Reversible inactivation of serpins at acidic pH.
ABSTRACT The inhibitory activity of the serpins alpha(1)-proteinase inhibitor, alpha(1)-antichymotrypsin, alpha(2)-antiplasmin, antithrombin and C(1)-esterase inactivator is rapidly lost at pH 3 but slowly recovers at pH 7.4 with variable first-order rates (t(1/2)=1.4-19.2 min). All except alpha(1)-antichymotrypsin undergo a variation in intrinsic fluorescence intensity upon acidification (midpoint ca. 4.5) with a slow bi-exponential return to the initial intensity at pH 7.4 (mean t(1/2)=2.3-23 min). No correlation was found between the time of fluorescence recovery and that of reactivation. The acid-treated serpins are proteolyzed at neutral pH by their target proteinases. alpha(1)-Proteinase inhibitor was studied in more detail. Its acidification at pH 3 has a mild effect on its secondary structure, strongly disorders its tertiary structure, changes the microenvironment of Cys(232) and causes a very fast change in ellipticity at 225 nm (t(1/2)=1.6s). Neutralization of the acid-treated alpha(1)-proteinase inhibitor is an exothermic phenomenon. It leads to a much faster recovery of activity (t(1/2)=4+/-1 min) than of fluorescence intensity (t(1/2)=23+/-19 min), ellipticity (t(1/2)=32+/-4 min) and change in total energy, indicating that the inhibitory activity of alpha(1)-proteinase inhibitor does not require a fully native structure.
Article: pH-dependent stability of neuroserpin is mediated by histidines 119 and 138; implications for the control of beta-sheet A and polymerization.[show abstract] [hide abstract]
ABSTRACT: Neuroserpin is a member of the serpin superfamily. Point mutations in the neuroserpin gene underlie the autosomal dominant dementia, familial encephalopathy with neuroserpin inclusion bodies. This is characterized by the retention of ordered polymers of neuroserpin within the endoplasmic reticulum of neurons. pH has been shown to affect the propensity of several serpins to form polymers. In particular, low pH favors the formation of polymers of both alpha(1)-antitrypsin and antithrombin. We report here opposite effects in neuroserpin, with a striking resistance to polymer formation at acidic pH. Mutation of specific histidine residues showed that this effect is not attributable to the shutter domain histidine as would be predicted by analogy with other serpins. Indeed, mutation of the shutter domain His338 decreased neuroserpin stability but had no effect on the pH dependence of polymerization when compared with the wild-type protein. In contrast, mutation of His119 or His138 reduced the polymerization of neuroserpin at both acidic and neutral pH. These residues are at the lower pole of neuroserpin and provide a novel mechanism to control the opening of beta-sheet A and hence polymerization. This mechanism is likely to have evolved to protect neuroserpin from the acidic environment of the secretory granules.Protein Science 12/2009; 19(2):220-8. · 2.80 Impact Factor