Abstract
Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase-type plasminogen activator (uPA). We used X-ray crystal structure analysis, site-directed mutagenesis, liquid state NMR, surface plasmon resonance analysis, and isothermal titration calorimetry and wild type and engineered variants of murine and human uPA. We demonstrate that Arg6 inserts into the S1 specificity pocket, its carbonyl group aligning improperly relative to Ser195 and the oxyanion hole, explaining why the peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with novel unnatural Arg analogues with aliphatic or aromatic ring structures led to an increased affinity, depending on changes in both P1 - S1 and exosite interactions. Site-directed mutagenesis showed that exosite interactions, while still supporting high affinity binding, differed substantially between different uPA variants. Surprisingly, high affinity binding was facilitated by Ala-substitution of Asp9 of the peptide, in spite of a less favorable binding entropy and loss of a polar interaction. We conclude that increased flexibility of the peptide allows more favorable exosite interactions, which, in combination with the use of novel Arg analogues as P1 residues, can be used to manipulate the affinity and specificity of this peptidic inhibitor, a concept different from conventional attempts at improving inhibitor affinity by reducing the entropic burden.
Highlights
Peptides are of considerable interest as drug candidates
The contact distances between the residues of the peptides and residues of human uPA (huPA)-H99Y are shown in S3-S5 Tables
We describe studies of the inhibition mechanism and the binding mechanism of derivatives of the serine protease inhibitor mupain-1, which was originally selected from a phage-displayed peptide library for binding to murine uPA (muPA) [12]
Summary
Peptides binding to specific protein targets can be selected from phage-displayed peptide libraries with a diversity of up to 106 different sequences. Peptides directly selected from phage-displayed peptide libraries usually bind their targets with KD values in the mM range, but the affinities can be improved by construction of focused libraries or chemical modification, like introduction of unnatural amino acids. Serine proteases of the trypsin family (clan SA) have many physiological and pathophysiological functions [4,5,6]. One interesting member of the trypsin family of serine proteases is urokinase-type plasminogen activator (uPA), which catalyses the conversion of the zymogen plasminogen into the active protease plasmin through cleavage of plasminogen’s Arg15–Val bond (using the chymotrypsin numbering [8]). Plasmin generated by uPA participates in the turnover of extracellular matrix proteins in physiological and pathophysiological tissue remodeling [9, 10]. Abnormal expression of uPA is responsible for tissue damage in several pathological conditions, including rheumatoid arthritis, allergic vasculitis, and xeroderma pigmentosum, and in particular, is a key factor for the invasive capacity of malignant tumors [11]. uPA is a potential therapeutic target
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