PDBsum entry 5wxf

Hydrolase/hydrolase inhibitor

PDB id: 5wxf

Contents

Protein chains

246 a.a.

12 a.a.

Ligands

SO4

Waters ×180

PDB id:

5wxf

Name:

Hydrolase/hydrolase inhibitor

Title:

Crystal structure of upa in complex with upain-2-2

Structure:

Urokinase-type plasminogen activator chain b. Chain: u. Synonym: upa. Engineered: yes. Mutation: yes. Upain-2-2 peptide. Chain: p. Engineered: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: plau. Expressed in: komagataella pastoris. Expression_system_taxid: 4922. Synthetic: yes. Phage display vector ptdisp. Organism_taxid: 279974

Resolution:

1.46Å R-factor: 0.211 R-free: 0.245

Authors:

L.Jiang,M.Huang

Key ref:

L.Jiang et al. (2018). Cleavage of peptidic inhibitors by target protease is caused by peptide conformational transition. Biochim Biophys Acta, 1862, 2017-2023. PubMed id: 29959058 DOI: 10.1016/j.bbagen.2018.06.016

Date:

07-Jan-17 Release date: 11-Jul-18

Protein chain

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

Seq: 431 a.a.

Struc: 246 a.a.*

Protein chain

No UniProt id for this chain

Struc: 12 a.a.

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

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

DOI no: 10.1016/j.bbagen.2018.06.016

Biochim Biophys Acta 1862:2017-2023 (2018)

PubMed id: 29959058

Cleavage of peptidic inhibitors by target protease is caused by peptide conformational transition.

L.Jiang, E.Oldenburg, T.Kromann-Hansen, P.Xu, J.K.Jensen, P.A.Andreasen, M.Huang.

ABSTRACT

Some peptide sequences can behave as either substrates or inhibitors of serine proteases. Working with a cyclic peptidic inhibitor of the serine protease urokinase-type plasminogen activator (uPA), we have now demonstrated a new mechanism for an inhibitor-to-substrate switch. The peptide, CSWRGLENHAAC (upain-2), is a competitive inhibitor of human uPA, but is also slowly converted to a substrate in which the bond between Arg⁴ and Gly⁵ (the P1-P1' bond) is cleaved. Substituting the P2 residue Trp³ to an Ala or substituting the P1 Arg⁴ residue with 4-guanidino-phenylalanine strongly increased the substrate cleavage rate. We studied the structural basis for the inhibitor-to-substrate switch by determining the crystal structures of the various peptide variants in complex with the catalytic domain of uPA. While the slowly cleaved peptides bound clearly in inhibitory mode, with the oxyanion hole blocked by the side chain of the P3' residue Glu⁷, peptides behaving essentially as substrates with a much accelerated rate of cleavage was observed to be bound to the enzyme in substrate mode. Our analysis reveals that the inhibitor-to-substrate switch was associated with a 7 Å translocation of the P2 residue, and we conclude that the inhibitor-to-substrate switch of upain-2 is a result of a major conformational change in the enzyme-bound state of the peptide. This conclusion is in contrast to findings with so-called standard mechanism inhibitors in which the inhibitor-to-substrate switch is linked to minor conformational changes in the backbone of the inhibitory peptide stretch.