PDBsum entry 4x1r

Hydrolase inhibitor/hydrolase

PDB id: 4x1r

Contents

Protein chain

247 a.a.

Ligands

CYS-PRO-ALA-TYR-SER-ALA-TYR-LEU-ASP-CYS

PL0

Waters ×32

PDB id:

4x1r

Name:

Hydrolase inhibitor/hydrolase

Title:

The crystal structure of mupain-1-12 in complex with murinised human upa at ph7.4

Structure:

Mupain-1-12. Chain: p. Engineered: yes. Urokinase-type plasminogen activator. Chain: u. Fragment: catalytic domain (unp residues 179-425). Synonym: upa. Engineered: yes. Mutation: yes

Source:

Synthetic: yes. Synthetic construct. Organism_taxid: 32630. Homo sapiens. Human. Organism_taxid: 9606. Gene: plau. Expressed in: komagataella pastoris. Expression_system_taxid: 4922

Resolution:

2.10Å R-factor: 0.213 R-free: 0.273

Authors:

L.Jiang,B.Zhao,P.Xu,P.Andreasen,M.Huang

Key ref:

B.Zhao et al. (2014). A cyclic peptidic serine protease inhibitor: increasing affinity by increasing peptide flexibility. Plos One, 9, e115872. PubMed id: 25545505 DOI: 10.1371/journal.pone.0115872

Date:

25-Nov-14 Release date: 25-Mar-15

Protein chain

P00749  (UROK_HUMAN) -  Urokinase-type plasminogen activator from Homo sapiens

Seq: 431 a.a.

Struc: 247 a.a.*

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.4.21.73 - u-plasminogen activator.
• Reaction: Specific cleavage of Arg-|-Val bond in plasminogen to form plasmin.

DOI no: 10.1371/journal.pone.0115872

Plos One 9:e115872 (2014)

PubMed id: 25545505

A cyclic peptidic serine protease inhibitor: increasing affinity by increasing peptide flexibility.

B.Zhao, P.Xu, L.Jiang, B.Paaske, T.Kromann-Hansen, J.K.Jensen, H.P.Sørensen, Z.Liu, J.T.Nielsen, A.Christensen, M.Hosseini, K.K.Sørensen, N.C.Nielsen, K.J.Jensen, M.Huang, P.A.Andreasen.

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.