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ABSTRACT: Members of the serpin family of serine proteinase inhibitors play important roles in the inflammatory, coagulation, fibrinolytic, and complement cascades. An inherent part of their function is the ability to undergo a structural rearrangement, the stressed (S) to relaxed (R) transition, in which an extra strand is inserted into the central A β-sheet. In order for this transition to take place, the A sheet has to be unusually flexible. Malfunctions in this flexibility can lead to aberrant protein linkage, serpin inactivation, and diseases as diverse as cirrhosis, thrombosis, angioedema, emphysema, and dementia. The development of agents that control this conformational rearrangement requires a high resolution structure of an active serpin. We present here the topology of the archetypal serpin α1-antitrypsin to 2 Å resolution. This structure allows us to define five cavities that are potential targets for rational drug design to develop agents that will prevent conformational transitions and ameliorate the associated disease.
Protein Science 12/1999; 9(7):1274 - 1281. · 2.80 Impact Factor
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ABSTRACT: The Z (Glu342 → Lys) and Siiyama (Ser53 → Phe) deficiency variants of α1-antitrypsin result in the retention of protein in the endoplasmic reticulum of the hepatocyte by loop-sheet polymerization
in which the reactive center loop of one molecule is inserted into a β-pleated sheet of a second. We show here that antitrypsin
Mmalton (Phe52-deleted), which is associated with the same liver inclusions, is also retained at an endoglycosidase H-sensitive stage of
processing in the Xenopus oocyte and spontaneously forms polymers in vivo. These polymers, obtained from the plasma of an Mmalton/QO (null) bolton heterozygote, were much shorter than other antitrypsin
polymers and contained a reactive center loop-cleaved species. Monomeric mutant antitrypsin was also isolated from the plasma.
The monomeric component had a normal unfolding transition on transverse urea gradient gel electrophoresis and formed polymers
in vitro more readily than M, but less readily than Z, antitrypsin. The A β-sheet accommodated a reactive center loop peptide much
less readily than Z antitrypsin, which in turn was less receptive than native M antitrypsin. The nonreceptive conformation
of the A sheet in antitrypsin Mmalton had little effect on kinetic parameters, the formation of SDS-stable complexes, the
S to R transition, and the formation of the latent conformation.
Comparison of the results with similar findings of short chain polymers associated with the antithrombin variant Rouen VI
(Bruce, D., Perry, D., Borg, J.-Y., Carrell, R. W., and Wardell, M. R.(1994) J. Clin. Invest. 94, 2265-2274) suggests that polymerization is more complicated than the mechanism proposed earlier. The Z, Siiyama, and
Mmalton mutations favor a conformational change in the antitrypsin molecule to an intermediate between the native and latent
forms. This would involve a partial overinsertion of the reactive loop into the A sheet with displacement of strand 1C and
consequent loop-C sheet polymerization.
Journal of Biological Chemistry 07/1995; 270(28):16864-16870. · 4.77 Impact Factor
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ABSTRACT: Members of the serine proteinase inhibitor or serpin superfamily have a common molecular architecture based on a dominant
five-membered A β-pleated sheet and a mobile reactive center loop. The reactive center loop has been shown to adopt a range
of conformations from the three turn α-helix of ovalbumin to the cleaved or latent inhibitor in which the reactive center
loop is fully inserted into the A sheet of the molecule. While the cleaved state can be achieved in all inhibitory serpins
only plasminogen activator inhibitor-1 and, more recently, antithrombin have been shown to adopt the latent conformation.
We show here that the archetypal serpin, α1-antitrypsin, can also be induced to adopt the latent conformation by heating at high temperatures in 0.7 M citrate for 12
h. The resulting species elutes at a lower sodium chloride concentration on an anion-exchange column and has a more cathodal
electrophoretic mobility on non-denaturing polyacrylamide gel electrophoresis and isoelectric focusing than native M antitrypsin.
Latent antitrypsin is inactive as an inhibitor of bovine α-chymotrypsin, is stable to unfolding with 8 M urea, and is more
resistant to heat-induced loop-sheet polymerization than native but less resistant than cleaved antitrypsin. The reactive
center loop of latent antitrypsin is inaccessible to proteolytic cleavage, and its occupancy of the A sheet prevents the molecule
accepting an exogenous reactive center loop peptide. The activity of latent antitrypsin may be increased from <1% to approximately
35% by refolding from 6 M guanidinium chloride.
Journal of Biological Chemistry 03/1995; 270(10):5282-5288. · 4.77 Impact Factor
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ABSTRACT: α1-Antitrypsin is the archetypal member of the serine proteinase inhibitor or serpin superfamily. Members of the family show structural homology based on a dominant A β-sheet and a mobile reactive centre loop. Our recent crystal structure of α1-antitrypsin stabilized with a point mutation showed the loop to be in a canonical inhibitory conformation in the absence of significant insertion into the A β-sheet. It could be argued that the stabilizing mutation may induce the reactive centre loop to adopt an artificial, and unrepresentative, conformation and the finding seems to be at variance with studies assessing rates of peptide insertion into the A β-sheet and limited proteolysis of the reactive loop. Here we present a 2.9 Å structure of recombinant wild-type α1-antitrypsin with no stabilizing mutations. Again, the reactive loop is in a canonical conformation in the absence of significant insertion into the A β-sheet. A stabilizing salt bridge between P5 glutamate and arginine residue 196, 223 and 281, already identified in the mutant, provides strong evidence that this conformation is not an artefact of crystallization but represents the conformation of the circulating inhibitor in vivo. Comparison with the structure of α1-antitrypsin stabilized with the Phe51Leu mutation indicates that the increased thermal stability of the mutant results from enhanced packing of aromatic residues in the hydrophobic core of the molecule. The structure of wild-type α1-antitrypsin reveals a hydrophobic pocket between s2A and helices D and E that is filled on reactive loop insertion and the formation of biologically relevant loop-sheet polymers. This pocket may provide a target for rational drug design to prevent the formation of polymers and the associated plasma deficiency, liver cirrhosis and emphysema.
Journal of Molecular Biology.
