PDBsum entry 1oyh

Blood clotting

PDB id: 1oyh

Contents

Protein chain

416 a.a. *

Ligands

NAG-NAG ×2

NAG-NAG-BMA-MAN-MAN

NAG ×4

Waters ×54

* Residue conservation analysis

PDB id:

1oyh

Name:

Blood clotting

Title:

Crystal structure of p13 alanine variant of antithrombin

Structure:

Antithrombin-iii. Chain: i. Synonym: atiii. Pro0309. Engineered: yes. Mutation: yes. Other_details: e381a mutant on the beta glycoform background of s137a. Antithrombin-iii. Chain: l.

Source:

Homo sapiens. Human. Organism_taxid: 9606. Organ: blood. Tissue: plasma. Expressed in: cricetulus griseus. Expression_system_taxid: 10029. Expression_system_cell: baby hampster kidney cells. Other_details: plasma alpha antithrombin was converted to latent by

Resolution:

2.62Å R-factor: 0.221 R-free: 0.258

Authors:

D.J.D.Johnson,J.A.Huntington

Key ref:

D.J.Johnson and J.A.Huntington (2004). The influence of hinge region residue Glu-381 on antithrombin allostery and metastability. J Biol Chem, 279, 4913-4921. PubMed id: 14623882 DOI: 10.1074/jbc.M311644200

Date:

04-Apr-03 Release date: 13-Apr-04

Protein chains

P01008  (ANT3_HUMAN) -  Antithrombin-III from Homo sapiens

Seq: 464 a.a.

Struc: 416 a.a.*

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

DOI no: 10.1074/jbc.M311644200

J Biol Chem 279:4913-4921 (2004)

PubMed id: 14623882

The influence of hinge region residue Glu-381 on antithrombin allostery and metastability.

D.J.Johnson, J.A.Huntington.

ABSTRACT

Antithrombin becomes an efficient inhibitor of factor Xa and thrombin by binding a specific pentasaccharide sequence found on a small fraction of the heparan sulfate proteoglycans lining the microvaculature. In the structure of native antithrombin, the reactive center loop is restrained due to the insertion of its hinge region into the main beta-sheet A, whereas in the heparin-activated state the reactive center loop is freed from beta-sheet A. In both structures, hinge region residue Glu-381 makes several stabilizing contacts. To determine the role of these contacts in the allosteric mechanism of antithrombin activation, we replaced Glu-381 with an alanine. This variant is less active toward its target proteases than control antithrombin, due to a perturbation of the equilibrium between the two forms, and to an increase in stoichiometry of inhibition. Pentasaccharide binding affinity is reduced 4-fold due to an increase in the off-rate. These data suggest that the main role of Glu-381 is to stabilize the activated conformation. Stability studies also showed that the E381A variant is resistant to continued insertion of its reactive center loop upon incubation at 50 degrees C, suggesting new stabilizing interactions in the native structure. To test this hypothesis, and to aid in the interpretation of the kinetic data we solved to 2.6 A the structure of the variant. We conclude that wild-type Glu-381 interactions stabilize the activated state and decreases the energy barrier to full loop insertion.

Selected figure(s)

Figure 1.
FIG. 1. The induced-fit heparin binding mechanism of antithrombin. a, antithrombin (ribbon diagram) interacts with heparin (ball-and-stick) in an apparent three step, four state mechanism. The native state is in the low activity and low affinity conformation with its reactive center loop (yellow and green) partially inserted into the main -sheet A (red). The specific heparin pentasaccharide interacts with antithrombin primarily via helices A (green) and D (cyan). Although kinetic data fit a model with only one weak-binding intermediate, I[1], a recent crystallographic structure suggests that there may be an additional intermediate that has undergone all conformational changes save expulsion of the reactive center loop and helix D elongation, I[2]. Full activation occurs only after expulsion of the hinge region of the reactive center loop, and the closing of -sheet A to the five stranded form (Activated). b-e, stereo representations of the main chain and selected side chains of hinge region and surrounding residues reveal important interactions in several crystal forms of antithrombin. b-e correspond to native ( -glycoform, 1e05 [PDB] ), pentasaccharide-activated (1e03 [PDB] ), S380C-fluorescein-derivatized (1dzh [PDB] ), and the heparin-bound intermediate I2 (1nq9 [PDB] ), respectively. In native AT (b) the P13 Glu (381) bridges strands 3, 4, and 5A to helix F residues (Arg-197 and Glu-195) through a series of salt-bridges and hydrogen bonds. In the activated conformation (c) strands 3 and 5A have annealed with P13 Glu interacting with strands 2 and 3A and helix F. A fluorescein moiety at the P14 position (d) blocked the native P13 interactions leading to an activated conformation in solution. One of the major conformational responses to heparin binding in the intermediate I[2] (e) is the repositioning of Tyr-220 so that it no longer interacts with P13 Glu. Residue numbers are given for some of the residues for clarity.

Figure 5.
FIG. 5. Stereo representations of C traces of AT colored according to root mean squared deviation from native AT. a, the C trace of the P13 Ala (ball) variant reveals surprisingly profound main chain differences when compared with the -glycoform of AT. Although perturbation of the hinge region might be expected, the effect seems to have propagated to the heparin binding region. To determine which conformational changes are significant, and which reflect the flexible nature of the molecule, the structure of the -glycoform compared with the -form is shown in b. The color scheme reflects the RMSD from light gray to red for 0-3 Å. The distance between Lys-220 and -139 C is shown as a dashed green line, and is increases from 5.1 to 7.8 Å in response to the E381A mutation.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2004, 279, 4913-4921) copyright 2004.

Figures were selected by the author.