title: The binding mechanism of a peptidic cyclic serine protease inhibitor.
abstract: Serine proteases are classical objects for studies of catalytic and inhibitory mechanisms as well as interesting as therapeutic targets. Since small-molecule serine protease inhibitors generally suffer from specificity problems, peptidic inhibitors, isolated from phage-displayed peptide libraries, have attracted considerable attention. Here, we have investigated the mechanism of binding of peptidic inhibitors to serine protease targets. Our model is upain-1 (CSWRGLENHRMC), a disulfide-bond-constrained competitive inhibitor of human urokinase-type plasminogen activator with a noncanonical inhibitory mechanism and an unusually high specificity. Using a number of modified variants of upain-1, we characterised the upain-1-urokinase-type plasminogen activator complex using X-ray crystal structure analysis, determined a model of the peptide in solution by NMR spectroscopy, and analysed binding kinetics and thermodynamics by surface plasmon resonance and isothermal titration calorimetry. We found that upain-1 changes both main-chain conformation and side-chain orientations as it binds to the protease, in particular its Trp3 residue and the surrounding backbone. The properties of upain-1 are strongly influenced by the addition of three to four amino acids long N-terminal and C-terminal extensions to the core, disulfide-bond-constrained sequence: The C-terminal extension stabilises the solution structure compared to the core peptide alone, and the protease-bound structure of the peptide is stabilised by intrapeptide contacts between the N-terminal extension and the core peptide around Trp3. These results provide a uniquely detailed description of the binding of a peptidic protease inhibitor to its target and are of general importance in the development of peptidic inhibitors with high specificity and new inhibitory mechanisms.
authors.affiliation: Danish-Chinese Centre for Proteases and Cancer, Aarhus University, DK-8000 Aarhus C, Denmark.
authors.forename: Longguang
authors.initials: L
authors.lastname: Jiang
authors.forename: Anna S P
authors.initials: AS
authors.lastname: Svane
authors.forename: Hans Peter
authors.initials: HP
authors.lastname: Sørensen
authors.forename: Jan K
authors.initials: JK
authors.lastname: Jensen
authors.forename: Masood
authors.initials: M
authors.lastname: Hosseini
authors.forename: Zhuo
authors.initials: Z
authors.lastname: Chen
authors.forename: Caroline
authors.initials: C
authors.lastname: Weydert
authors.forename: Jakob T
authors.initials: JT
authors.lastname: Nielsen
authors.forename: Anni
authors.initials: A
authors.lastname: Christensen
authors.forename: Cai
authors.initials: C
authors.lastname: Yuan
authors.forename: Knud J
authors.initials: KJ
authors.lastname: Jensen
authors.forename: Niels Chr
authors.initials: NC
authors.lastname: Nielsen
authors.forename: Anders
authors.initials: A
authors.lastname: Malmendal
authors.forename: Mingdong
authors.initials: M
authors.lastname: Huang
authors.forename: Peter A
authors.initials: PA
authors.lastname: Andreasen
mesh_terms: D000595:Amino Acid Sequence; D006801:Humans; D008969:Molecular Sequence Data; D010456:Peptides, Cyclic / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000378:metabolism*; D020349:Surface Plasmon Resonance
chemical_list: D010456:Peptides, Cyclic; D015842:Serine Proteinase Inhibitors