Investigating serpin-enzyme complex formation and stability via single and multiple residue reactive centre loop substitutions in heparin cofactor II.
BACKGROUND
Following thrombin cleavage of the reactive centre (P1-P1'; L444-S445) of the serpin heparin cofactor II (HCII), HCII traps thrombin (IIa) in a stable inhibitory complex. To compare HCII to other serpins we substituted: the P13-P5' residues of HCII with those of alpha(1)-proteinase inhibitor (alpha(1)-PI), alpha(1)-PI (M358R), or antithrombin (AT); the P4-P1, P3-P1, and P2-P1 residues of HCII with those of AT; and made L444A/H/K/M or R point mutations. We also combined L444R with changes in the glycosaminoglycan binding domain collectively termed MutD.


METHODS
Variants were made by site-directed mutagenesis, expressed in bacteria, purified and characterized electrophoretically and kinetically.


CONCLUSIONS
Of the P13-P5' mutants, only the alpha(1)-PI-loop variant retained anti-IIa activity, but less than the corresponding L444M. Heparin-catalyzed rate constants for IIa inhibition were reduced vs. wild-type (WT) by at most three-fold for all P1 mutants save L444A (reduced 20-fold). L444R and L444K inhibited IIa>50- and >6-fold more rapidly than WT in heparin-free reactions, but stoichiometries of inhibition were increased for all variants. HCII-IIa complexes of all P1 variants were stable in the absence of heparin, but those of the L444K and L444R variants released active IIa over time with heparin. Limited proteolysis of these two groups of HCII-IIa complexes produced different fragmentation patterns consistent with conformational differences. The combination of either substituted AT residues at P2, P3, and P4, or the MutD mutations with L444R resulted in complex instability with or without heparin. This is the first description of HCII-IIa complexes of transient stability forming in the absence of heparin, and may explain the extent to which the reactive centre loop of HCII differs from that of AT.
Sutherland
Jason S
JS
Canadian Blood Services, Research and Development Department, Canada.
Bhakta
Varsha
V
Sheffield
William P
WP
D019943:Amino Acid Substitution; D001665:Binding Sites; D004795:Enzyme Stability; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology*; D015844:Heparin Cofactor II / Q000737:chemistry* / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D015843:Serpins / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship
D015843:Serpins; D015844:Heparin Cofactor II; D010447:Peptide Hydrolases