Abstract
The Z mutation (E342K) of α1-antitrypsin (α1-AT), carried by 4% of Northern Europeans, predisposes to early onset of emphysema due to decreased functional α1-AT in the lung and to liver cirrhosis due to accumulation of polymers in hepatocytes. However, it remains unclear why the Z mutation causes intracellular polymerization of nascent Z α1-AT and why 15% of the expressed Z α1-AT is secreted into circulation as functional, but polymerogenic, monomers. Here, we solve the crystal structure of the Z-monomer and have engineered replacements to assess the conformational role of residue Glu-342 in α1-AT. The results reveal that Z α1-AT has a labile strand 5 of the central β-sheet A (s5A) with a consequent equilibrium between a native inhibitory conformation, as in its crystal structure here, and an aberrant conformation with s5A only partially incorporated into the central β-sheet. This aberrant conformation, induced by the loss of interactions from the Glu-342 side chain, explains why Z α1-AT is prone to polymerization and readily binds to a 6-mer peptide, and it supports that annealing of s5A into the central β-sheet is a crucial step in the serpins' metastable conformational formation. The demonstration that the aberrant conformation can be rectified through stabilization of the labile s5A by binding of a small molecule opens a potential therapeutic approach for Z α1-AT deficiency.
Highlights
The mechanism by which the E342K mutation causes polymerization of Z ␣1-AT is not fully understood, and various models of serpin polymerization have been proposed (12, 14 –18)
Our findings reveal how the mutation of Glu-342 would lead to an aberrant conformation of Z ␣1-AT and explain how the Z mutation will disrupt a key step in the folding pathway of ␣1-AT leading to its pathological polymerization
Aberrant Conformation of Z ␣1-AT—It has long been proposed by several groups that Z ␣1-AT likely exists in an abnormal conformation [7, 23, 30, 41, 42]
Summary
Role of Residue 342 in ␣1-AT Folding—The Z mutation (E342K) will result in the direct loss of the stabilizing interactions of Glu-342 but will perturb the nearby packing due to the positive charge of the lysine side chain. This is consistent with previous observations that Z ␣1-AT derived from human plasma or recombinant Z ␣1-AT is largely active in inhibiting protease, with increased SIs (30 –32) These data indicate that, once the ␣1-AT mutant is folded, the positive charge at residue 342 has little effect on the inhibitory activity of ␣1-AT because both E342C and E342C-mod behave toward protease (Table 1). E342A and E342C mutants form a binary complex with the 6-mer peptide much faster than wild type ␣1-AT (Fig. 3a) This suggests that all the Glu-342 mutants, regardless of the side chain of residue 342, can adopt an aberrant conformation, similar to that of plasma Z ␣1-AT, which binds the 6-mer peptide preferentially. The reaction was followed by SDSPAGE analysis Both mutants with P14 cysteine can be readily modified by mPEG (Fig. 4a) at a similar rate, indicating that the P14 residue is surface-exposed in both wild type ␣1-AT and E342A variant. P21 a, b, c ϭ 74.13, 53.67, 110.14 ␣, , ␥ ϭ 90, 96.88, 90 44 0.9763 74.54–3.3; 3.48–3.3 55,690; 7261 12,855; 1803 4.3; 4.0 7.7; 1.5 98.9; 96.1 0.136; 0.946
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