STRUCTURE OF THE GLYCOSYLATED PLANT ASPARTIC PROTEINASE CARDOSIN A, TO 1.72 Å RESOLUTION

Bento, I.1, Frazão, C.1, Coelho, R.1, Costa1, J., Faro, C.2, Veríssimo, P.2, Pires, E.2, Cooper, J.3, Dauter, Z.4 ,Wilson, K.4, and Carrondo M. A.1.

1ITQB, Oeiras, Portugal,
2Univ. Coimbra, Portugal,
3Birkbeck College, London, UK,
4 EMBL, c/o DESY, Hamburg, Germany, Present address - Dept. Chemistry, Univ. of York, York, U.K.

Keywords: aspartic proteinase, glycoprotein, cryocrystallography.

Plant aspartic proteinases (AP), although ubiquitous in the vegetal kingdom, have not yet been structurally studied so intensively as in viruses, fungi and animals, as it proved very difficult to obtain good quality diffracting crystals. Cardosin A is the most abundant of the two plant AP's isolated from the stigmas of Cynara cardunculus L. [1]. It is a 45 kDa vacuolar protein, glycosylated in both of its two subunits (30 kDa and 15 kDa), with a sugar content of 9.6% [2]. As in all others known plant AP, it is expressed with an insertion of 100 residues within its aminoacid sequence [3].

Seeding techniques were used for crystal optimisation. Three sets of X-ray data were collected. The first data set was collected at room temperature using synchrotron radiation, Rmerge=7.6%, completeness 99% and redundancy 3.4. The crystal did not diffract beyond 2.85 Å resolution and was prone to radiation damage. The second data set, collected with in house equipment under cryogenic conditions, minimised X-ray damage and hinted for a substantial improvement on data quality and resolution: the crystal diffracted up to 2.85 Å with a Rmerge=10.3%, completeness 97.5%, redundancy 8.8. A third crystal diffracted to 1.72 Å at a synchrotron source and under cryogenic conditions, Rmerge=6.2%, completeness 96.3% and redundancy 3.6. Crystals belong to the space group C2, a=116.9(2), b=87.20(8), c=81.3(1), b=104.44(4).

The human cathepsin D structure [4] was used as search model (sequence identity of 44.4 %) for molecular replacement of the two molecules in the asymmetric unit. Refinement was carried out with X-PLOR [5] in a first stage, using simulating annealing with constrained non-crystallographic symmetry. SHELXL97 [6] was used afterwards restraining non-crystallographic symmetry and isotropic atomic displacement parameters (actual R=20% and Rfree=25% considering ca. 300 solvent molecules). The crystal structure of cardosin A shows the 3D-folding characteristic of the pepsin family with both glycosylation sites localised away from the aspartic proteinase catalytic site, and from the specificity determining cleft.

[1] Veríssimo P., Pires E., et al. (1996) Eur. J. Biochem 235, 762-768.
[2] Costa, J, Carrondo, M. A. et al. (1997). Eur. J. Biochem. 235, 762-768.
[3] Faro, C. J., Pires, E. (1995). Aspartic Proteinases: Structure, Function, Biology, and Biomedical Implications, edited by K. Takahashi, pp. 373-377, Plenum Press, New York.
[4] Baldwin E., et al. (1993) Proc. Nat. Acad. Sci. USA 90, 6796-6800
[5] Brünger A., (1993). X-PLOR, Version 3.1. Yale Univ., New Haven.
[6] Sheldrick G. and Schneider T., (1997), Methods. Enzymol., 277 Part B: 319-343, Academic Press, London, UK.