J. Mol. Biol. (1992) 225, 1135-1136
Crystallization and Preliminary Crystallographic Study of Neuraminidase from Micromonospora viridifaciens Garry Taylor?, Liz Dineley Department Claverton
of Biochemistry, University of Bath Down, Bath BA2 7A Y, U.K.
Marek Gfowka Institute
of General Chemistry, Technical 90-924 E&k!, Poland
University
and Graeme Laver John Curtin Medical School The Australian National University GPO Box 334, Canberra, ACT 2601, Australia (Received 20 February
1992; accepted 2 March
1992)
Single crystals of neuraminidase from the bacterium Micromonoapora viridifaciens were obtained using the hanging drop vapour diffusion method and polyethylene glycol as precipitant at pH 50 or 5.5. The crystals belong to the orthorhombic space group P2,212,, with unit cell dimensions a = 48.14 A, b = 8273 A, c = 8475 A and with one molecule in the asymmetric unit. Diffraction extends to at least 1.7 A. Keywords:
neuraminidase;
sialidase;
crystallography;
Neuraminidase cleaves terminal sialic acid residues from glycoproteins, glycolipids and oligosaccharides. Neuraminidase can be found as one of two glycoproteins on the surface of influenza viruses, where its role is thought to be in releasing progeny virus particles from infected cells as well as being involved in the breakdown of the mucosal lining of the upper respiratory tract during the initial infection. The structures of several subtypes of influenza neuraminidase are known (Varghese & Colman, 1991; Tulip et al., 1991; Burmeister et al., 1992). Neuraminidase is also produced by a variety of pathogenic microorganisms, such as Clostridium Corynebacterium diptheriae, Vibrio perfringens, cholerae, Bacteroidacae, Streptococci, and nonpathogenic bacteria of Arthrobacter species. The structures of the bacterial neuraminidases are unknown, and there is no overall sequence homology between the viral and bacterial enzymes. Neuraminidase is also expressed by Trypanosoma cruzi on its surface during certain developmental stages, where it can chemically modify, by desialy-
7 Author addressed.
to whom
all correspondence
should
be
Micromonospora
viridifaciens
lation, the surfaces of myocardial and vascular endothelial cells (Pereira et al., 1991). Neuraminidases from Actinomycetes have been well characterized and, in particular, a marked neuraminidase activity was found in the culture broth of a non-pathogenic Actinomycete, Micromonospora viridifaciens, where it was detected only when either the substrate colominic acid, or the product N-acetylneuraminic acid was added to the culture medium (Aisaka et al., 1991). This is consistent with previous findings that sialic acids act as inducers of microbial neuraminidases. This neuraminidase was reported to have a molecular mass of 41,000 daltons, and determination of the amino acid sequence is in progress. The M. viridifaciens enzyme is similar in molecular mass and activity to that of C. perfringens whose sequence is known. Comparison of the N-terminal sequence of T. cruzi neuraminidase with that of C. perfringens suggests a structural homology between these two (Pereira et of the structure of al., 1991). Determination M. viridifaciens neuraminidase should therefore enable other pathogenic neuraminidases to be modelled. The protein was supplied as a crude ammonium sulphate precipitation extract, which was first
1135 0022%2836/92/121135-02
$03.00/O
0
1992 Academic
Press Limited
1136
G. Taylor
dialysed against 10 mw-Tris. HCl buffer at pH 8.0. After concentration, the protein solution was run down an f.p.1.c.t Superdex 200 gel filtration column, equilibrated with the same buffer. Pooled fractions were run down an f.p.1.c. Mono-Q anion exchange column equilibrated with 50 mM-NaC1 at pH 8.0, and eluted with an NaCl gradient from 0 to 1 IVI. The pooled, active fractions were combined and run down the Superdex 200 column equilibrated with 10 mM-Tris . HCl at pH 8.0. Crystals were grown by vapour diffusion using the hanging drop method around the isoelectric point of the protein, 5.6, using PEG 3350 in a sodium cacodylate buffer. Crystals appeared overnight as long prisms with dimensions up to 1.6 mm x 0.6 mm x 0.6 mm. The best crystals were obtained at pH 50 or 5.5 and 8% PEG 3350. The enzyme in the crystals was shown to have neuraminidase activity by two methods: a fluoromet,ric assay with 2’-(4-methylumbelliferyl)a-D-N-acetylneuraminic acid, or by the production using thiobarbituric acid of a chromophore (Aymard-Henry et al., 1973). Diffraction data were collected on a Siemens area detector mounted on a Siemens rotating anode X-ray source operating at 50 kV and 80 mA. Frames of data were recorded while the crystal was oscillated through 0.25 degree steps. The unit cell was obtained with the XENGEN suite of software Genex Corporation, (A. Howard, 1988; Gaithersburg, MD, U.S.A.). The intensities were integrated using the XDS program (Kabsch, 1988aJ). Diffraction extended to beyond 1.7 d (1 a = 0.1 nm) resolution. A native dataset, lOOo/o complete to 2.0 a and 77% complete to 1% 8, with an Rmerg(l) = 5.5% was collected from one crystal:
The crystals belong to space group PZ12,2,, as judged by systematically absent or weak (F < 2.50) axial reflections. The unit cell parameters are With a a = 48.14 8, 6 = 82.73 8, c = 84.75 8. molecular mass of 41,000 daltons for the monomer, t Abbreviations chromatography;
used: f.p.l.c., fast protein PEG, polyethylene glycol.
liquid
et al.
a vm value of 2.06 X3/Da is obtained consistent with a monomer in the asymmetric unit and within the reported range for protein crystals (Matthews, 1968). A search for suitable heavy atom derivatives is in progress. We thank Dr T. Uwajima, formerly of Kyowa Kogyo Co. Ltd., Tokyo, Japan for the gift neuraminidase.
References Aisaka, K., Igarashi, A. & Uwajima, T. (1991). Purification, crystallization, and characterization of neuraminidase from Micromonospora viridifaciens.
Agric. Biol. Chem. 55, 997-1004. Aymard-Henry, M., Coleman, M. T.; Dowdle, W. R.; Laver, W. G., Schild, G. C. & Webster, R. G. (1973). Influenza virus neuraminidase and neuraminidaseinhibition test procedures. Bull. World Health Org. 48, 199-202. Burmeister, W. P., Ruigrok, R. W. H. & Cusack, S. (1992). The 2.2 $ resolution crystal structure of influenza B neuraminidase and its complex with sialie acid. EMBO J. 11, 49-56. Kabsch, W. J. (1988a). Automatic indexing of rotation diffraction patterns. J. Appl. Crystallogr. 21, 67-71. Kabsch, W. J. (19886). Evaluation of single-crystal X-ray diffraction dat,a from a position-sensitive detector. J. Appl. Crystallogr. 21, 916-924. Matthews, B. W. (1968). Solvent content of protein crystals. J. Mol. Biol. 33, 491-497. M. E. A., Mejia, J. S., Ortega-Barria, E., Pereira, Matzilevich, D. & Prioli, R. P. (1991). The Trypanosoma cruzi neura,minidase contains sequences similar to bacterial neuraminidases, YWTD repeats of the low density lipoprotein receptor, and type III modules of fibronectin. J. Ezp. Med. 174; 179-191. Tulip, W. R., Varghese, J. h’., Baker, A. T., van Donkelaar, A., Laver, W. G., Webster, R. G. & Colman, P. M. (1991). Refined a,tomic st’ructures of N9 subtype influenza virus neuraminidase and escape mutants. J. Mol. Biol. 221, 487-497. Varghese, J. N. & Colman, P. M. (1991). Threedimensional structure of the neuraminidase of influenza virus A/Tokyo/3/67 at 2.2 A resolution.
J. Mol. Biol. 221, 473-486.
Edited
Hal&o of the
by A. Klug
Crystallization and Preliminary Crystallographic Study of Neuraminidase from Micromonospora viridifaciens Garry Taylor?, Liz Dineley Department Claverton
of Biochemistry, University of Bath Down, Bath BA2 7A Y, U.K.
Marek Gfowka Institute
of General Chemistry, Technical 90-924 E&k!, Poland
University
and Graeme Laver John Curtin Medical School The Australian National University GPO Box 334, Canberra, ACT 2601, Australia (Received 20 February
1992; accepted 2 March
1992)
Single crystals of neuraminidase from the bacterium Micromonoapora viridifaciens were obtained using the hanging drop vapour diffusion method and polyethylene glycol as precipitant at pH 50 or 5.5. The crystals belong to the orthorhombic space group P2,212,, with unit cell dimensions a = 48.14 A, b = 8273 A, c = 8475 A and with one molecule in the asymmetric unit. Diffraction extends to at least 1.7 A. Keywords:
neuraminidase;
sialidase;
crystallography;
Neuraminidase cleaves terminal sialic acid residues from glycoproteins, glycolipids and oligosaccharides. Neuraminidase can be found as one of two glycoproteins on the surface of influenza viruses, where its role is thought to be in releasing progeny virus particles from infected cells as well as being involved in the breakdown of the mucosal lining of the upper respiratory tract during the initial infection. The structures of several subtypes of influenza neuraminidase are known (Varghese & Colman, 1991; Tulip et al., 1991; Burmeister et al., 1992). Neuraminidase is also produced by a variety of pathogenic microorganisms, such as Clostridium Corynebacterium diptheriae, Vibrio perfringens, cholerae, Bacteroidacae, Streptococci, and nonpathogenic bacteria of Arthrobacter species. The structures of the bacterial neuraminidases are unknown, and there is no overall sequence homology between the viral and bacterial enzymes. Neuraminidase is also expressed by Trypanosoma cruzi on its surface during certain developmental stages, where it can chemically modify, by desialy-
7 Author addressed.
to whom
all correspondence
should
be
Micromonospora
viridifaciens
lation, the surfaces of myocardial and vascular endothelial cells (Pereira et al., 1991). Neuraminidases from Actinomycetes have been well characterized and, in particular, a marked neuraminidase activity was found in the culture broth of a non-pathogenic Actinomycete, Micromonospora viridifaciens, where it was detected only when either the substrate colominic acid, or the product N-acetylneuraminic acid was added to the culture medium (Aisaka et al., 1991). This is consistent with previous findings that sialic acids act as inducers of microbial neuraminidases. This neuraminidase was reported to have a molecular mass of 41,000 daltons, and determination of the amino acid sequence is in progress. The M. viridifaciens enzyme is similar in molecular mass and activity to that of C. perfringens whose sequence is known. Comparison of the N-terminal sequence of T. cruzi neuraminidase with that of C. perfringens suggests a structural homology between these two (Pereira et of the structure of al., 1991). Determination M. viridifaciens neuraminidase should therefore enable other pathogenic neuraminidases to be modelled. The protein was supplied as a crude ammonium sulphate precipitation extract, which was first
1135 0022%2836/92/121135-02
$03.00/O
0
1992 Academic
Press Limited
1136
G. Taylor
dialysed against 10 mw-Tris. HCl buffer at pH 8.0. After concentration, the protein solution was run down an f.p.1.c.t Superdex 200 gel filtration column, equilibrated with the same buffer. Pooled fractions were run down an f.p.1.c. Mono-Q anion exchange column equilibrated with 50 mM-NaC1 at pH 8.0, and eluted with an NaCl gradient from 0 to 1 IVI. The pooled, active fractions were combined and run down the Superdex 200 column equilibrated with 10 mM-Tris . HCl at pH 8.0. Crystals were grown by vapour diffusion using the hanging drop method around the isoelectric point of the protein, 5.6, using PEG 3350 in a sodium cacodylate buffer. Crystals appeared overnight as long prisms with dimensions up to 1.6 mm x 0.6 mm x 0.6 mm. The best crystals were obtained at pH 50 or 5.5 and 8% PEG 3350. The enzyme in the crystals was shown to have neuraminidase activity by two methods: a fluoromet,ric assay with 2’-(4-methylumbelliferyl)a-D-N-acetylneuraminic acid, or by the production using thiobarbituric acid of a chromophore (Aymard-Henry et al., 1973). Diffraction data were collected on a Siemens area detector mounted on a Siemens rotating anode X-ray source operating at 50 kV and 80 mA. Frames of data were recorded while the crystal was oscillated through 0.25 degree steps. The unit cell was obtained with the XENGEN suite of software Genex Corporation, (A. Howard, 1988; Gaithersburg, MD, U.S.A.). The intensities were integrated using the XDS program (Kabsch, 1988aJ). Diffraction extended to beyond 1.7 d (1 a = 0.1 nm) resolution. A native dataset, lOOo/o complete to 2.0 a and 77% complete to 1% 8, with an Rmerg(l) = 5.5% was collected from one crystal:
The crystals belong to space group PZ12,2,, as judged by systematically absent or weak (F < 2.50) axial reflections. The unit cell parameters are With a a = 48.14 8, 6 = 82.73 8, c = 84.75 8. molecular mass of 41,000 daltons for the monomer, t Abbreviations chromatography;
used: f.p.l.c., fast protein PEG, polyethylene glycol.
liquid
et al.
a vm value of 2.06 X3/Da is obtained consistent with a monomer in the asymmetric unit and within the reported range for protein crystals (Matthews, 1968). A search for suitable heavy atom derivatives is in progress. We thank Dr T. Uwajima, formerly of Kyowa Kogyo Co. Ltd., Tokyo, Japan for the gift neuraminidase.
References Aisaka, K., Igarashi, A. & Uwajima, T. (1991). Purification, crystallization, and characterization of neuraminidase from Micromonospora viridifaciens.
Agric. Biol. Chem. 55, 997-1004. Aymard-Henry, M., Coleman, M. T.; Dowdle, W. R.; Laver, W. G., Schild, G. C. & Webster, R. G. (1973). Influenza virus neuraminidase and neuraminidaseinhibition test procedures. Bull. World Health Org. 48, 199-202. Burmeister, W. P., Ruigrok, R. W. H. & Cusack, S. (1992). The 2.2 $ resolution crystal structure of influenza B neuraminidase and its complex with sialie acid. EMBO J. 11, 49-56. Kabsch, W. J. (1988a). Automatic indexing of rotation diffraction patterns. J. Appl. Crystallogr. 21, 67-71. Kabsch, W. J. (19886). Evaluation of single-crystal X-ray diffraction dat,a from a position-sensitive detector. J. Appl. Crystallogr. 21, 916-924. Matthews, B. W. (1968). Solvent content of protein crystals. J. Mol. Biol. 33, 491-497. M. E. A., Mejia, J. S., Ortega-Barria, E., Pereira, Matzilevich, D. & Prioli, R. P. (1991). The Trypanosoma cruzi neura,minidase contains sequences similar to bacterial neuraminidases, YWTD repeats of the low density lipoprotein receptor, and type III modules of fibronectin. J. Ezp. Med. 174; 179-191. Tulip, W. R., Varghese, J. h’., Baker, A. T., van Donkelaar, A., Laver, W. G., Webster, R. G. & Colman, P. M. (1991). Refined a,tomic st’ructures of N9 subtype influenza virus neuraminidase and escape mutants. J. Mol. Biol. 221, 487-497. Varghese, J. N. & Colman, P. M. (1991). Threedimensional structure of the neuraminidase of influenza virus A/Tokyo/3/67 at 2.2 A resolution.
J. Mol. Biol. 221, 473-486.
Edited
Hal&o of the
by A. Klug
