PDBsum entry 2ra3

Hydrolase/hydrolase inhibitor

PDB id: 2ra3

Contents

Protein chains

224 a.a. *

58 a.a. *

Ligands

SO4 ×24

Metals

_CA ×2

Waters ×665

* Residue conservation analysis

PDB id:

2ra3

Name:

Hydrolase/hydrolase inhibitor

Title:

Human cationic trypsin complexed with bovine pancreatic trypsin inhibitor (bpti)

Structure:

Trypsin-1. Chain: a, b. Synonym: trypsin i, cationic trypsinogen, serine protease 1. Engineered: yes. Mutation: yes. Pancreatic trypsin inhibitor. Chain: i, c. Synonym: basic protease inhibitor, bpi, bpti, aprotinin

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: prss1, trp1, try1, tryp1. Expressed in: escherichia coli. Expression_system_taxid: 562. Bos taurus. Cattle. Organism_taxid: 9913.

Resolution:

1.46Å R-factor: 0.154 R-free: 0.178

Authors:

M.A.Salameh,A.S.Soares,E.S.Radisky

Key ref:

M.A.Salameh et al. (2007). Structural basis for accelerated cleavage of bovine pancreatic trypsin inhibitor (BPTI) by human mesotrypsin. J Biol Chem, 283, 4115-4123. PubMed id: 18077447 DOI: 10.1074/jbc.M708268200

Date:

14-Sep-07 Release date: 11-Dec-07

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.M708268200

J Biol Chem 283:4115-4123 (2007)

PubMed id: 18077447

Structural basis for accelerated cleavage of bovine pancreatic trypsin inhibitor (BPTI) by human mesotrypsin.

M.A.Salameh, A.S.Soares, A.Hockla, E.S.Radisky.

ABSTRACT

Human mesotrypsin is an isoform of trypsin that displays unusual resistance to polypeptide trypsin inhibitors, and has been observed to cleave several such inhibitors as substrates. While substitution of arginine for the highly conserved glycine-193 in the trypsin active site has been implicated as a critical factor in the inhibitor resistance of mesotrypsin, how this substitution leads to accelerated inhibitor cleavage is not clear. Bovine pancreatic trypsin inhibitor (BPTI) forms an extremely stable and cleavage-resistant complex with trypsin, and thus provides a rigorous challenge of mesotrypsin catalytic activity toward polypeptide inhibitors. Here, we report kinetic constants for mesotrypsin and the highly homologous (but inhibitor sensitive) human cationic trypsin, describing inhibition by and cleavage of BPTI, as well as crystal structures of the mesotrypsin-BPTI and human cationic trypsin-BPTI complexes. We find that mesotrypsin cleaves BPTI with a rate constant accelerated 350-fold over that of human cationic trypsin and 150,000-fold over that of bovine trypsin. From the crystal structures, we see that small conformational adjustments limited to several side chains enable mesotrypsin-BPTI complex formation, surmounting the predicted steric clash introduced by Arg-193. Our results show that the mesotrypsin-BPTI interface favors catalysis through (a) electrostatic repulsion between the closely spaced mesotrypsin Arg-193 and BPTI Arg-17, and (b) elimination of two hydrogen bonds between the enzyme and the amine leaving group portion of BPTI. Our model predicts that these deleterious interactions accelerate leaving group dissociation and deacylation.

Selected figure(s)

Figure 3.
FIGURE 3. Mesotrypsin conformational changes upon BPTI binding. A, superposition of the mesotrypsin-BPTI complex (mesotrypsin in orange and BPTI in blue/cyan) with the mesotrypsin-benzamidine complex (mesotrypsin in beige and benzamidine in red) highlights several differences induced by BPTI binding. Significant movements in the positions of side chains, illustrated by the black arrows, include the upward displacement of Arg-193 by 6 Å displacing a water molecule, adoption of an alternate rotamer by His-40, and displacement of Asp-153 by 1 Å to enable formation of a water-bridged ionic interaction with Arg-193. B, environment surrounding the BPTI-bound position of mesotrypsin Arg-193 is shown with 2F[o]-F[c] map contoured at 1.5 sigma. Arg-193 forms direct H-bonds to the carbonyl oxygens of Trp-141 and Pro-152, and water bridged interactions with the side chains of Asp-153 and BPTI Arg-17.

Figure 4.
FIGURE 4. Differences between the mesotrypsin-BPTI and human cationic trypsin-BPTI interfaces. In this superposition of the two complexes, mesotrypsin and BPTI are colored orange and blue/cyan as in the previous figure, while cationic trypsin and its bound BPTI are colored dark and light gray, respectively. Three H-bonds present only in the cationic trypsin complex (see text) are shown as dotted lines.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2007, 283, 4115-4123) copyright 2007.

Figures were selected by the author.