Rajesh Shenoy - Academia.edu (original) (raw)
Papers by Rajesh Shenoy
PLoS Genetics, 2013
Sexually reproducing organisms halve their cellular ploidy during gametogenesis by undergoing a s... more Sexually reproducing organisms halve their cellular ploidy during gametogenesis by undergoing a specialized form of cell division known as meiosis. During meiosis, a single round of DNA replication is followed by two rounds of nuclear divisions (referred to as meiosis I and II). While sister kinetochores bind to microtubules emanating from opposite spindle poles during mitosis, they bind to microtubules originating from the same spindle pole during meiosis I. This phenomenon is referred to as mono-orientation and is essential for setting up the reductional mode of chromosome segregation during meiosis I. In budding yeast, mono-orientation depends on a four component protein complex referred to as monopolin which consists of two nucleolar proteins Csm1 and Lrs4, meiosis-specific protein Mam1 of unknown function and casein kinase Hrr25. Monopolin complex binds to kinetochores during meiosis I and prevents bipolar attachments. Although monopolin associates with kinetochores during meiosis I, its binding site(s) on the kinetochore is not known and its mechanism of action has not been established. By carrying out an imaging-based screen we have found that the MIND complex, a component of the central kinetochore, is required for monopolin association with kinetochores during meiosis. Furthermore, we demonstrate that interaction of monopolin subunit Csm1 with the N-terminal domain of MIND complex subunit Dsn1, is essential for both the association of monopolin with kinetochores and for monopolar attachment of sister kinetochores during meiosis I. As such this provides the first functional evidence for a monopolin-binding site at the kinetochore.
Nucleic Acids Research, 2010
Sgm (Sisomicin-gentamicin methyltransferase) from antibiotic-producing bacterium Micromonospora z... more Sgm (Sisomicin-gentamicin methyltransferase) from antibiotic-producing bacterium Micromonospora zionensis is an enzyme that confers resistance to aminoglycosides like gentamicin and sisomicin by specifically methylating G1405 in bacterial 16S rRNA. Sgm belongs to the aminoglycoside resistance methyltransferase (Arm) family of enzymes that have been recently found to spread by horizontal gene transfer among disease-causing bacteria. Structural characterization of Arm enzymes is the key to understand their mechanism of action and to develop inhibitors that would block their activity. Here we report the structure of Sgm in complex with cofactors S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) at 2.0 and 2.1 Å resolution, respectively, and results of mutagenesis and rRNA footprinting, and protein-substrate docking. We propose the mechanism of methylation of G1405 by Sgm and compare it with other m 7 G methyltransferases, revealing a surprising diversity of active sites and binding modes for the same basic reaction of RNA modification. This analysis can serve as a stepping stone towards developing drugs that would specifically block the activity of Arm methyltransferases and thereby re-sensitize pathogenic bacteria to aminoglycoside antibiotics.
A proteomic study of mitotic phase-specific interactors of EB1 reveals a role for SXIP mediated p... more A proteomic study of mitotic phase-specific interactors of EB1 reveals a role for SXIP mediated protein interactions in anaphase onset. Tamura N, Simon JE, Nayak A, Shenoy RT, Hiroi N, Boilot V, Funahashi A, Draviam VM. Biol Open. 2015 Jan 16;4(2):15569.
Monopolin subunit Csm1 associates with MIND complex to establish monopolar attachment of sister kinetochores at meiosis I. Sarkar S, Shenoy RT(coauthor), Dalgaard JZ, Newnham L, Hoffmann E, Millar JB, Arumugam P. PLoS Genet . 2013 Jul;9(7):e1003610.
Structural basis for dual inhibition mechanism of a nonclassical Kazal-type serine protease inhibitor from horseshoe crab in complex with subtilisin. Shenoy RT, Thangamani S, Velazquez Campoy A, Ho B, Ding JL, Sivaraman J. PLoS One . 2011 Apr 26;6(4):e18838.
Structural basis for reversible and irreversible inhibition of human cathepsin L by their respective dipeptidyl glyoxal
and diazomethylketone inhibitors. Shenoy RT, Sivaraman J. J Struct Biol . 2011 Jan; 173(1):149.
Structural basis for the methylation of G1405 in 16S rRNA by aminoglycoside resistance methyltransferase Sgm from an antibiotic producer: a diversity of active sites in m7G methyltransferases. Husain N, Tkaczuk KL, Shenoy RT, Kaminska KH, Cubrilo S, Maravic Vlahovicek G, Bujnicki JM, Sivaraman J. Nucleic Acids Res. 2010 Jul; 8(12):412032.
Modifying the substrate specificity of Carcinoscorpius rotundicauda serine protease inhibitor domain 1 to target thrombin. Giri PK, Tang X, Thangamani S, Shenoy RT, Ding JL, Swaminathan K, Sivaraman J. PLoS One. 2010 Dec 20;5(12):e15258.
Crystallization of a nonclassical Kazal-type Carcinoscorpius rotundicauda serine protease inhibitor, CrSPI1,
complexed with subtilisin. Shenoy RT, Thangamani S, Ho B, Sivaraman J, Ding JL. Acta Crystallogr Sect F Struct Biol
Cryst Commun. 2009 May 1;65(Pt 5):5335.
A combined crystallographic and molecular dynamics study of cathepsin L retrobinding inhibitors. Shenoy RT, Chowdhury SF, Kumar S, Joseph L, Purisima EO, Sivaraman J. J Med Chem . 2009 Oct 22;52(20):633546.
Exploring inhibitor binding at the S' subsites of cathepsin L. Chowdhury SF, Joseph L, Kumar S, S henoy RT, Bhat S, Ziomek E, Ménard R, Sivaraman J, Purisima EO. J Med Chem . 2008 Mar 13; 51(5):13618.
Biology open, 2015
Microtubules execute diverse mitotic events that are spatially and temporally separated; the unde... more Microtubules execute diverse mitotic events that are spatially and temporally separated; the underlying regulation is poorly understood. By combining drug treatments, large-scale immunoprecipitation and mass spectrometry, we report the first comprehensive map of mitotic phase-specific protein interactions of the microtubule-end binding protein, EB1. EB1 interacts with some, but not all, of its partners throughout mitosis. We show that the interaction of EB1 with Astrin-SKAP complex, a key regulator of chromosome segregation, is enhanced during prometaphase, compared to anaphase. We find that EB1 and EB3, another EB family member, can interact directly with SKAP, in an SXIP-motif dependent manner. Using an SXIP defective mutant that cannot interact with EB, we uncover two distinct pools of SKAP at spindle microtubules and kinetochores. We demonstrate the importance of SKAP's SXIP-motif in controlling microtubule growth rates and anaphase onset, without grossly disrupting spindle ...
Journal of Structural Biology, 2011
Cathepsin L plays a key role in many pathophysiological conditions including rheumatoid arthritis... more Cathepsin L plays a key role in many pathophysiological conditions including rheumatoid arthritis, tumor invasion and metastasis, bone resorption and remodeling. Here we report the crystal structures of two analogous dipeptidyl inhibitor complexes which inhibit human cathepsin L in reversible and irreversible modes, respectively. To-date, there are no crystal structure reports of complexes of proteases with their glyoxal inhibitors or complexes of cathepsin L and their diazomethylketone inhibitors. These two inhibitors -inhibitor 1, an a-keto-b-aldehyde and inhibitor 2, a diazomethylketone, have different groups in the S1 subsite. Inhibitor 1 [Z-Phe-Tyr (OBut)-COCHO], with a K i of 0.6 nM, is the most potent, reversible, synthetic peptidyl inhibitor of cathepsin L reported to-date. The structure of the inhibitor 1 complex was refined up to 2.2 Å resolution. The structure of the complex of the inhibitor 2 [Z-Phe-Tyr (t-Bu)-diazomethylketone], an irreversible inhibitor that can inactivate cathepsin L at lM concentrations, was refined up to 1.76 Å resolution. These two inhibitors have substrate-like interactions with the active site cysteine (Cys25). Inhibitor 1 forms a tetrahedral hemithioacetal adduct, whereas the inhibitor 2 forms a thioester with Cys25. The inhibitor 1 b-aldehyde group is shown to make a hydrogen bond with catalytic His163, whereas the ketone carbonyl oxygen of the inhibitor 2 interacts with the oxyanion hole. tert-Butyl groups of both inhibitors are found to make several non-polar contacts with S 0 subsite residues of cathepsin L. These studies, combined with other complex structures of cathepsin L, reveal the structural basis for their potency and selectivity.
PLoS ONE, 2011
Serine proteases play a crucial role in host-pathogen interactions. In the innate immune system o... more Serine proteases play a crucial role in host-pathogen interactions. In the innate immune system of invertebrates, multidomain protease inhibitors are important for the regulation of host-pathogen interactions and antimicrobial activities. Serine protease inhibitors, 9.3-kDa CrSPI isoforms 1 and 2, have been identified from the hepatopancreas of the horseshoe crab, Carcinoscorpius rotundicauda. The CrSPIs were biochemically active, especially CrSPI-1, which potently inhibited subtilisin (Ki = 1.43 nM). CrSPI has been grouped with the non-classical Kazal-type inhibitors due to its unusual cysteine distribution. Here we report the crystal structure of CrSPI-1 in complex with subtilisin at 2.6 Å resolution and the results of biophysical interaction studies. The CrSPI-1 molecule has two domains arranged in an extended conformation. These two domains act as heads that independently interact with two separate subtilisin molecules, resulting in the inhibition of subtilisin activity at a ratio of 1:2 (inhibitor to protease). Each subtilisin molecule interacts with the reactive site loop from each domain of CrSPI-1 through a standard canonical binding mode and forms a single ternary complex. In addition, we propose the substrate preferences of each domain of CrSPI-1. Domain 2 is specific towards the bacterial protease subtilisin, while domain 1 is likely to interact with the host protease, Furin. Elucidation of the structure of the CrSPI-1: subtilisin (1:2) ternary complex increases our understanding of host-pathogen interactions in the innate immune system at the molecular level and provides new strategies for immunomodulation.
PLoS ONE, 2010
Protease inhibitors play a decisive role in maintaining homeostasis and eliciting antimicrobial a... more Protease inhibitors play a decisive role in maintaining homeostasis and eliciting antimicrobial activities. Invertebrates like the horseshoe crab have developed unique modalities with serine protease inhibitors to detect and respond to microbial and host proteases. Two isoforms of an immunomodulatory two-domain Kazal-like serine protease inhibitor, CrSPI-1 and CrSPI-2, have been recently identified in the hepatopancreas of the horseshoe crab, Carcinoscorpius rotundicauda. Full length and domain 2 of CrSPI-1 display powerful inhibitory activities against subtilisin. However, the structure and function of CrSPI-1 domain-1 (D1) remain unknown. Here, we report the crystal structure of CrSPI-1-D1 refined up to 2.0 Å resolution. Despite the close structural homology of CrSPI-1-D1 to rhodniin-D1 (a known thrombin inhibitor), the CrSPI-1-D1 does not inhibit thrombin. This prompted us to modify the selectivity of CrSPI-1-D1 specifically towards thrombin. We illustrate the use of structural information of CrSPI-1-D1 to modify this domain into a potent thrombin inhibitor with IC 50 of 26.3 nM. In addition, these studies demonstrate that, besides the rigid conformation of the reactive site loop of the inhibitor, the sequence is the most important determinant of the specificity of the inhibitor. This study will lead to the significant application to modify a multi-domain inhibitor protein to target several proteases.
Journal of Medicinal Chemistry, 2009
These authors contributed equally to the work.
Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2009
Serine proteases play a major role in host-pathogen interactions. The innate immune system is kno... more Serine proteases play a major role in host-pathogen interactions. The innate immune system is known to respond to invading pathogens in a nonspecific manner. The serine protease cascade is an essential component of the innate immune system of the horseshoe crab. The serine protease inhibitor CrSPI isoform 1 (CrSPI-1), a unique nonclassical Kazal-type inhibitor of molecular weight 9.3 kDa, was identified from the hepatopancreas of the horseshoe crab Carcinoscorpius rotundicauda. It potently inhibits subtilisin and constitutes a powerful innate immune defence against invading microbes. Here, the cloning, expression, purification and cocrystallization of CrSPI-1 with subtilisin are reported. The crystals diffracted to 2.6 Å resolution and belonged to space group P2 1 , with unit-cell parameters a = 73.8, b = 65.0, c = 111.9 Å , = 95.4 . The Matthews coefficient (V M = 2.64 Å 3 Da À1 , corresponding to 53% solvent content) and analysis of the preliminary structure solution indicated the presence of one heterotrimer (1:2 ratio of CrSPI-1:subtilisin) and one free subtilisin molecule in the asymmetric unit.
PLoS Genetics, 2013
Sexually reproducing organisms halve their cellular ploidy during gametogenesis by undergoing a s... more Sexually reproducing organisms halve their cellular ploidy during gametogenesis by undergoing a specialized form of cell division known as meiosis. During meiosis, a single round of DNA replication is followed by two rounds of nuclear divisions (referred to as meiosis I and II). While sister kinetochores bind to microtubules emanating from opposite spindle poles during mitosis, they bind to microtubules originating from the same spindle pole during meiosis I. This phenomenon is referred to as mono-orientation and is essential for setting up the reductional mode of chromosome segregation during meiosis I. In budding yeast, mono-orientation depends on a four component protein complex referred to as monopolin which consists of two nucleolar proteins Csm1 and Lrs4, meiosis-specific protein Mam1 of unknown function and casein kinase Hrr25. Monopolin complex binds to kinetochores during meiosis I and prevents bipolar attachments. Although monopolin associates with kinetochores during meiosis I, its binding site(s) on the kinetochore is not known and its mechanism of action has not been established. By carrying out an imaging-based screen we have found that the MIND complex, a component of the central kinetochore, is required for monopolin association with kinetochores during meiosis. Furthermore, we demonstrate that interaction of monopolin subunit Csm1 with the N-terminal domain of MIND complex subunit Dsn1, is essential for both the association of monopolin with kinetochores and for monopolar attachment of sister kinetochores during meiosis I. As such this provides the first functional evidence for a monopolin-binding site at the kinetochore.
Nucleic Acids Research, 2010
Sgm (Sisomicin-gentamicin methyltransferase) from antibiotic-producing bacterium Micromonospora z... more Sgm (Sisomicin-gentamicin methyltransferase) from antibiotic-producing bacterium Micromonospora zionensis is an enzyme that confers resistance to aminoglycosides like gentamicin and sisomicin by specifically methylating G1405 in bacterial 16S rRNA. Sgm belongs to the aminoglycoside resistance methyltransferase (Arm) family of enzymes that have been recently found to spread by horizontal gene transfer among disease-causing bacteria. Structural characterization of Arm enzymes is the key to understand their mechanism of action and to develop inhibitors that would block their activity. Here we report the structure of Sgm in complex with cofactors S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) at 2.0 and 2.1 Å resolution, respectively, and results of mutagenesis and rRNA footprinting, and protein-substrate docking. We propose the mechanism of methylation of G1405 by Sgm and compare it with other m 7 G methyltransferases, revealing a surprising diversity of active sites and binding modes for the same basic reaction of RNA modification. This analysis can serve as a stepping stone towards developing drugs that would specifically block the activity of Arm methyltransferases and thereby re-sensitize pathogenic bacteria to aminoglycoside antibiotics.
A proteomic study of mitotic phase-specific interactors of EB1 reveals a role for SXIP mediated p... more A proteomic study of mitotic phase-specific interactors of EB1 reveals a role for SXIP mediated protein interactions in anaphase onset. Tamura N, Simon JE, Nayak A, Shenoy RT, Hiroi N, Boilot V, Funahashi A, Draviam VM. Biol Open. 2015 Jan 16;4(2):15569.
Monopolin subunit Csm1 associates with MIND complex to establish monopolar attachment of sister kinetochores at meiosis I. Sarkar S, Shenoy RT(coauthor), Dalgaard JZ, Newnham L, Hoffmann E, Millar JB, Arumugam P. PLoS Genet . 2013 Jul;9(7):e1003610.
Structural basis for dual inhibition mechanism of a nonclassical Kazal-type serine protease inhibitor from horseshoe crab in complex with subtilisin. Shenoy RT, Thangamani S, Velazquez Campoy A, Ho B, Ding JL, Sivaraman J. PLoS One . 2011 Apr 26;6(4):e18838.
Structural basis for reversible and irreversible inhibition of human cathepsin L by their respective dipeptidyl glyoxal
and diazomethylketone inhibitors. Shenoy RT, Sivaraman J. J Struct Biol . 2011 Jan; 173(1):149.
Structural basis for the methylation of G1405 in 16S rRNA by aminoglycoside resistance methyltransferase Sgm from an antibiotic producer: a diversity of active sites in m7G methyltransferases. Husain N, Tkaczuk KL, Shenoy RT, Kaminska KH, Cubrilo S, Maravic Vlahovicek G, Bujnicki JM, Sivaraman J. Nucleic Acids Res. 2010 Jul; 8(12):412032.
Modifying the substrate specificity of Carcinoscorpius rotundicauda serine protease inhibitor domain 1 to target thrombin. Giri PK, Tang X, Thangamani S, Shenoy RT, Ding JL, Swaminathan K, Sivaraman J. PLoS One. 2010 Dec 20;5(12):e15258.
Crystallization of a nonclassical Kazal-type Carcinoscorpius rotundicauda serine protease inhibitor, CrSPI1,
complexed with subtilisin. Shenoy RT, Thangamani S, Ho B, Sivaraman J, Ding JL. Acta Crystallogr Sect F Struct Biol
Cryst Commun. 2009 May 1;65(Pt 5):5335.
A combined crystallographic and molecular dynamics study of cathepsin L retrobinding inhibitors. Shenoy RT, Chowdhury SF, Kumar S, Joseph L, Purisima EO, Sivaraman J. J Med Chem . 2009 Oct 22;52(20):633546.
Exploring inhibitor binding at the S' subsites of cathepsin L. Chowdhury SF, Joseph L, Kumar S, S henoy RT, Bhat S, Ziomek E, Ménard R, Sivaraman J, Purisima EO. J Med Chem . 2008 Mar 13; 51(5):13618.
Biology open, 2015
Microtubules execute diverse mitotic events that are spatially and temporally separated; the unde... more Microtubules execute diverse mitotic events that are spatially and temporally separated; the underlying regulation is poorly understood. By combining drug treatments, large-scale immunoprecipitation and mass spectrometry, we report the first comprehensive map of mitotic phase-specific protein interactions of the microtubule-end binding protein, EB1. EB1 interacts with some, but not all, of its partners throughout mitosis. We show that the interaction of EB1 with Astrin-SKAP complex, a key regulator of chromosome segregation, is enhanced during prometaphase, compared to anaphase. We find that EB1 and EB3, another EB family member, can interact directly with SKAP, in an SXIP-motif dependent manner. Using an SXIP defective mutant that cannot interact with EB, we uncover two distinct pools of SKAP at spindle microtubules and kinetochores. We demonstrate the importance of SKAP's SXIP-motif in controlling microtubule growth rates and anaphase onset, without grossly disrupting spindle ...
Journal of Structural Biology, 2011
Cathepsin L plays a key role in many pathophysiological conditions including rheumatoid arthritis... more Cathepsin L plays a key role in many pathophysiological conditions including rheumatoid arthritis, tumor invasion and metastasis, bone resorption and remodeling. Here we report the crystal structures of two analogous dipeptidyl inhibitor complexes which inhibit human cathepsin L in reversible and irreversible modes, respectively. To-date, there are no crystal structure reports of complexes of proteases with their glyoxal inhibitors or complexes of cathepsin L and their diazomethylketone inhibitors. These two inhibitors -inhibitor 1, an a-keto-b-aldehyde and inhibitor 2, a diazomethylketone, have different groups in the S1 subsite. Inhibitor 1 [Z-Phe-Tyr (OBut)-COCHO], with a K i of 0.6 nM, is the most potent, reversible, synthetic peptidyl inhibitor of cathepsin L reported to-date. The structure of the inhibitor 1 complex was refined up to 2.2 Å resolution. The structure of the complex of the inhibitor 2 [Z-Phe-Tyr (t-Bu)-diazomethylketone], an irreversible inhibitor that can inactivate cathepsin L at lM concentrations, was refined up to 1.76 Å resolution. These two inhibitors have substrate-like interactions with the active site cysteine (Cys25). Inhibitor 1 forms a tetrahedral hemithioacetal adduct, whereas the inhibitor 2 forms a thioester with Cys25. The inhibitor 1 b-aldehyde group is shown to make a hydrogen bond with catalytic His163, whereas the ketone carbonyl oxygen of the inhibitor 2 interacts with the oxyanion hole. tert-Butyl groups of both inhibitors are found to make several non-polar contacts with S 0 subsite residues of cathepsin L. These studies, combined with other complex structures of cathepsin L, reveal the structural basis for their potency and selectivity.
PLoS ONE, 2011
Serine proteases play a crucial role in host-pathogen interactions. In the innate immune system o... more Serine proteases play a crucial role in host-pathogen interactions. In the innate immune system of invertebrates, multidomain protease inhibitors are important for the regulation of host-pathogen interactions and antimicrobial activities. Serine protease inhibitors, 9.3-kDa CrSPI isoforms 1 and 2, have been identified from the hepatopancreas of the horseshoe crab, Carcinoscorpius rotundicauda. The CrSPIs were biochemically active, especially CrSPI-1, which potently inhibited subtilisin (Ki = 1.43 nM). CrSPI has been grouped with the non-classical Kazal-type inhibitors due to its unusual cysteine distribution. Here we report the crystal structure of CrSPI-1 in complex with subtilisin at 2.6 Å resolution and the results of biophysical interaction studies. The CrSPI-1 molecule has two domains arranged in an extended conformation. These two domains act as heads that independently interact with two separate subtilisin molecules, resulting in the inhibition of subtilisin activity at a ratio of 1:2 (inhibitor to protease). Each subtilisin molecule interacts with the reactive site loop from each domain of CrSPI-1 through a standard canonical binding mode and forms a single ternary complex. In addition, we propose the substrate preferences of each domain of CrSPI-1. Domain 2 is specific towards the bacterial protease subtilisin, while domain 1 is likely to interact with the host protease, Furin. Elucidation of the structure of the CrSPI-1: subtilisin (1:2) ternary complex increases our understanding of host-pathogen interactions in the innate immune system at the molecular level and provides new strategies for immunomodulation.
PLoS ONE, 2010
Protease inhibitors play a decisive role in maintaining homeostasis and eliciting antimicrobial a... more Protease inhibitors play a decisive role in maintaining homeostasis and eliciting antimicrobial activities. Invertebrates like the horseshoe crab have developed unique modalities with serine protease inhibitors to detect and respond to microbial and host proteases. Two isoforms of an immunomodulatory two-domain Kazal-like serine protease inhibitor, CrSPI-1 and CrSPI-2, have been recently identified in the hepatopancreas of the horseshoe crab, Carcinoscorpius rotundicauda. Full length and domain 2 of CrSPI-1 display powerful inhibitory activities against subtilisin. However, the structure and function of CrSPI-1 domain-1 (D1) remain unknown. Here, we report the crystal structure of CrSPI-1-D1 refined up to 2.0 Å resolution. Despite the close structural homology of CrSPI-1-D1 to rhodniin-D1 (a known thrombin inhibitor), the CrSPI-1-D1 does not inhibit thrombin. This prompted us to modify the selectivity of CrSPI-1-D1 specifically towards thrombin. We illustrate the use of structural information of CrSPI-1-D1 to modify this domain into a potent thrombin inhibitor with IC 50 of 26.3 nM. In addition, these studies demonstrate that, besides the rigid conformation of the reactive site loop of the inhibitor, the sequence is the most important determinant of the specificity of the inhibitor. This study will lead to the significant application to modify a multi-domain inhibitor protein to target several proteases.
Journal of Medicinal Chemistry, 2009
These authors contributed equally to the work.
Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2009
Serine proteases play a major role in host-pathogen interactions. The innate immune system is kno... more Serine proteases play a major role in host-pathogen interactions. The innate immune system is known to respond to invading pathogens in a nonspecific manner. The serine protease cascade is an essential component of the innate immune system of the horseshoe crab. The serine protease inhibitor CrSPI isoform 1 (CrSPI-1), a unique nonclassical Kazal-type inhibitor of molecular weight 9.3 kDa, was identified from the hepatopancreas of the horseshoe crab Carcinoscorpius rotundicauda. It potently inhibits subtilisin and constitutes a powerful innate immune defence against invading microbes. Here, the cloning, expression, purification and cocrystallization of CrSPI-1 with subtilisin are reported. The crystals diffracted to 2.6 Å resolution and belonged to space group P2 1 , with unit-cell parameters a = 73.8, b = 65.0, c = 111.9 Å , = 95.4 . The Matthews coefficient (V M = 2.64 Å 3 Da À1 , corresponding to 53% solvent content) and analysis of the preliminary structure solution indicated the presence of one heterotrimer (1:2 ratio of CrSPI-1:subtilisin) and one free subtilisin molecule in the asymmetric unit.