Characterization of a Human α1-Antitrypsin Variant That Is as Stable as Ovalbumin

Lee, Kee Nyung; Im, Ha Na; Kang, Sang Won; Yu, Myeong Hee

doi:10.1074/jbc.273.5.2509

Cited by 35 publications

(35 citation statements)

References 50 publications

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“…4). In contrast, the increase in the stabilization energy up to 9 kcal mol Ϫ1 in the hydrophobic core by combining seven stabilizing single amino acid substitutions (F51L, T59A, T68A, A70G, M374I, S381A, and K387R; the mutant was named Multi-7) did not affect the inhibitory activity of ␣ 1 AT toward target elastases (28). The results imply that local stability of the serpin is critical to inhibitory activity.…”

Section: Importance Of Local Strain In the Inhibitory Functionmentioning

confidence: 53%

“…Complex Formation with a Protease-Complex formation of mutant ␣ 1 AT with porcine pancreatic elastase was examined by monitoring the SDS-resistant ␣ 1 AT-proteinase complex (28). The active concentration of porcine pancreatic elastase was determined as described above.…”

Section: Methodsmentioning

confidence: 99%

“…Determination of the Stoichiometry of Inhibition-The stoichiometry of inhibition was determined by titration reactions as described (28). The active concentration of porcine pancreatic elastase was determined by measuring the initial rates of hydrolysis of 1 mM N-succinyl-(Ala) 3 p-nitroanilide.…”

Section: Methodsmentioning

confidence: 99%

“…Unlike group I mutations, which decreased the size of side chains, G164V and A183V increased the size of side chains. The hydrophobic core mutation reported previously, Multi-7, did not show any significant activity change even with a stability increase up to 9 kcal mol Ϫ1 (28). It is also included in the plot for comparison, and the relationship between stability increase and activity change is also indicated (Fig.…”

Section: Figmentioning

confidence: 99%

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Metastability in the Inhibitory Mechanism of Human α1-Antitrypsin

Seo

1999

Journal of Biological Chemistry

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Metastability of the native form of proteins has been recognized as a mechanism of biological regulation. The energy-loaded structure of the fusion protein of influenza virus and the strained native structure of serpins (serine protease inhibitors) are typical examples. To understand the structural basis and functional role of the native metastability of inhibitory serpins, we characterized stabilizing mutations of ␣ 1 -antitrypsin in a region presumably involved in complex formation with a target protease. We found various unfavorable interactions such as overpacking of side chains, polar-nonpolar interactions, and cavities as the structural basis of the native metastability. For several stabilizing mutations, there was a concomitant decrease in the inhibitory activity. Remarkably, some substitutions at Lys-335 increased the stability over 6 kcal mol ؊1 with simultaneous loss of activity over 30% toward porcine pancreatic elastase. Considering the location and energetic cost of Lys-335, we propose that this lysine plays a pivotal role in conformational switch during complex formation. Our current results are quite contradictory to those of previously reported hydrophobic core mutations, which increased the stability up to 9 kcal mol ؊1 without any significant loss of activity. It appears that the local strain of inhibitory serpins is critical for the inhibitory activity.Facile conversion of the metastable native structure of proteins into an alternative more stable form, accompanying the execution of their functions, has been recognized as a mechanism of biological regulation. The energy-loaded structure of the fusion protein of influenza virus (1), the strained native structure of plasma serpins (serine protease inhibitors) (2), and possibly the surface glycoprotein of human immunodeficiency virus (HIV) 1 (3) are typical examples. The native strain of serpins is considered to be crucial to their physiological functions, such as plasma protease inhibition (2, 4), hormone delivery (5), Alzheimer filament assembly (6, 7), and extracellular matrix remodeling (8). The inhibitory serpins, which include ␣ 1 -antitrypsin (␣ 1 AT), antithrombin III, ␣ 1 -antichymotrypsin, and plasminogen activator inhibitor-1, serve as a good model system to study the native metastability; several crystal structures of both the strained native (9 -13) and the relaxed cleaved forms (14 -16) are available. In addition, the inhibitory activity that presumably is related to the native metastability is easy to assay.The serpin structure is composed of three ␤-sheets and several ␣-helices (Fig. 1). Upon binding a target protease, the reactive center loop of inhibitory serpins is thought to be inserted into the major ␤-sheet, A sheet, to form a very stable complex between the inhibitor and the protease (17, 18). Various biochemical (19,20) and structural (21-23) studies suggest that the loop insertion is necessary for the formation of a stable complex but not sufficient to confer inhibitory activity. Instead, the rate of loop insertion is tho...

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Section: Importance Of Local Strain In the Inhibitory Functionmentioning

confidence: 53%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Figmentioning

confidence: 99%

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Metastability in the Inhibitory Mechanism of Human α1-Antitrypsin

Seo

1999

Journal of Biological Chemistry

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“…Determination of the Stoichiometry of Inhibition-The SI was determined as described (25). The active concentration of PPE was determined by measuring the initial rates of hydrolysis of 1 mM N-succinyl-(Ala) 3 -p-nitroanilide.…”

Section: Methodsmentioning

confidence: 99%

Distribution of the Native Strain in Human α1-Antitrypsin and Its Association with Protease Inhibitor Function

Seo¹,

Im²,

Maeng³

et al. 2000

Journal of Biological Chemistry

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Serine protease inhibitors (serpins) are metastable in their native state. This strain, which is released upon binding to target proteases, is essential for the inhibitory activity of serpins. To understand the structural basis of the native strain, we previously characterized stabilizing mutations of ␣ 1 -antitrypsin, a prototypical inhibitory serpin, in regions such as the hydrophobic core. The present study evaluates the effects of single point mutations throughout the molecule on stability and protease inhibitory activity. We identified stabilizing mutations in most secondary structures, suggesting that the native strain is distributed throughout the molecule. Examination of the substitution patterns and the structures of the mutation sites revealed surface hydrophobic pockets as a component of the native strain in ␣ 1 -antitrypsin, in addition to the previously identified unusual interactions such as side chain overpacking and cavities. Interestingly, many of the stabilizing substitutions did not affect the inhibitory activity significantly. Those that affected the activity were confined in the regions that are mobilized during the complex formation with a target enzyme. The results of our study should be useful for designing proteins with strain and for regulating the stability and functions of serpins.1 is a prototype of the serpin (serine protease inhibitor) superfamily that shares a common tertiary structure composed of three ␤-sheets and several ␣-helices (1). Serpins include protease inhibitors in blood plasma such as ␣ 1 AT, ␣ 1 -antichymotrypsin, antithrombin III, plasminogen activator inhibitor-I, C1 inhibitor, and ␣ 2 -antiplasmin, as well as non-inhibitory members such as ovalbumin and angiotensinogen (1, 2). One salient feature of the inhibitory serpin structure is the strain in the native conformation (3-7), which is necessary for biological functions such as protease inhibition and ligand binding (1,2,8,9). The inhibition process of serpins can be described as a suicide substrate mechanism (10 -12) in which serpins, upon binding proteases, partition between cleaved serpins and stable serpin-enzyme complexes. The stoichiometry of inhibition (SI; the number of moles of inhibitor required to completely inhibit 1 mol of a target protease) is designated as 1 ϩ k substrate /k inhibition , in which k substrate is the rate constant for the substrate pathway toward the cleaved serpin and k inhibition is the rate constant for the inhibitory pathway toward the complex formation. For cognate target proteases, most serpin molecules partition into the complex formation, bringing the SI values close to 1. During the complex formation, the reactive center loop (RCL) of inhibitory serpins is cleaved (13-15) and inserted into the major ␤-sheet, A sheet, forming a stable complex between the serpin and the protease (11,12,(15)(16)(17). It has been suggested that the rate of loop insertion is critical for inhibitory function; retardation of the loop insertion would alter the partitioning between the inhibitory and...

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Engineering the serpin α₁‐antitrypsin: A diversity of goals and techniques

Scott

Sheffield

2019

Protein Science

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α1‐Antitrypsin (α1‐AT) serves as an archetypal example for the serine proteinase inhibitor (serpin) protein family and has been used as a scaffold for protein engineering for >35 years. Techniques used to engineer α1‐AT include targeted mutagenesis, protein fusions, phage display, glycoengineering, and consensus protein design. The goals of engineering have also been diverse, ranging from understanding serpin structure–function relationships, to the design of more potent or more specific proteinase inhibitors with potential therapeutic relevance. Here we summarize the history of these protein engineering efforts, describing the techniques applied to engineer α1‐AT, specific mutants of interest, and providing an appended catalog of the >200 α1‐AT mutants published to date.

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Characterization of a Human α1-Antitrypsin Variant That Is as Stable as Ovalbumin

Cited by 35 publications

References 50 publications

Metastability in the Inhibitory Mechanism of Human α1-Antitrypsin

Metastability in the Inhibitory Mechanism of Human α1-Antitrypsin

Distribution of the Native Strain in Human α1-Antitrypsin and Its Association with Protease Inhibitor Function

Engineering the serpin α₁‐antitrypsin: A diversity of goals and techniques

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Characterization of a Human α1-Antitrypsin Variant That Is as Stable as Ovalbumin

Cited by 35 publications

References 50 publications

Metastability in the Inhibitory Mechanism of Human α1-Antitrypsin

Metastability in the Inhibitory Mechanism of Human α1-Antitrypsin

Distribution of the Native Strain in Human α1-Antitrypsin and Its Association with Protease Inhibitor Function

Engineering the serpin α1‐antitrypsin: A diversity of goals and techniques

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Product

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Engineering the serpin α₁‐antitrypsin: A diversity of goals and techniques