PDBsum entry 2f9o

Hydrolase

PDB id: 2f9o

Contents

Protein chain

242 a.a. *

Ligands

NAG-FUC-NAG ×2

Waters ×747

* Residue conservation analysis

PDB id:

2f9o

Name:

Hydrolase

Title:

Crystal structure of the recombinant human alpha i tryptase mutant d216g

Structure:

Tryptase alpha-1. Chain: a, b, c, d. Fragment: residues 31-275. Synonym: tryptase-1. Engineered: yes. Mutation: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: tpsab1, tps1. Expressed in: spodoptera frugiperda. Expression_system_taxid: 7108.

Biol. unit:

Dimer (from PQS)

Resolution:

2.10Å R-factor: 0.216 R-free: 0.245

Authors:

K.B.Rohr,T.Selwood,U.Marquardt,R.Huber,N.M.Schechter,W.Bode,M.E.Than

Key ref:

K.B.Rohr et al. (2006). X-ray structures of free and leupeptin-complexed human alphaI-tryptase mutants: indication for an alpha-->beta-tryptase transition. J Mol Biol, 357, 195-209. PubMed id: 16414069 DOI: 10.1016/j.jmb.2005.12.037

Date:

06-Dec-05 Release date: 31-Jan-06

Protein chains

Q15661  (TRYB1_HUMAN) -  Tryptase alpha/beta-1 from Homo sapiens

Seq: 275 a.a.

Struc: 242 a.a.*

* PDB and UniProt seqs differ at 15 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.4.21.59 - tryptase.
• Reaction: Preferential cleavage: Arg-|-, Lys-|-, but with more restricted specificity than trypsin.

DOI no: 10.1016/j.jmb.2005.12.037

J Mol Biol 357:195-209 (2006)

PubMed id: 16414069

X-ray structures of free and leupeptin-complexed human alphaI-tryptase mutants: indication for an alpha-->beta-tryptase transition.

K.B.Rohr, T.Selwood, U.Marquardt, R.Huber, N.M.Schechter, W.Bode, M.E.Than.

ABSTRACT

Tryptases alpha and beta are trypsin-like serine proteinases expressed in large amounts by mast cells. Beta-tryptase is a tetramer that has enzymatic activity, but requires heparin binding to maintain functional and structural stability, whereas alpha-tryptase has little, if any, enzymatic activity but is a stable tetramer in the absence of heparin. As shown previously, these differences can be mainly attributed to the different conformations of the 214-220 segment. Interestingly, the replacement of Asp216 by Gly, which is present in beta-tryptase, results in enzymatically active but less stable alpha-tryptase mutants. We have solved the crystal structures of both the single (D216G) and the double (K192Q/D216G) mutant forms of recombinant human alphaI-tryptase in complex with the peptide inhibitor leupeptin, as well as the structure of the non-inhibited single mutant. The inhibited mutants exhibited an open functional substrate binding site, while in the absence of an inhibitor, the open (beta-tryptase-like) and the closed (alpha-tryptase-like) conformations were present simultaneously. This shows that both forms are in a two-state equilibrium, which is influenced by the residues in the vicinity of the active site and by inhibitor/substrate binding. Novel insights regarding the observed stability differences as well as a potential proteolytic activity of wild-type alpha-tryptase, which may possess a cryptic active site, are discussed.

Selected figure(s)

Figure 2.
Figure 2. Stereo ribbon representation of the rHTaQGI tetramer surrounded by a semitransparent surface. The four monomers A to D are represented in gray, orange, yellow and blue. Sugar chains, visible only in monomers B and D, and the four leupeptin molecules binding to the active sites are represented as ball-and-stick models with nitrogen blue, oxygen red and carbonyl atoms black.

Figure 7.
Figure 7. The uncomplexed tryptase single mutant rHTaG. (a) Stick representation of the active site of rHTaG, showing the open conformation ( vert, similar 75% occupancy, blue) and the closed conformation ( vert, similar 25% occupancy, light blue) as thick sticks. Additionally, the 214-220 segments of aI-tryptase (orange) and bII-tryptase (green) are superimposed as thin stick models to show the high level of similarity of both conformations with the two conformations seen in free active rHTaG. (b) B-factors of the inhibited single mutant rHTaGI (continuous line), of the inhibited double mutant rHTaQGI (dotted line) and of the uncomplexed single mutant rHTaG (broken line) are plotted against residue number (chymotrypsinogen numbering). A striking increase of the B-factors can be seen for the 214-220 segment of rHTaG. (c) Electron density of an averaged kicked omit map (orange mesh) shown together with the open (blue) and the closed (light blue) conformation of the 214-220 segment. The map was calculated for residues 214-220 in the closed conformation and contoured at 1.8s. The active site residues are shown in green.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2006, 357, 195-209) copyright 2006.

Figures were selected by the author.