The Bβ-sheet in the PAI-1 Molecule Plays an Important Role for Its Stability

Summary

We have investigated the B β-sheet in PAI-1 regarding its role for the stability of the molecule. The residues from His219 to Tyr241 (except for Gly²³⁰ and Pro²⁴⁰), covering the s2B and s3B strands, and in addition His¹⁸⁵ and His¹⁹⁰ were substituted by amino acids with opposite properties. The 23 generated single-site changed mutants and also wild type PAI-1 (wtPAI-1) were expressed in E. coli. Subsequently they were purified by heparin-Sepharose and anhydrotrypsin agarose affinity chromatographies. The stability of the purified PAI-1 variants was analyzed at 37° C and at different pHs (5.5, 6.5 or 7.5). At pH 7.5 and 37° C, single substitutions of the residues in the central portions of both strands 2 and 3 in the B β-sheet (Ile²²³ to Leu²²⁶ on s2B and Met²³⁵ to Ile²³⁷ on s3B), caused a significant decrease in stability, yielding half-lives of about 10–25% as compared to wtPAI-1. On the other hand, mutations at both sides of the central portion of the B β-sheet (Tyr²²¹, Asp²²², Tyr²²⁸ and Thr²³²) frequently resulted in an increased PAI-1 stability (up to 7-fold). While wtPAI-1 exhibited prolonged half-lives at pH 6.5 and 5.5, the PAI-1 variant Y228S was more stable at neutral pH (half-life of 9.6 h at pH 7.5) as compared to its half-life at pH 5.5 (1.1 h). One of the 4 modified histidine residues (His²²⁹) resulted in a variant with a clearly affected stability as a function of pH, suggesting that it may, at least in part, be of importance for the pH dependence of the PAI-1 stability. Thus, our data demonstrate that the B β-sheet is of great importance for the stability of the molecule. Modifications in this part causes decreased or increased stability in a certain pattern, suggesting effects on the insertion rate of the reactive center loop into the A β-sheet of the molecule.

Keywords

PAI-1 - site-directed mutagenesis - serine proteinase inhibitor

¹ Current address: Dr. G.-C. Sui, WAB-120, Dept. of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA

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