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Expression levels of CX3CR1 (C-X3-C motif chemokine receptor 1) on immune cells have significant importance in maintaining tissue homeostasis under physiological and pathological conditions. The factors implicated in the regulation of CX3CR1 and its specific ligand CX3CL1 (fractalkine) expression remain largely unknown. Recent studies provide evidence that host's misfolded proteins occurring in the forms of polymers or amyloid fibrils can regulate CX3CR1 expression. Herein, a novel example demonstrates that polymers of human ZZ alpha-1 antitrypsin (Z-AAT) protein, resulting from its conformational misfolding due to the Z (Glu342Lys) mutation in SERPINA1 gene, strongly lower CX3CR1 mRNA expression in human peripheral blood mononuclear cells (PBMCs). This parallels with increase of intracellular levels of CX3CR1 and Z-AAT proteins. Presented data indicate the involvement of the CX3CR1 pathway in the Z-AAT-related disorders and further support the role of misfolded proteins in CX3CR1 regulation., Competing Interests: ST, MB, SW, MK, ML, RL, BM, EK, DD, NJ, MF, SJ No competing interests declared, TW reports grants from German Ministry of Research and Education, during the conduct of the study; personal fees from Grifols, CSL Behring, outside the submitted work, PS reports grants and personal fees from CSL Behring, grants and personal fees from Grifols Inc, personal fees from Dicerna Inc, grants from Vertex, grants from Arrowhead, outside the submitted work, JS reports grants from CSL Behring, grants from Kamada LtD, during the conduct of the study, (© 2021, Tumpara et al.)

Alpha1-antitrypsin (AAT) deficiency predisposes individuals to emphysema and liver diseases such as cirrhosis and hepatocellular carcinoma. The deficiency results from mutations in the SERPIN1A gene encoding AAT molecules that cause hepatotoxic retention within the endoplasmic reticulum. Since the E342K mutation is the basis for destabilization leading to lung and liver pathologies, we used the crystal structure of the mutated AAT as the basis for molecular docking selection of candidate compounds that may bind and stabilize the 342K structural pocket. We identified compounds that inhibited intracellular accumulation of AAT in hepatocytes in vitro. These data suggest that drug binding to a structural site encoded by a mutation associated with AAT deficiency has the potential for clinical utility by modulating conformational transitions., (© 2019 Federation of European Biochemical Societies.)

HIV-1 entry into cells is mediated by the envelope glycoprotein (Env) and represents an attractive target for therapeutic intervention. Two drugs that inhibit HIV entry are approved for clinical use: the membrane fusion-inhibitor T20 (Fuzeon, enfuvirtide) and the C-C chemokine receptor type 5 (CCR5) blocker maraviroc (Selzentry). Another class of entry inhibitors supposedly target the fusion peptide (FP) and are termed anchor inhibitors. These include the VIRIP peptide and VIRIP derivatives such as VIR165, VIR353, and VIR576. Here, we investigated the mechanism of inhibition by VIR165. We show that substitutions within the FP modulate sensitivity to VIR165, consistent with the FP being the drug target. Our results also revealed that VIR165 acts during an intermediate post-CD4-binding entry step that is overlapping but not identical to the step inhibited by fusion inhibitors such as T20. We found that some but not all resistance mutations to heptad repeat 2 (HR2)-targeting fusion inhibitors can provide cross-resistance to VIR165. In contrast, resistance mutations in the HR1-binding site for the fusion inhibitors did not cause cross-resistance to VIR165. However, Env with mutations located outside this binding site and thought to affect fusion kinetics, exhibited decreased sensitivity to VIR165. Although we found a strong correlation between Env stability and resistance to HR2-based fusion inhibitors, such correlation was not observed for Env stability and VIR165 resistance. We conclude that VIRIP analogs target the FP during an intermediate, post-CD4-binding entry step that overlaps with but is distinct from the step(s) inhibited by HR2-based fusion inhibitors., (© 2019 Eggink et al.)

Conserpin is an engineered protein that represents the consensus of a sequence alignment of eukaryotic serpins: protease inhibitors typified by a metastable native state and a structurally well-conserved scaffold. Previously, this protein has been found to adopt a native inhibitory conformation, possess an atypical reversible folding pathway and exhibit pronounced resistance to inactivation. Here we have designed a version of conserpin, cAT, with the inhibitory specificity of α 1 -antitrypsin, and generated single-tryptophan variants to probe its folding pathway in more detail. cAT exhibited similar thermal stability to the parental protein, an inactivation associated with oligomerisation rather a transition to the latent conformation, and a native state with pronounced kinetic stability. The tryptophan variants reveal the unfolding intermediate ensemble to consist of an intact helix H, a distorted helix F and 'breach' region structurally similar to that of a mesophilic serpin intermediate. A combination of intrinsic fluorescence, circular dichroism, and analytical gel filtration provide insight into a highly cooperative folding pathway with concerted changes in secondary and tertiary structure, which minimises the accumulation of two directly-observed aggregation-prone intermediate species. This functional conserpin variant represents a basis for further studies of the relationship between structure and stability in the serpin superfamily.

Severe alpha-1-antitrypsin deficiency (AATD) is most frequently associated with the alpha-1-antitrypsin (AAT) Z variant (E342K). ZZ homozygotes exhibit accumulation of AAT as polymers in the endoplasmic reticulum of hepatocytes. This protein deposition can lead to liver disease, with the resulting low circulating levels of AAT predisposing to early-onset emphysema due to dysregulation of elastinolytic activity in the lungs. An increasing number of rare AAT alleles have been identified in patients with severe AATD, typically in combination with the Z allele. Here we report a new mutation (E75V) in a patient with severe plasma deficiency, which we designate Trento. In contrast to the Z mutant, Trento AAT was secreted efficiently when expressed in cellular models but showed compromised conformational stability. Polyacrylamide gel electrophoresis (PAGE) and ELISA-based analyses of the secreted protein revealed the presence of oligomeric species with electrophoretic and immunorecognition profiles different from those of Z and S (E264V) AAT polymers, including reduced recognition by conformational monoclonal antibodies 2C1 and 4B12. This altered recognition was not due to direct effects on the epitope of the 2C1 monoclonal antibody which we localized between helices E and F. Structural analyses indicate the likely basis for polymer formation is the loss of a highly conserved stabilizing interaction between helix C and the posthelix I loop. These results highlight this region as important for maintaining native state stability and, when compromised, results in the formation of pathological polymers that are different from those produced by Z and S AAT., (© 2017 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Misfolding, polymerization, and defective secretion of functional alpha-1 antitrypsin underlies the predisposition to severe liver and lung disease in alpha-1 antitrypsin deficiency. We have identified a novel (Ala336Pro, Baghdad) deficiency variant and characterized it relative to the wild-type (M) and Glu342Lys (Z) alleles. The index case is a homozygous individual of consanguineous parentage, with levels of circulating alpha-1 antitrypsin in the moderate deficiency range, but is a biochemical phenotype that could not be classified by standard methods. The majority of the protein was present as functionally inactive polymer, and the remaining monomer was 37% active relative to the wild-type protein. These factors combined indicate an 85 to 95% functional deficiency, similar to that seen with ZZ homozygotes. Biochemical, biophysical, and computational studies further defined the molecular basis of this deficiency. These studies demonstrated that native Ala336Pro alpha-1 antitrypsin could populate the polymerogenic intermediate-and therefore polymerize-more readily than either wild-type alpha-1 antitrypsin or the Z variant. In contrast, folding was far less impaired in Ala336Pro alpha-1 antitrypsin than in the Z variant. The data are consistent with a disparate contribution by the "breach" region and "shutter" region of strand 5A to folding and polymerization mechanisms. Moreover, the findings demonstrate that, in these variants, folding efficiency does not correlate directly with the tendency to polymerize in vitro or in vivo. They therefore differentiate generalized misfolding from polymerization tendencies in missense variants of alpha-1 antitrypsin. Clinically, they further support the need to quantify loss-of-function in alpha-1 antitrypsin deficiency to individualize patient care.

α1-Antitrypsin (α1AT) deficiency, the most common serpinopathy, results in both emphysema and liver disease. Over 90% of all clinical cases of α1AT deficiency are caused by the Z variant in which Glu342, located at the top of s5A, is replaced by a Lys which results in polymerization both in vivo and in vitro. The Glu342Lys mutation removes a salt bridge and a hydrogen bond but does not effect the thermodynamic stability of Z α1AT compared to the wild type protein, M α1AT, and so it is unclear why Z α1AT has an increased polymerization propensity. We speculated that the loss of these interactions would make the native state of Z α1AT more dynamic than M α1AT and that this change renders the protein more polymerization prone. We have used hydrogen/deuterium exchange combined with mass spectrometry (HXMS) to determine the structural and dynamic differences between native Z and M α1AT to reveal the molecular basis of Z α1AT polymerization. Our HXMS data shows that the Z mutation significantly perturbs the region around the site of mutation. Strikingly the Z mutation also alters the dynamics of regions distant to the mutation such as the B, D and I helices and specific regions of each β-sheet. These changes in global dynamics may lead to an increase in the likelihood of Z α1AT sampling a polymerogenic structure thereby causing disease.

The human serine protease inhibitor (serpin) α-1 antitrypsin (α1-AT) protects tissues from proteases of inflammatory cells. The most common disease-causing mutation in α1-AT is the Z-mutation (E342K) that results in an increased propensity of α1-AT to polymerize in the ER of hepatocytes, leading to a lack of secretion into the circulation. The structural consequences of this mutation, however, remain elusive. We report a comparative molecular dynamics investigation of the native states of wild-type and Z α1-AT, revealing a striking contrast between their structures and dynamics in the breach region at the top of β-sheet A, which is closed in the wild-type simulations but open in the Z form. Our findings are consistent with experimental observations, notably the increased solvent exposure of buried residues in the breach region in Z, as well as polymerization via domain swapping, whereby the reactive center loop is rapidly inserted into an open A-sheet before proper folding of the C-terminal β-strands, allowing C-terminal domain swapping with a neighboring molecule. Taken together, our experimental and simulation data imply that mutations at residue 342 that either stabilize an open form of the top of β-sheet A or increase the local flexibility in this region, may favor polymerization and hence aggregation., (Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Mutations in the SERPINA1 gene can cause deficiency in the circulating serine protease inhibitor α(1)-Antitrypsin (α(1)AT). α(1)AT deficiency is the major contributor to pulmonary emphysema and liver disease in persons of European ancestry, with a prevalence of 1 in 2500 in the USA. We present the discovery and characterization of a novel SERPINA1 mutant from an asymptomatic Middle Eastern male with circulating α(1)AT deficiency. This 49 base pair deletion mutation (T379Δ), originally mistyped by IEF, causes a frame-shift replacement of the last sixteen α(1)AT residues and adds an extra twenty-four residues. Functional analysis showed that the mutant protein is not secreted and prone to intracellular aggregation.

Human alpha(1)-proteinase inhibitor (alpha(1)-PI) is the most abundant protease inhibitor found in the blood and expression of biologically active recombinant alpha(1)-PI has great potential in therapeutic applications. We report here the expression of a synthetic alpha(1)-PI gene and its variants in Escherichia coli. Modified alpha(1)-PI gene and its single amino acid variants were cloned in pMAL-c2X vector, which allowed expression of recombinant protein(s) as a fusion of maltose-binding protein (MBP) with factor Xa protease recognition site between the fusion partners. The synthetic gene(s) were expressed in different E. coli strains and maximum expression of recombinant alpha(1)-PI and variants up to 24% of total soluble protein (TSP) was achieved with engineered strain carrying extra copies of tRNAs for rare codons. Recombinant alpha(1)-PI protein(s) were purified by amylose affinity chromatography with high homogeneity and overall yield of about 7-9 mg l(-1) of bacterial culture (approximately 5.2 g wet cell mass). E. coli expressed recombinant alpha(1)-PI showed specific anti-elastase activity and appeared as a single band of approximately 45.0 kDa on SDS-PAGE. Primary structure of purified protein and integrity of N-terminus has been verified by mass spectrometric analysis. Recombinant alpha(1)-PI expressed in E. coli was fully intact having molecular mass similar to native unglycosylated protein purified from human plasma. Increased thermostability and specific activities of purified alpha(1)-PI variant proteins confirmed the stabilizing effect of incorporated mutations. Our results demonstrate efficient expression and purification of stable and biologically active alpha(1)-PI and its variants in E. coli for further therapeutic applications., (2010 Elsevier B.V. All rights reserved.)

The serpins are an unusual class of protease inhibitors which fold to a metastable form and subsequently undergo a massive conformational change to a stable form when they inhibit their target proteases. The driving force for this conformational change has been extensively investigated by site directed mutagenesis, and it has been found that mutations which stabilize the metastable form frequently result in activity deficiency. Here, we employ hydrogen/deuterium exchange to probe the effects of a cavity filling mutant of alpha(1)AT. The Gly117 --> Phe substitution fills a cavity between the F-helix and the face of beta-sheet A, stabilizes the metastable form of alpha(1)AT by approximately 4 kcal/mol and results in a 60% reduction in inhibitory activity against elastase. Globally, the G117F substitution alters the unfolding mechanism by eliminating the molten globule intermediate that is seen in wild type unfolding. Remarkably, this is accomplished primarily by destabilizing the molten globule rather than stabilizing the metastable native state. Locally, conformational flexibility in the native state is reduced in specific regions: the top of the F-helix, beta-strands 5A, 1C, and 4C, and helix D. Except for strand 4C, all of these regions mediate or propagate conformational changes. The F-helix and strand 5A must be displaced during protease inhibition, displacement of strand 1C is required for polymer formation, and helix D is a site (in antithrombin) of allosteric regulation. Our results indicate that these functionally important regions form a delocalized network of residues that are dynamically coupled and that both local and global stability mediate inhibitory activity.

A 65-year-old man with a colonic adenocarcinoma and a single small liver nodule underwent surgery. The nodule showed the histological features of a bile duct adenoma. Large eosinophilic, periodic acid-Schiff-positive diastase-resistant intracytoplasmic globules conferred a signet-ring appearance to the cells. Similar globules were found in the biliary epithelium in perinodular portal tracts, whereas smaller globules were detected in periportal hepatocytes. All globules showed strong immunoreactivity for alpha-1-antitrypsin, and the sequence of the alpha-1-antitrypsin gene showed a heterozygous M(malton) genotype. The selective recurrence of large alpha-1-antitrypsin globules within the bile ducts may indicate a neoplastic rather than a reactive or hamartomatous nature of the nodule.

Furin proteolytically cleaves a wide variety of proprotein substrates mainly within the trans-Golgi network (TGN) but also at the cell membrane and in endosomal compartments where pH is more acidic. Incorporation of furin recognition sequences within the reactive site loop (RSL) of alpha(1)-antitrypsin (AT) leads to the production of furin inhibitors. In an attempt to design more stable, potent, and specific serpin-based inhibitors, we constructed a series of AT and alpha(1)-antichymotrypsin (ACT) mutants by modifying the P(7)-P(1) region of their RSLs. The biochemical properties of these variants were assessed by evaluating their propensity to establish SDS-resistant complexes with furin in a variety of conditions (pH 6.0-9.0) and by measuring their association rate constants. The effect of pH during the initial steps of complex formation was minimal, suggesting that the acylation step is not rate-limiting. The decrease in stoichiometry of inhibition (SI) values observed in AT variants at high pHs was a result of the reduced pH-dependent deacylation rate, which is rate-limiting in this mechanism and which suggests increased complex stability. Conversely, the SI values for ACT mutants had a tendency to be lower at acidic pH. Transiently transfecting HEK293 cells with these mutants abolished processing of the pro-von Willebrand factor precursor but, interestingly, only the ACT variants were secreted in the media as uncleaved forms. Our results suggest that reengineering the reactive site loops of serpins to accommodate and target furin or other serine proteases must take into account the intrinsic physicochemical properties of the serpin.

The native conformation of proteins in the serpin superfamily is metastable. In order to understand why serpins attain the native state instead of more stable conformations we have begun investigations into the equilibrium-unfolding of alpha(1)-antitrypsin. alpha(1)-Antitrypsin contains two tryptophan residues, Trp194 and Trp238, situated on the A and B beta-sheets, respectively. Site-directed mutagenesis was used to construct two single-tryptophan variants. Both variants were fully active and had similar secondary structure and stabilities to alpha(1)-antitrypsin. The denaturation of alpha(1)-antitrypsin and its variants was extremely similar when followed by far-UV CD, indicating the presence of a single intermediate. Fluorescence analysis of the unfolding behavior of each single tryptophan variant indicated that the sole tryptophan residue reported the structural changes within its immediate environment. These data suggest that the A beta-sheet is expanded in the intermediate state whilst no structural change around the B beta-sheet has occurred. In the urea-induced unfolded state, Trp238 does not become fully solvated, suggesting the persistence of structure around this residue. The implications of these data on the folding, misfolding and function of the serpin superfamily are discussed., (Copyright 2001 Academic Press.)

The classical form of alpha 1-antitrypsin (alpha 1-AT) deficiency is associated with a mutant alpha 1-ATZ molecule that polymerizes in the endoplasmic reticulum (ER) of liver cells. A subgroup of individuals homozygous for the protease inhibitor (PI) Z allele develop chronic liver injury and are predisposed to hepatocellular carcinoma. In this study we evaluated the primary structure of alpha 1-AT in a family in which three affected members had severe liver disease associated with alpha 1-AT deficiency. We discovered that one sibling was a compound heterozygote with one PI Z allele and a second allele, the PI Z + saar allele, bearing the mutation that characterizes alpha 1-ATZ as well as the mutation that characterizes alpha 1-AT Saarbrucken (alpha 1-AT saar). The mutation in PI saar introduces a premature termination codon resulting in an alpha 1-AT protein truncated for 19 amino acids at its carboxyl terminus. Studies of a second sib with severe liver disease and other living family members did not reveal the presence of the alpha 1-AT saar mutation and therefore do not substantiate a role for this mutation in the liver disease phenotype of this family. However, studies of alpha 1-AT saar and alpha 1-ATZ + saar expressed in heterologous cells show that there is prolonged intracellular retention of these mutants even though they do not have polymerogenic properties. These results therefore have important implications for further understanding the fate of mutant alpha 1-AT molecules, the mechanism of ER retention, and the pathogenesis of liver injury in alpha 1-AT deficiency.

The structure of native alpha1-antitrypsin, the most abundant protease inhibitor in human plasma, is characterised primarily by a reactive loop containing the centre of proteinase inhibition, and a beta-sheet composed of five strands. Mobility of the reactive loop is confined as a result of electrostatic interactions between side chains of Glu342 and Lys290, both located at the junction of the reactive loop and the beta structure. The most common mutation in the protein, resulting in its inactivation, is Glu342-->Lys, named the Z mutation. The main goal of this work was to investigate the influence of the Z mutation on the structure of alpha1-antitrypsin. Commonly used molecular modelling methods have been applied in a comparative study of two protein models: the wild type and the Z mutant. The results indicate that the Z mutation introduces local instabilities in the region of the reactive loop. Moreover, even parts of the protein located far apart from the mutation region are affected. The Z mutation causes a relative change in the total energy of about 3%. Relatively small root mean square differences between the optimised structures of the wild type and the Z mutant, together with detailed analysis of 'conformational searching' process, lead to the hypothesis that the Z mutation principally induces a change in the dynamics of alpha1-antitrypsin.

The metastability of inhibitory serpins (serine proteinase inhibitors) is thought to play a key role in the facile conformational switch and the insertion of the reactive center loop into the central beta-sheet, A-sheet, during the formation of a stable complex between a serpin and its target proteinase. We have examined the folding and inhibitory activity of a very stable variant of human alpha1-antitrypsin, a prototype inhibitory serpin. A combination of seven stabilizing single amino acid substitutions of alpha1-antitrypsin, designated Multi-7, increased the midpoint of the unfolding transition to almost that of ovalbumin, a non-inhibitory but more stable serpin. Compared with the wild-type alpha1-antitrypsin, Multi-7 retarded the opening of A-sheet significantly, as revealed by the retarded unfolding and latency conversion of the native state. Surprisingly, Multi-7 alpha1-antitrypsin could form a stable complex with a target elastase with the same kinetic parameters and the stoichiometry of inhibition as the wild type, indicating that enhanced A-sheet closure conferred by Multi-7 does not affect the complex formation. It may be that the stability increase of Multi-7 alpha1-antitrypsin is not sufficient to influence the rate of loop insertion during the complex formation.

The Z (Glu342-->Lys) and Siiyama (Ser53-->Phe) deficiency variants of alpha 1-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 beta-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 beta-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.

A thermostable mutation, F51L, at the hydrophobic core of human alpha 1-antitrypsin (alpha 1AT) increased the conformational stability of the molecule by decreasing the unfolding rate significantly without altering the refolding rate. The mutation specifically influenced the transition between the native state and a compact intermediate, which retained approximately 70% of the far-UV CD signal, but which had most of the fluorescence signal already dequenched. The mutant alpha 1AT protein was more resistant than the wild-type protein to the insertion of the tetradecapeptide mimicking the sequence of the reactive center loop, indicating that the mutation increases the closing of the central beta-sheet, the A-sheet, in the native state. The F51L mutation enhanced the folding efficiency of the Z-type (E342K) genetic variation, which causes aggregation of the molecule in the liver. It has been shown previously that the aggregation of the Z protein occurs via loop-sheet polymerization, in which the reactive center loop of one molecule is inserted into the opening of the A-sheet of another molecule. Our results strongly suggest that the hydrophobic core of alpha 1AT regulates the opening-closing of the A-sheet and that certain genetic variations that cause opening of the A-sheet can be corrected by inserting an additional stable mutation into the hydrophobic core.

Antitrypsin Siiyama is a rare example of the deficiency variants of antitrypsin that accumulate in the endoplasmic reticulum of the hepatocyte. The common example is Z antitrypsin, which has a mutation (Glu342-->Lys) at the junction of the head of the fifth strand of the A sheet and the base of the reactive center loop. It was previously shown that Z antitrypsin spontaneously polymerizes due to the insertion of the reactive center loop of one molecule into the A sheet of a second. The mutation in antitrypsin Siiyama (Ser53-->Phe) affects a residue that provides a ridge for the sliding movement that opens the A sheet, and it had been predicted that this would result in the same type of loop-sheet polymerization observed with the Z variant. We confirm this here and show that virtually all the plasma antitrypsin in a homozygote for the Siiyama variant was polymerized due to non-covalent bonding with a loss of accessibility of the reactive center loop. The common basis of the polymerization of Z and Siiyama antitrypsin is supported by identical findings on electron microscopy. Taken together these results confirm that loop-sheet polymerization is a general mechanism and as such is likely to be responsible for the intracellular inclusions associated with liver pathology.

A major feature of the structure of alpha 1-antitrypsin is a five-stranded A-sheet into which the reactive center loop inserts after cleavage. We describe here the effect of the Z mutation (342Glu to Lys) at the head of the fifth strand of the A-sheet on the mobility of the reactive center loop and hence on the physical properties of the antitrypsin molecule. The mutant Z but not the normal M antitrypsin spontaneously polymerizes at 37 degrees C by a mechanism involving the insertion of the reactive center loop of one molecule into the A-sheet of a second. It is demonstrated that Z antitrypsin polymerized after incubation with 1.0 M guanidinium chloride at 37 degrees C at the same rate as M antitrypsin. Reducing the temperature to 4 degrees C favored the formation of the L-state in M antitrypsin in which the loop is stably incorporated into the A-sheet, but resulted in loop-sheet polymerization in Z antitrypsin. Z, like M antitrypsin, undergoes the S to R transition, but we show that the accompanying change in thermal stability results from loop-sheet polymerization (S) which can be prevented by the insertion of the cleaved strand of the reactive center loop into the A-sheet (R). Z antitrypsin has a reduced association rate constant with neutrophil elastase [(5.3 +/- 0.06) x 10(7) and (1.2 +/- 0.02) x 10(7) M-1 s-1 for M and Z, respectively], but both M and Z antitrypsin had Ki values of less than 5 pM.(ABSTRACT TRUNCATED AT 250 WORDS)