PDBsum entry 4wwy

Hydrolase/hydrolase inhibitor

PDB id: 4wwy

Contents

Protein chains

224 a.a.

58 a.a.

Ligands

SO4 ×8

Metals

_CA

Waters ×399

PDB id:

4wwy

Name:

Hydrolase/hydrolase inhibitor

Title:

Human cationic trypsin g193r mutant in complex with bovine pancreatic trypsin inhibitor

Structure:

Trypsin-1. Chain: a, b. Fragment: unp residues 24-247. Synonym: beta-trypsin,cationic trypsinogen,serine protease 1,trypsin i. Engineered: yes. Mutation: yes. Pancreatic trypsin inhibitor. Chain: c, i.

Source:

Homo sapiens. Human. Organism_taxid: 9606. Organ: pancreas. Gene: prss1, trp1, try1, tryp1. Expressed in: escherichia coli. Expression_system_taxid: 469008. Bos taurus. Bovine.

Resolution:

1.70Å R-factor: 0.173 R-free: 0.207

Authors:

A.Alloy,O.Kayode,A.S.Soares,R.Wang,E.S.Radisky

Key ref:

A.P.Alloy et al. (2015). Mesotrypsin Has Evolved Four Unique Residues to Cleave Trypsin Inhibitors as Substrates. J Biol Chem, 290, 21523-21535. PubMed id: 26175157 DOI: 10.1074/jbc.M115.662429

Date:

12-Nov-14 Release date: 22-Jul-15

Protein chains

P07477  (TRY1_HUMAN) -  Serine protease 1 from Homo sapiens

Seq: 247 a.a.

Struc: 224 a.a.*

Protein chains

P00974  (BPT1_BOVIN) -  Pancreatic trypsin inhibitor from Bos taurus

Seq: 100 a.a.

Struc: 58 a.a.

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

Enzyme reactions

• Enzyme class: Chains A, B: E.C.3.4.21.4 - trypsin.
• Reaction: Preferential cleavage: Arg-|-Xaa, Lys-|-Xaa.

DOI no: 10.1074/jbc.M115.662429

J Biol Chem 290:21523-21535 (2015)

PubMed id: 26175157

Mesotrypsin Has Evolved Four Unique Residues to Cleave Trypsin Inhibitors as Substrates.

A.P.Alloy, O.Kayode, R.Wang, A.Hockla, A.S.Soares, E.S.Radisky.

ABSTRACT

Human mesotrypsin is highly homologous to other mammalian trypsins, and yet it is functionally unique in possessing resistance to inhibition by canonical serine protease inhibitors and in cleaving these inhibitors as preferred substrates. Arg-193 and Ser-39 have been identified as contributors to the inhibitor resistance and cleavage capability of mesotrypsin, but it is not known whether these residues fully account for the unusual properties of mesotrypsin. Here, we use human cationic trypsin as a template for engineering a gain of catalytic function, assessing mutants containing mesotrypsin-like mutations for resistance to inhibition by bovine pancreatic trypsin inhibitor (BPTI) and amyloid precursor protein Kunitz protease inhibitor (APPI), and for the ability to hydrolyze these inhibitors as substrates. We find that Arg-193 and Ser-39 are sufficient to confer mesotrypsin-like resistance to inhibition; however, compared with mesotrypsin, the trypsin-Y39S/G193R double mutant remains 10-fold slower at hydrolyzing BPTI and 2.5-fold slower at hydrolyzing APPI. We identify two additional residues in mesotrypsin, Lys-74 and Asp-97, which in concert with Arg-193 and Ser-39 confer the full catalytic capability of mesotrypsin for proteolysis of BPTI and APPI. Novel crystal structures of trypsin mutants in complex with BPTI suggest that these four residues function cooperatively to favor conformational dynamics that assist in dissociation of cleaved inhibitors. Our results reveal that efficient inhibitor cleavage is a complex capability to which at least four spatially separated residues of mesotrypsin contribute. These findings suggest that inhibitor cleavage represents a functional adaptation of mesotrypsin that may have evolved in response to positive selection pressure.