1. Identification of serpin determinants of specificity and selectivity for furin inhibition through studies of α1PDX (α1-protease inhibitor Portland)-serpin B8 and furin active-site loop chimeras.
- Author
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Izaguirre G, Qi L, Lima M, and Olson ST
- Subjects
- Amino Acid Motifs genetics, Amino Acid Sequence, Biocatalysis drug effects, Catalytic Domain genetics, Electrophoresis, Polyacrylamide Gel, Furin genetics, Furin metabolism, Humans, Kinetics, Models, Molecular, Molecular Sequence Data, Mutation, Proprotein Convertases antagonists & inhibitors, Proprotein Convertases genetics, Proprotein Convertases metabolism, Protein Structure, Secondary, Protein Structure, Tertiary, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Sequence Homology, Amino Acid, Serpins genetics, Serpins metabolism, alpha 1-Antitrypsin genetics, alpha 1-Antitrypsin metabolism, Furin antagonists & inhibitors, Recombinant Fusion Proteins antagonists & inhibitors, Serpins pharmacology, alpha 1-Antitrypsin pharmacology
- Abstract
α1-Protease inhibitor Portland (α1PDX) is an engineered serpin family inhibitor of the proprotein convertase (PC), furin, that exhibits high specificity but limited selectivity for inhibiting furin over other PC family proteases. Here, we characterize serpin B8, a natural inhibitor of furin, together with α1PDX-serpin B8 and furin-PC chimeras to identify determinants of serpin specificity and selectivity for furin inhibition. Replacing reactive center loop (RCL) sequences of α1PDX with those of serpin B8 demonstrated that both the P4-P1 RXXR recognition sequence as well as the P1'-P5' sequence are critical determinants of serpin specificity for furin. Alignments of PC catalytic domains revealed four variable active-site loops whose role in furin reactivity with serpin B8 was tested by engineering furin-PC loop chimeras. The furin(298-300) loop but not the other loops differentially affected furin reactivity with serpin B8 and α1PDX in a manner that depended on the serpin RCL-primed sequence. Modeling of the serpin B8-furin Michaelis complex identified serpin exosites in strand 3C close to the 298-300 loop whose substitution in α1PDX differentially affected furin reactivity depending on the furin loop and serpin RCL-primed sequences. These studies demonstrate that RCL-primed residues, strand 3C exosites, and the furin(298-300) loop are critical determinants of serpin reactivity with furin, which may be exploited in the design of specific and selective α1PDX inhibitors of PCs.
- Published
- 2013
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