Crystal Structure of the Complex of Plasminogen Activator Inhibitor 2 with a Peptide Mimicking the Reactive Center Loop

Abstract

The structure of the serpin, plasminogen activator inhibitor type-2 (PAI-2), in a complex with a peptide mimicking its reactive center loop (RCL) has been determined at 1.6-Å resolution. The structure shows the relaxed state serpin structure with a prominent sixstranded ␤-sheet. Clear electron density is seen for all residues in the peptide. The P1 residue of the peptide binds to a well defined pocket at the base of PAI-2 that may be important in determining the specificity of protease inhibition. The stressed-to-relaxed state (S 3 R) transition in PAI-2 can be modeled as the relative motion between a quasirigid core domain and a smaller segment comprising helix hF and ␤-strands s1A, s2A, and s3A. A comparison of the Ramachandran plots of the stressed and relaxed state PAI-2 structures reveals the location of several hinge regions connecting these two domains. The hinge regions cluster in three locations on the structure, ensuring a cooperative S 3 R transition. We hypothesize that the hinge formed by the conserved Gly 206 on ␤-strand s3A in the breach region of PAI-2 effects the S 3 R transition by altering its backbone torsion angles. This torsional change is due to the binding of the P14 threonine of the RCL to the open breach region of PAI-2.The serpins are an unusual class of protein in that they fold into a metastable structure (the stressed state) that can usually undergo a major structural change to an extremely stable form (the relaxed state) on cleavage by a target protease (1). Most serpins are serine (or cysteine) protease inhibitors that act in a suicide fashion, using the stability of the relaxed state to trap the protease in a nonfunctional covalent serpin-protease complex. The target protease cuts the accessible reactive center loop (RCL) 1 on the serpin to produce a covalent acyl intermediate (2, 3). The RCL then inserts into ␤-sheet 2 A to form the relaxed state. Attached to the RCL, the target protease moves to the base of the serpin, where it is partially unfolded, thus inhibiting its activity (4, 5). This mode of action, whereby one protein uses the increased stability of an altered conformer to partially unfold a second, covalently bound target protein, is unprecedented in biology.Plasminogen activator inhibitor 2 (PAI-2) is a serine protease inhibitor that belongs to the ov-serpin branch of the serpin superfamily (6). PAI-2 is an effective inhibitor of urinary plasminogen activator (urokinase or uPA) and, to a lesser extent, an inhibitor of tissue-type plasminogen activator. PAI-2 has several features that distinguish it from the more widely studied serpins. PAI-2 lacks an N-terminal secretory signal but contains a relatively inefficient internal signal sequence (7). This produces a distribution where a majority (ϳ70%) of the protein is nonglycosylated and intracellular, with the rest in a glycosylated, extracellular form. PAI-2 has a unique insertion of ϳ30 residues between helices hC and hD (CD loop), which is the site of glycosylation and the site of cross-linking to the cell membrane a...

PZP and PAI-2: Structurally-diverse, functionally similar pregnancy proteins?

“…, Asn 339 ). Image shows the 3D structure of PAI-2 based on PDB ID: 1JRR (Jankova et al, 2001). PAI-2 conforms to the general tertiary structure conserved across all known serpins (Silverman et al, 2004).…”

Interaction between the P14 Residue and Strand 2 of β-Sheet B Is Critical for Reactive Center Loop Insertion in Plasminogen Activator Inhibitor-2

“…The recently solved crystal structure of the complex between PAI-2 and 14-mer RCL peptide (21) (Fig. 4, C and E) allows a detailed examination of the mechanisms underlying the apparent importance of P14 for RCL insertion in PAI-2.…”

“…The P14 residue (Thr 367 in PAI-2, Thr 345 in LEI) forms backbone hydrogen bonds with the adjacent glycine residue on s3A (Gly 206 in PAI-2, Gly 192 in LEI), whereas the P13 residue (Glu 368 ) bonds to Asp 361 on s5A (21). Hydrogen bond rearrangements in the PAI-2/14-mer complex involving residues immediately N-terminal to P14 (P15-P17) are difficult to interpret.…”

“…Structural determination of the binary complex between PAI-2 (CD-loop deletion mutant) and 14-mer RCL peptide (Protein Data Bank code 1JRR) is described in the accompanying paper (21). Alignment of the 225 serpin amino acid sequences available in the SwissProt and TrEMBL data bases (releases 37 and 10, respectively) (22) was generated by ProDom99.2 (23) using PSI-BLAST with a profile built from the seed alignment of the PF00079 serpin entry of Pfam-A3.4.…”

“…Similarly, the distance between Gly 206 (s3A) and Tyr 258 (s2B) increases from 5.7 Å in stressed PAI-2 to 6.2 Å in relaxed PAI-2. A complete description of the structural differences between stressed and relaxed PAI-2 is provided in the accompanying paper (21).…”