Structural studies of latexin, A NOVEL carboxypeptidase inhibitor

Anna Aagaard,^a Pawel Listwan,^b Nathan Cowieson,^a Thomas Huber,^c Christine Wells,^a Timothy Ravasi,^a Bostjan Kobe,^a,b David Hume^a and Jennifer Martin^a

^aInstitute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia; ^bDepartment of Biochemistry and Molecular Biology, The University of Queensland, Brisbane, QLD 4072, Australia; ^cAdvanced Computational Modelling Centre, The University of Queensland, Brisbane, QLD 4072, Australia (a.aagaard@imb.uq.edu.au).

Latexin is a 222 amino acid protein possessing inhibitory activity against carboxypeptidase A1 (CPA1) and CPA2 as well as mast-cell CPA[1]. It is unusual in the sense that it is by far the largest proteinaceous carboxypeptidase inhibitor so far reported[2]and it shows no sequence similarity to any of the other known carboxypeptidase inhibitors. Furthermore, it is the only known CPA protein inhibitor of mammalian origin[1]. Studies on rat and human latexin showed that it is expressed in many different tissues including heart, prostate, ovary and kidney[3].

We found that latexin is also expressed at high levels in mouse macrophages and is further inducible in these cells by lipopolysaccharide. Based on these results, latexin was selected as a target for structural studies as part of a focused high throughput crystallography project at The University of Queensland. The gene was cloned and the protein expressed in E. coli. We used an autoinduction method to produce tens of milligrams of native and selenomethionine labelled protein from 500 ml cultures. Crystals of both the native and labelled protein were obtained by means of hanging-drop vapour diffusion using a 96-well format crystallisation tray. Conditions for freezing crystals have been identified and frozen crystals diffract to 2.2 Å on our in-house X-ray equipment. Beamtime has been allocated for both MAD and native data collection at the Advanced Light Source in Berkeley early in 2003. We expect that the structure of the protein will be solved from the MAD data. Results from these experiments will be presented at the conference.

References

1 Normant, E., et al., Proceedings of the National Academy of Sciences of the United States of America, 1995. 92(26): p. 12225-12229.

2 Vendrell, J., E. Querol, and F.X. Aviles.Biochimica Et Biophysica Acta-Protein Structure and Molecular Enzymology, 2000. 1477(1-2): p. 284-298.

3 Liu, Q., et al. Molecular Biology Reports, 2000. 27(4): p. 241-246.