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.
Highlights
Furin is a member of the proprotein convertase (PC)2 family of calcium-dependent serine proteases that have structural
We demonstrate that in addition to the P4 Arg-X-X-P1 Arg recognition sequence in the serpin reactive center loop (RCL), the primed RCL sequence plays a critical role in ␣1PDX reactivity with furin and its ability to discriminate among different PC family members
Reactions performed at increasing serpin B8 concentrations produced a family of inhibition progress curves that were characterized by increasing rates of furin inhibition and decreasing fluorescence amplitudes (Fig. 1)
Summary
Proteins—cDNA encoding serpin B8 was kindly provided by Dr Walter Kisiel (University of New Mexico, Albuquerque, NM). Protein concentration was determined from the 280 nm absorbance using an extinction coefficient of 32,900 MϪ1 cmϪ1 calculated from the amino acid sequence [23]. Both serpin B8 forms migrated on SDS-PAGE as monomeric forms with the expected molecular weight under both nonreducing and reducing conditions, and proved to be very stable. The slope of linear regression fits of the data (solid lines) provided the apparent second order association rate constants (ka,app) for the inhibition reaction. The abscissa intercept of the linear regression fit of the data yielded the functional furin concentration. Kinetic constants and stoichiometries of inhibition for reactions of furin with wild-type and variant forms of serpin B8 and ␣1PDX. The SI for the serpin B8 R342A mutant was assumed to be 1 based on the absence of detectable cleaved serpin product in the reaction by SDS-PAGE analysis (Fig. 2)
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