Crystallographic analysis of serpins and bacterial response regulator.

摘要

Proteins undergo conformational changes during their course of action and those changes are often very important for their functional performance. In the case of the family of serpins proteolytic cleavage of the peptide bond in the reactive center loop causes irreversible conformational change resulting in the separation of the previously neighboring amino acids to a distance of over 70 A. However, in the case of bacterial response regulators a small conformational change in the loop determines whether the molecule is in the active or in the inactive conformation. Structural changes can be in many instances analyzed by protein crystallography.; Herein, crystal structures of several serpins have been determined along with the structure of the bacterial response regulator, CheY. The crystal structure of the native alpha1-PI Pittsburgh variant is determined. The comparative analysis with the heparin activated antithrombin structure as well as modeling of the non-covalent complex with thrombin offered insights about differences in inhibitory potency between alpha1-PI Pittsburgh and antithrombin. Further, the crystal structure of the cowpox virus serpin crmA in cleaved form is determined to 2.9 A. The structure suggested that crmA may employ the same mechanism for inhibition of both serine and cysteine proteinases. In addition, comparison between two non-isomorphous structures of cleaved crmA revealed a physiologically important site of this serpin outside of the reactive center loop. The crystal structure of the non-inhibitory serpin PEDF, the most potent anti-angiogenic factor in human eye was solved. This structure showed the location of a peptide previously identified as having some of the neurotrophic properties of intact PEDF, but suggested that only restricted portions of this peptide are likely to be candidates for receptor interactions. Also, the structure showed an asymmetric charge distribution that suggested another possible site of interaction with cofactor proteins or receptors. Finally, the 1.08 A resolution crystal structure of wild-type apo-CheY shows the beta4-alpha4 loop in two distinctly different conformations that sterically correlate with the two rotameric positions of the tyrosine 106 side-chain. One of these conformational states of CheY is the inactive form, and the other is a metaactive form, responsible for the active properties seen in apo-CheY.