus (123) Google Scholar, 4Buttle D.J. Burnett D. Abrahamson M. Scand. J. Clin. Lab. Investig. 1990; 50: 509-516Crossref PubMed Google Scholar). The cystatins constitute a superfamily of related proteins. The mammalian superfamily members are of three major types (1Abrahamson M. Alvarez-Fernandez M. Nathanson C.M. Biochem. Soc. Symp. 2003; 70: 179-199Crossref PubMed Scopus (350) Google Scholar, 5Barrett A.J. Rawlings N.D. Davies M.E. Machleidt W. Salvesen G. Turk V. Barrett A.J. Salvesen G. Proteinase Inhibitors. 12. Elsevier Science Publishers BV, New York1986: 515-569Google Scholar, 6Rawlings N.D. Barrett A.J. J. Mol. Evol. 1990; 30: 60-71Crossref PubMed Scopus (270) Google Scholar). Type 1 cystatins (also called stefins) are primarily cytoplasmatic, single-domain proteins composed of ∼100 amino acid residues, with no disulfide bridges and no signal peptide. Type 2 cystatins are secreted inhibitors and are also single-domain proteins (but are about 120 residues long) and present two well-conserved disulfide bridges and typical signal peptides. Type 3 cystatins, or kininogens, are multidomain proteins presenting three tandemly repeated type 2 cystatin-like domains. Chicken egg-white (CEW) 1The abbreviations used are: CEW, chicken egg-white; BSL, backside loop; r.m.s.d., root mean square deviation; PDB, Protein Data Bank. cystatin, an avian type 2 cystatin, was the first cysteine peptidase inhibitor for which the three-dimensional structure was determined by x-ray crystallography (7Bode W. Engh R. Musil D. Thiele U. Huber R. Karshikov A. Brzin J. Kos J. Turk V. EMBO J. 1988; 7: 2593-2599Crossref PubMed Scopus (547) Google Scholar). The structures of two type 1 cystatins have also been determined: human cystatin A (or stefin A), determined by NMR spectroscopy (8Martin J.R. Craven C.J. Jerala R. Kroon-Zitko L. Zerovnik E. Turk V. Waltho J.P. J. Mol. Biol. 1995; 246: 331-343Crossref PubMed Scopus (102) Google Scholar) and recently by x-ray crystallography of a complex with cathepsin H (9Jenko S. Dolenc I. Guncar G. Dobersek A. Podobnik M. Turk D. J. Mol. Biol. 2003; 326: 875-885Crossref PubMed Scopus (97) Google Scholar); and human cystatin B (or stefin B) in complex with papain, determined by x-ray crystallography (10Stubbs M.T. Laber B. Bode W. Huber R. Jerala R. Lenarcic B. Turk V. EMBO J. 1990; 9: 1939-1947Crossref PubMed Scopus (470) Google Scholar). All three cystatins show the same overall structure, with a five-stranded anti-parallel β-sheet wrapped around a five-turn α-helix. In these cystatin structures the papain-binding site is a tripartite, wedge-shaped edge, formed by the N-terminal segment and the first and second hairpin loops, which are called L1 and L2. There is also a NMR model for the plant inhibitor oryzacystatin, which shows the same “cystatin fold” as the animal cystatins (11Nagata K. Kudo N. Abe K. Arai S. Tanokura M. Biochemistry. 2000; 39: 14753-14760Crossref PubMed Scopus (113) Google Scholar). In addition, the structure of a dimeric form of human cystatin C has been published (12Janowski R. Kozak M. Jankowska E. Grzonka Z. Grubb A. Abrahamson M. Jaskolski M. Nat. Struct. Biol. 2001; 8: 316-320Crossref PubMed Scopus (349) Google Scholar). Although this dimeric form of cystatin C is inactive as a papain inhibitor due to shedding of the binding site, each of the two domains formed by three-dimensional subdomain swapping the cystatin these structural of the specificity of cystatins is Cystatin D is a type 2 cystatin found in human saliva and tear J.P. M. Abrahamson M. G. Grubb A. J. Biol. PubMed Google Scholar). is as a of amino acid residues, of which the first residues constitute a typical signal J.P. Abrahamson M. I. G. Grubb A. J. Biol. PubMed Google Scholar). Cystatin D was found as the of a segment a of to the human cystatin C M. I. A. M. Grubb A. Biochem. J. 1990; PubMed Scopus Google Scholar). amino acid residues compared with the cystatin C with to for cysteine peptidase inhibition J.P. Abrahamson M. I. G. Grubb A. J. Biol. PubMed Google Scholar). the inhibition profile of cystatin D for human family C1 peptidases is of cystatin C (1Abrahamson M. Alvarez-Fernandez M. Nathanson C.M. Biochem. Soc. Symp. 2003; 70: 179-199Crossref PubMed Scopus (350) Google Scholar, M. A. Grubb A. Abrahamson M. J. Biol. PubMed Google Scholar). the cystatin D is to inhibit cathepsin B. shows a preferential inhibition of cathepsin S cathepsins H and L M. A. Grubb A. Abrahamson M. J. Biol. PubMed Google Scholar). a to residues in the N-terminal of cystatin D and has been these residues with the of the enzymes in a A. I. Grubb A. Abrahamson M. Biochemistry. PubMed Google Scholar). was N-terminal differences the specificity differences cystatins D and by of cystatins, was structural differences in the of the cystatin also have a on the inhibitory specificity of cystatin D A. I. Grubb A. Abrahamson M. Biochemistry. PubMed Google Scholar). was recently some type 2 cystatins inhibit mammalian a cysteine peptidase of family a site on the to the papain-binding site M. Barrett A.J. B. J. Abrahamson M. J. Biol. PubMed Scopus Google in a to as the site in tight inhibition of pig and is on human cystatins and but on cystatins B, and D. also with to inhibition of family cystatin D a restricted and inhibition profile other type 2 In the present we have crystallized and determined the three-dimensional structure of recombinant human cystatin with the of the structural reasons for its at and of Cystatin Arg26-cystatin D of the two present in in the M. J.P. Abrahamson M. G. Grubb A. PubMed Scopus Google Scholar) was in an as J.P. M. Abrahamson M. G. Grubb A. J. Biol. PubMed Google Scholar). the was by on a by on a to a The was 1 as and the was with The of and The was a with a of to a of Protein determined by spectroscopy at as A. I. Grubb A. Abrahamson M. Biochemistry. PubMed Google Scholar). The of the in was determined by and in PubMed Scopus Google Scholar) and B. Clin. PubMed Scopus Google respectively. the in of at was the 1 and 2 J. J. Scopus Google Scholar, R. S. K. A. Scholar) used in the of and of and used in in and at Data and temperature on a on a at in a for A was a conditions for as cryo The was with the in the of for a The was in a and in a at about K. at at the a A typical was with All the and Z. W. PubMed Scopus Google Scholar). structure of cystatin D was solved by the room temperature this was by cystatin (7Bode W. Engh R. Musil D. Thiele U. Huber R. Karshikov A. Brzin J. Kos J. Turk V. EMBO J. 1988; 7: 2593-2599Crossref PubMed Scopus (547) Google Scholar) as a such as and The J. 50: Scopus Google Scholar) and NMR J. M. Scopus Google Scholar) used for the of the and The was the on the room temperature to The room temperature structure was used as model for the on the cryo and of cystatins with structures was by the The was the structure with the of Protein in The structures used in the the Protein Data J. Z. G. 2000; PubMed Scopus Google cystatin, (7Bode W. Engh R. Musil D. Thiele U. Huber R. Karshikov A. Brzin J. Kos J. Turk V. EMBO J. 1988; 7: 2593-2599Crossref PubMed Scopus (547) Google Scholar); dimeric human cystatin (12Janowski R. Kozak M. Jankowska E. Grzonka Z. Grubb A. Abrahamson M. Jaskolski M. Nat. Struct. Biol. 2001; 8: 316-320Crossref PubMed Scopus (349) Google Scholar); cystatin (8Martin J.R. Craven C.J. Jerala R. Kroon-Zitko L. Zerovnik E. Turk V. Waltho J.P. J. Mol. Biol. 1995; 246: 331-343Crossref PubMed Scopus (102) Google Scholar); cystatin B, (10Stubbs M.T. Laber B. Bode W. Huber R. Jerala R. Lenarcic B. Turk V. EMBO J. 1990; 9: 1939-1947Crossref PubMed Scopus (470) Google Scholar); and oryzacystatin, (11Nagata K. Kudo N. Abe K. Arai S. Tanokura M. Biochemistry. 2000; 39: 14753-14760Crossref PubMed Scopus (113) Google Scholar). the amino acid used is of human cystatin cystatin C A. U. S. A. PubMed Scopus Google Scholar) is used for cystatin D and other cystatins in this as used for cystatin D and other human type 2 cystatins J.P. M. Abrahamson M. G. Grubb A. J. Biol. PubMed Google Scholar, J. Abrahamson M. M. Alvarez-Fernandez Grubb A. J. D. L. S. R. N. M. R. J. Biol. PubMed Scopus Google Scholar, J. L. J. Grubb A. J. R. Abrahamson M. J. Biol. PubMed Scopus Google Scholar). with the R. M. K. J. Mol. PubMed Scopus Google Scholar) and A. B. PubMed Scopus Google Scholar). of Cystatin D and Data human cystatin D the first in 1 with and 2 at on this at the same temperature by or as at a and and by the and and typical as or of the used for structure at in with and A room temperature was a with of about The and a a A second in was used to a at about K, and of of of in a was used as to the structure of cystatin D the room temperature was the structure of cystatin as J. 50: Scopus Google Scholar) and with and we the same which of which model was the two as the and both for The the The A for and New Scholar) with the room temperature to the to respectively. The was for The and and the residues of the model to the to model A of residues, to cystatin C and are in the room temperature structure was for the residues in the N-terminal segment in The around residues was to in the to the model and the was The was to the room temperature of cystatins with determined The is on a structural by of the human cystatin D structure and for type 1 and 2 cystatins cystatin human cystatin B, cystatin, human cystatin C the structure, and the plant cystatin, and are in and respectively. The papain-binding site is by in The the of the which is for the two disulfide bridges in type 2 Human cystatin C amino acid was with the residues in the cystatin D structure compared with the other cystatins The residues in at structural are in this but structural of cystatin D residues for residues in cystatin and human cystatin of the three cystatin D residues as the with in the L1 of cystatin residues to of the other two type 2 cystatins, with the in a to of cystatin D residues and The structure was for the cryo the room temperature structure was The cryo structure but in the The room temperature model was used for the on the cryo in the to was to the by and to a of to the cryo model the on the of the the room temperature and cryo structures to with root mean square on the of and an overall with of the C1 peptidase binding the when the cryo and room temperature structures are and for the of the N-terminal acid residues the L1 and the respectively. The for is to the the binding site, the for the of the is The the room temperature and cryo structures also a cystatin D the cystatin as its with structures (7Bode W. Engh R. Musil D. Thiele U. Huber R. Karshikov A. Brzin J. Kos J. Turk V. EMBO J. 1988; 7: 2593-2599Crossref PubMed Scopus (547) Google Scholar, J.R. Craven C.J. Jerala R. Kroon-Zitko L. Zerovnik E. Turk V. Waltho J.P. J. Mol. Biol. 1995; 246: 331-343Crossref PubMed Scopus (102) Google Scholar, M.T. Laber B. Bode W. Huber R. Jerala R. Lenarcic B. Turk V. EMBO J. 1990; 9: 1939-1947Crossref PubMed Scopus (470) Google Scholar, K. Kudo N. Abe K. Arai S. Tanokura M. Biochemistry. 2000; 39: 14753-14760Crossref PubMed Scopus (113) Google Scholar). The structure is a five-stranded anti-parallel β-sheet of and is wrapped around a five-turn and with cystatin C and some differences in the overall structure of cystatin the at the of the is in cystatin, which the of the Cystatin D present a in the of the second of the β-sheet around There is no in the of cystatin D. presents a is also the for the domains in the structure of dimeric human cystatin C (12Janowski R. Kozak M. Jankowska E. Grzonka Z. Grubb A. Abrahamson M. Jaskolski M. Nat. Struct. Biol. 2001; 8: 316-320Crossref PubMed Scopus (349) Google Scholar). differences in the peptidase-interacting regions of cystatin D are of the of the two proteins Cystatin D has a and a of the in the the β-sheet in cystatin D. the two cystatins with to the on In cystatin, and are on the In cystatin the presents some other are Human cystatin D is present in two natural due to a M. J.P. Abrahamson M. G. Grubb A. PubMed Scopus Google Scholar). has been this the binding of the inhibitor has on M. A. Grubb A. Abrahamson M. J. Biol. PubMed Google but the structural of the have been The form crystallized Arg26-cystatin in which the of the cystatin D J.P. M. Abrahamson M. G. Grubb A. J. Biol. PubMed Google Scholar) is has a of M. J.P. Abrahamson M. G. Grubb A. PubMed Scopus Google Scholar). the amino acid of cystatin D is 1 in the the of cystatin the is in an of cystatin The other natural form of human cystatin D of has in this other type 2 cystatins, for which is The present structure shows the in cystatin D is in the second of the α-helix. Although to at the of the its is toward the formed by the as by the The is by a with the of or in the of the The for the in the of is or in both the cryo and the room temperature a in the of these by they the two formed by and the in cystatin to form a with the in in cystatin the in the type 2 cystatins and also form a with or found in The to an for of the of some type 2 In the cystatin D with as the the and the and its an for the the is to both in E. and saliva the M. A. Grubb A. Abrahamson M. J. Biol. PubMed Google Scholar). by M. A. Grubb A. Abrahamson M. J. Biol. PubMed Google the involved in disulfide with other proteins in The present the with its to The for papain-binding site in cystatins is by three well-conserved the N-terminal a hairpin in the and toward the of the M. A. Grubb A. Machleidt W. Barrett A.J. J. Biol. PubMed Google Scholar, M. D.J. Grubb A. K. Biochem. J. PubMed Scopus Google Scholar, G. I. Machleidt W. Engh J. Biochem. PubMed Scopus Google Scholar, I. I. Abrahamson M. Biochem. J. 1995; PubMed Scopus Google Scholar, I. I. E. E. K. Abrahamson M. Biochemistry. PubMed Scopus Google Scholar, A. Grubb A. Abrahamson M. Biochem. J. PubMed Scopus Google Scholar, A. K. Grubb A. Abrahamson M. J. Biol. 1995; PubMed Scopus Google Scholar, M. K. E. I. Biochem. J. PubMed Scopus Google Scholar, D. Abrahamson M. J.S. Biochemistry. PubMed Scopus Google Scholar, K. Abrahamson M. Biochem. J. PubMed Scopus Google Scholar). form a wedge-shaped (7Bode W. Engh R. Musil D. Thiele U. Huber R. Karshikov A. Brzin J. Kos J. Turk V. EMBO J. 1988; 7: 2593-2599Crossref PubMed Scopus (547) Google Scholar, J.R. Craven C.J. Jerala R. Kroon-Zitko L. Zerovnik E. Turk V. Waltho J.P. J. Mol. Biol. 1995; 246: 331-343Crossref PubMed Scopus (102) Google Scholar, M.T. Laber B. Bode W. Huber R. Jerala R. Lenarcic B. Turk V. EMBO J. 1990; 9: 1939-1947Crossref PubMed Scopus (470) Google Scholar, K. Kudo N. Abe K. Arai S. Tanokura M. Biochemistry. 2000; 39: 14753-14760Crossref PubMed Scopus (113) Google Scholar, L. M. Kos J. Turk V. R. J. Mol. Biol. PubMed Scopus Google Scholar) the site in a (10Stubbs M.T. Laber B. Bode W. Huber R. Jerala R. Lenarcic B. Turk V. EMBO J. 1990; 9: 1939-1947Crossref PubMed Scopus (470) Google Scholar, W. Engh R. Musil D. Laber B. M. Huber R. Turk V. Biol. 1990; PubMed Scopus Google Scholar). In the cystatin D structure, the papain-binding are in such a in with the other cystatin The for the N-terminal segment in cystatin D is and the structure model about the for the N-terminal segment of this cystatin and family C1 peptidases. In the room temperature the first for which is is In the cryo model for cystatin this is In the structures of cystatin and human cystatin the first residues with are in cystatin and (7Bode W. Engh R. Musil D. Thiele U. Huber R. Karshikov A. Brzin J. Kos J. Turk V. EMBO J. 1988; 7: 2593-2599Crossref PubMed Scopus (547) Google Scholar, R. Kozak M. Jankowska E. Grzonka Z. Grubb A. Abrahamson M. Jaskolski M. Nat. Struct. Biol. 2001; 8: 316-320Crossref PubMed Scopus (349) Google Scholar). a N-terminal segment in type 2 the other of the papain-binding site, are some differences in the cystatin D structure compared with of cystatin, The L1 of the binding site the cystatin and is and L1 in cystatin D is and a form in cystatin is a of the around in the is present in cystatin structures solved in human cystatins and and the plant cystatin, The a of 1 amino acid in the of cystatin when the of J. L. J. Grubb A. J. R. Abrahamson M. J. Biol. PubMed Scopus Google Scholar) to the three-dimensional structure on the present in at the of the papain-binding cystatin the is in the L1 its The of the binding site as for cystatin, a hairpin and The to the in the structure of cystatin a of of the of and the wedge-shaped formed by the three in the of the on the papain-binding site and of cystatin is In cystatin the of and in the structural and differences in the papain-binding site of cystatin D compared with other cystatins are the to the inhibition profile of cystatin D M. A. Grubb A. Abrahamson M. J. Biol. PubMed Google Scholar). In with other cystatins, is a of cystatin D papain by a M. K. E. I. Biochem. J. PubMed Scopus Google no structural in the inhibitor or the peptidase on a model of papain inhibition by cystatin by cystatin D in the structure of the cystatin B B) complex with papain (10Stubbs M.T. Laber B. Bode W. Huber R. Jerala R. Lenarcic B. Turk V. EMBO J. 1990; 9: 1939-1947Crossref PubMed Scopus (470) Google Scholar) and both cystatins the peptidase site in a cystatin D the of papain-like enzymes in an as cystatin B the N-terminal segment the as in the of the L1 in with the residues the in the and with the of the the in cystatin D with the of and in papain, as is the for the in other type 2 Cystatin D and papain the of the site of papain and the of cystatin D each other by the of cystatin D are the the cystatin D the site in the same as cystatin B in to the of with the we the of in the inhibitor to with the of the with cystatin D to the as as other cystatins in the other cystatin involved in binding and the peptidase the papain of cystatin D compared with cystatin and human cystatin C (1Abrahamson M. Alvarez-Fernandez M. Nathanson C.M. Biochem. Soc. Symp. 2003; 70: 179-199Crossref PubMed Scopus (350) Google Scholar). L1 as as the to as many with papain as by to the of binding of other have a form of human cystatin C the first residues of the N-terminal segment has 3 of for papain and other family C1 peptidases cystatin C M. D.J. Grubb A. K. Biochem. J. PubMed Scopus Google Scholar, A. Grubb A. Abrahamson M. Biochem. J. PubMed Scopus Google Scholar). The same in is for its A. K. Grubb A. Abrahamson M. J. Biol. 1995; PubMed Scopus Google Scholar). The of cystatin D for papain in M. Alvarez-Fernandez M. Nathanson C.M. Biochem. Soc. Symp. 2003; 70: 179-199Crossref PubMed Scopus (350) Google Scholar) is of for cystatin due to the L1 of cystatin D in this but also in the N-terminal in the In addition, the the in the inhibitor and the and we the structures of cystatin D and cathepsin B D. D. Turk D. Engh I. Huber R. Turk V. N. Bode W. EMBO J. PubMed Scopus Google the complex cystatin B and papain as for the to the inhibition of papain by cystatins, cathepsin B is in a M. S. J.S. Abrahamson M. I. PubMed Scopus Google Scholar). by M. S. J.S. Abrahamson M. I. PubMed Scopus Google the first is by the of the N-terminal of the cystatin in the S is by of the of cathepsin B as the cystatin in to the L1 and in the have the N-terminal segment of cystatin D is for this the N-terminal segment of cystatin C A. K. Grubb A. Abrahamson M. J. Biol. 1995; PubMed Scopus Google Scholar). In when the N-terminal segment of cystatin D was on to a cystatin C in a cystatin this for cathepsin B cystatin the A. K. Grubb A. Abrahamson M. J. Biol. 1995; PubMed Scopus Google Scholar). the other the of the N-terminal segment of cystatin C cystatin D in the of cystatin D to inhibit cathepsin B A. K. Grubb A. Abrahamson M. J. Biol. 1995; PubMed Scopus Google Scholar). The is due to the cystatin with the N-terminal segment of cystatin to the and to the peptidase with the other two of the inhibitory L1 and is due to the of in the inhibitor to in the of cathepsin B. the of cystatin D is by the of two in and in an by the also in the site of cathepsin B, cystatin D was to with the in the same as cystatin B B) with papain (10Stubbs M.T. Laber B. Bode W. Huber R. Jerala R. Lenarcic B. Turk V. EMBO J. 1990; 9: 1939-1947Crossref PubMed Scopus (470) Google Scholar). In the same cystatin and human cystatin C present In the of cystatin B, presents a in the its with cystatin the The for recently some type 2 cystatins are to inhibit mammalian M. Barrett A.J. B. J. Abrahamson M. J. Biol. PubMed Scopus Google a lysosomal cysteine of family M. Barrett A.J. Biochem. J. PubMed Scopus (113) Google which shows for an M. L. Barrett A.J. Biochem. J. PubMed Scopus Google Scholar). The at the of the A and was as involved in inhibition by these In for an inhibitory mechanism in which cystatins inhibit mammalian in a M. Barrett A.J. B. J. Abrahamson M. J. Biol. PubMed Scopus Google Scholar). the cystatins cystatins and and a type 2 cystatin the B. Biol. 2001; PubMed Scopus Google cystatin D presents an in this and cystatin D is the human type 2 cystatin inhibit mammalian M. Barrett A.J. B. J. Abrahamson M. J. Biol. PubMed Scopus Google Scholar). in other type 2 cystatins is involved in we and compared the structures in the of cystatin D and cystatin the of which is a tight binding inhibitor of pig Rawlings N.D. E. Barrett A.J. J. Biol. PubMed Scopus Google Scholar). was in an to a plausible of cystatin D is inactive as a cystatin the structural differences as the in cystatin D is at the of the is at a to in the other type 2 cystatin structures in toward the of the and is to at the of the is an of 1 amino acid in the The and the the and residues in the segment of cystatin and in cystatin is by the by the in the of the and in the of the a of this the the and the Cystatin D presents a at the to in of the to involved in inhibition in other type 2 cystatins M. Barrett A.J. B. J. Abrahamson M. J. Biol. PubMed Scopus Google Scholar). is toward the and is on the A and is for the of with an in the M. L. Barrett A.J. Biochem. J. PubMed Scopus Google for residues in Although for is for the in the is to show for the in cystatin D. by the is in the of cystatin D and as in cystatin C is cystatin D have as an inhibitor of enzymes with a for binding in The inhibition of by some cystatins enzymes with overall to of family with the binding for cystatin D enzymes the of family the which have a for in R. W. J. J. J. Biol. PubMed Google Scholar, N. J. V. J. J. Biol. PubMed Scopus Google Scholar). is to cystatin D as a inhibitor of these or enzymes in saliva and have a to inhibit the and of In the present we have determined the structure of recombinant human cystatin D by x-ray crystallography both room temperature and cryo conditions and resolution, type 2 cystatin is as in the as its cystatin but is restricted to saliva and tear J.P. M. Abrahamson M. G. Grubb A. J. Biol. PubMed Google Scholar). to a restricted of cystatin D of a papain-like lysosomal peptidases tumor or as has been for cystatin cystatin C is a inhibitory family C1 peptidases specificity, cystatin D shows a restricted inhibition profile with for cathepsin S > cathepsin H > cathepsin L and no inhibition of cathepsin B or pig in family M. A. Grubb A. Abrahamson M. J. Biol. PubMed Google Scholar, M. Barrett A.J. B. J. Abrahamson M. J. Biol. PubMed Scopus Google Scholar). restricted inhibition profile cystatin D a for at an of the inhibitory specificity of