CRYSTAL STRUCTURE OF HUMAN SALIVARY CYSTATIN. Narayanan Ramasubbu, Michael J. Levine, Department of Oral Biology and Dental Research Institute, School of Dental Medicine, State University of New York at Buffalo, Buffalo, NY 14214, USA
Human saliva contains several isoforms of thiol proteinase inhibitors known as cystatins which have potential antimicrobial and antiviral functions. These properties suggest an important role in maintaining oral health. Salivary cystatins contain 121 amino acids and have a molecular weight of ~13,500 daltons. There is considerable differences in the ability of salivary cystatins to inhibit papain and cathepsins.In an effort to define the role of cystatins in the oral cavity, we have initiated a structure-function-mutagenesis approach. In this regard, we have expressed and purified several recombinant salivary cystatin variants using an Escherichia coli expression system, pGEX-2T. One of the variants ([[Delta]]12-16) lacks a five-residues (G-G-I-Y-N) near the N-terminus. Interestingly, this variant possesses higher thiol-proteinase inhibitory activity towards papain compared to the full-length recombinant cystatin.
Cystatin [[Delta]]12-16 was crystallized by sitting drop vapor diffusion method in a silica gel environment using a protein concentration of 30 mg/mL. Crystals are of space group P622 and the unit cell constants of a = b = 85.41, c = 131.6 Å, [[alpha]] = [[beta]] = 90deg., [[gamma]] =120deg., with two molecules per asymmetric unit, Vm = 2.6 Å3/Da. Native data were collected to a resolution of 2.5 Å using a MAR imaging plate mounted on a Rigaku rotating anode. The complete native data set contains 9740 (I >2 [[sigma]](I); Rmerge 5.1%; ~94% complete). The structure was solved using the chicken egg white cystatin structure as a starting model by molecular replacement technique and refined to an R-factor of 25% using XPLOR. The refinement is still in progress. The details of the structure determination, refinement and its thiol proteinase activity will be described.
Work supported by USPHS grants DE10621 and DE08240.