Examination of the potential structure of human salivary cystatins based on computer modelling

The cystatin family of proteins exists in both excreted and intracellular forms, and appears to be involved in protective and regulatory roles, inhibiting a variety of bacterial, viral and intracellular proteases. The amino acid sequences of several human forms of cystatin are known, but currently only the structure of chicken cystatin (approx. 40% homologous to the human forms) has been experimentally determined. The objective of this study was to use the X-ray coordinates of chicken cystatin to construct computer models of the structures of three human salivary forms (SN, S and SA). These structures were energy-minimized and subjected to dynamic simulations. The resultant structures were compared to determine conformational differences. Global root mean square deviations between equivalent atoms ranged from 1.4 Å to 3.9 Å. The closest structural similarity to chicken cystatin involved cystatin SN, which also showed the highest (68%) functional sequence homology. Local secondary structure was examined in more detail. In comparisons of α-carbon position the third ß-strand (77% functional sequence conservation) and its preceding loop (60% conserved) showed the highest structural conservation in S, while ß-strand 4 showed the highest structural conservation in SN and SA. Throughout their structures, SN and SA were more structurally similar to chicken cystatin than to salivary cystatin S. There are two regions of conserved, negatively charged residues in the salivary cystatins, which appear to be spaced so that they are capable of interaction with hydroxyapatite. It is concluded that not only does structural modelling by analogy provide detailed models of salivary cystatins that can be tested by future experimentation, but also that examination of the models has revealed potential sites of interaction with hydroxyapatite.