SERA PROTEINS: are they CYSTEINE OR SERINE PROTEASES?

Robyn L. Malby,^a Anthony N. Hodder,^a Vidana C. Epa^b and Brendan S. Crabb^a

^aWalter and Eliza Hall Institute of Medical Research, Melbourne VIC 3050, Australia; ^bCSIRO Division of Health Sciences and Nutrition, Parkville VIC 3052, Australia (rmalby@wehi.edu.au)

The serine repeat antigen (SERA) of Plasmodium is a candidate for inclusion in a malaria vaccine. The full-length protein of ~120 kDa is processed into several smaller fragments, one of which (P50) has significant homology to the papain-like cysteine proteases [1, 2]. Various members of the SERA family exhibit intriguing differences in putative enzyme active site residues; for example, the dominantly expressed member of the P. falciparum gene family, SERA-5, has serine substituted for the active site cysteine. A three-dimensional model of the enzyme domain of SERA-5 has been constructed [3], by comparison with crystal structures of several cysteine proteases.

The enzyme domain of SERA-5 was produced as a recombinant protein in
E. coli, and purified by affinity and ion exchange chromatography. Small crystals
(ca. 80 mm ´ 40 mm ´ 20 mm) were grown, and were found to diffract to ~3Å. Diffraction data were collected [4] from a single cryo-cooled crystal on a Rigaku R-AXIS IV image plate detector, using a Rigaku RU-300 generator equipped with monocapillary optics [5]. We are currently attempting to solve the crystal structure by molecular replacement, with the SERA-5 model and related crystal structures as search models. We are also continuing to investigate both site-directed mutagenesis and altered crystallization conditions with the aim of optimizing crystal growth.

References

1 Eakin, A.E., Higaki, J.N., McKerrow, J.H. and Craik, C.S. (1989) Nature 342, 132.

2 Higgins, D.G., McConnell, D.J. and Sharp, P.M. (1989) Nature 340, 604.

3 Hodder, A.N. et al., manuscript in preparation.

4 We thank Bert van Donkelaar (CSIRO Division of Health Sciences and Nutrition) for assistance with data collection.

5 Balaic, D.X., Barnea, Z., Nugent, K.A., Garrett, R.F., Varghese, J.N. and Wilkins, S.W. (1996) J. Synchrotron Rad. 3, 289-295.