pH-dependent stability of neuroserpin is mediated by histidines 119 and 138; implications for the control of beta-sheet A and polymerization

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.

Figure 1

Neuroserpin was incubated at 45°C and 0.4 mg/mL and aliquots taken over time were analyzed by 3-12% w/v gradient nondenaturing PAGE. All lanes contain 2 μg of protein. A: Polymerization of wild-type neuroserpin at pH 6.0. B: Polymerization of wild-type neuroserpin at pH 7.0. C: Polymerization of wild-type neuroserpin at pH 8.0. Lanes 1-10 correspond to 0, 5, 10, 15, 20, 30, 45, 60, 120, and 180 min. D: Polymerization of neuroserpin at pH 6.0–7.0. Lanes 1-6 correspond to pH 6.0, 6.2, 6.4, 6.6, 6.8, and 7.0 at incubation times of 0, 2, 6, and 24 h. E: Complex formation between neuroserpin and tPA at different pH analyzed by 10% w/v SDS-PAGE. Lane 1, neuroserpin; lane 2, neuroserpin with two fold molar excess of tPA at pH 7.0; lane 3, neuroserpin incubated for 5 h at pH 5.0 and 45°C; lane 4, neuroserpin incubated for 5 h at pH 5.0 and 45°C, and then the pH was adjusted to 7.0 followed by the addition of two fold molar excess of tPA for 10 min; lane 5, tPA; lane 6, molecular mass markers. N, intact neuroserpin; Cl, reactive loop-cleaved neuroserpin; Cpx, the complex between neuroserpin and tPA. All lanes contain 1–2 μg of protein.

Figure 2

A: Far-UV spectra of wild-type neuroserpin at different pH. pH 6.0 (+ line), pH 7.0 (solid line), and pH 8.0 (× line). B: Transition temperature of neuroserpin with increasing pH as measured by circular dichroism. The transition temperature for the different pH values was calculated as described in the Materials and Methods section.

Figure 3

A: Schematic view of native neuroserpin (3FGQ). The reactive center loop is shown in red at the upper pole of the protein. The distances (in Å) between His338 and nearby residues in the shutter domain are shown (represented by the box on the left panel). B: Schematic view showing the interactions between strand 1A and helix E at the lower pole of native neuroserpin (3FGQ). The side chains of histidines (His138 and His119: yellow), the hydrophobic residues (Val114, Val120, Leu125, and Val136: orange), Asp327 (red), and Phe118 (cyan) are highlighted. His138 interacts with a water molecule (red sphere) by two hydrogen bonds (green dotted line), and the distance between NE2-His138 and O=C—Phe118 (black dotted line) is 2.50 Å. The distance between ND1-His119 and OD2-Asp327 is 2.64 Å. There is a π–π stacking interaction between the imidazole ring of His119 and O=C—Val120. For comparison, reactive loop-cleaved neuroserpin (3F02) and native neuroserpin obtained from different crystallizing conditions (3F5N) are superimposed, and His119 and Glu122 are shown with red (3F02) or blue letters (3F5N). The distance between NE2-His119 and OE1-Glu122 in 3F5N (blue dotted line) is 3.09 Å. C: Molecular surface of native neuroserpin (3FGQ: left) and reactive loop-cleaved neuroserpin (3F02: right). The side chains of His138, His119, Glu122, Asp327 and the hydrophobic residues (Val114, Val120, Leu125, and Val136) are highlighted with the same color as in panel B. Val114 is fully buried in native neuroserpin. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 4

A: Far UV spectra of wild-type neuroserpin, His119Gln, His138Gln, and His338Gln at pH 7.0. Wild-type neuroserpin (solid line), His119Gln (crossed line), His138Gln (dashed line), and His338Gln (dotted line). B: Transition temperature of wild-type neuroserpin, His119Gln, His138Gln, and His338Gln at different pH. Wild-type neuroserpin (O), His119Gln (□), His138Gln (Δ), and His338Gln (◊). The transition temperature for the different pH values was calculated as described in the Results section. N = 3 for all experiments. C: Complex formation between wild-type and mutant neuroserpin and tPA at pH 7.4 analyzed by 10% w/v SDS-PAGE. Lane M, molecular mass markers; Lane 1, wild-type neuroserpin; lane 2, wild-type neuroserpin with tPA at pH 7.4; lane 3, His119Gln neuroserpin; lane 4, His119Gln neuroserpin with tPA at pH 7.4; lane 5, His138Gln neuroserpin; lane 6, His138Gln neuroserpin with tPA at pH 7.4; lane 7, His338Gln neuroserpin; lane 8, His338Gln with tPA at pH 7.4; lane 9, tPA. N, intact neuroserpin; Cl, reactive loop-cleaved neuroserpin; Cpx, the complex between neuroserpin and tPA. All lanes contain 1-2 μg of protein.

Figure 5

Neuroserpin mutants were incubated at 45°C and 0.4 mg/mL and aliquots taken over time were analyzed by 3-12% w/v gradient nondenaturing PAGE. All lanes contained 1.2 μg of protein. A: Left: Polymerization of His338Gln neuroserpin at pH 6.0. Middle: Polymerization of His338Gln neuroserpin at pH 7.0. Right: Polymerization of His338Gln neuroserpin at pH 8.0. B: Left: Polymerization of His119Gln neuroserpin at pH 6.0. Middle: Polymerization of His119Gln neuroserpin at pH 7.0. Right: Polymerization of His119Gln neuroserpin at pH 8.0. C: Left: Polymerization of His138Gln neuroserpin at pH 6.0. Middle: Polymerization of His138Gln neuroserpin at pH 7.0. Right: Polymerization of His138Gln neuroserpin at pH 8.0. Lanes 1-10 correspond to 0, 5, 10, 15, 20, 30, 45, 60, 120, and 180 min.