PDBsum entry 2beh

Blood clotting

PDB id: 2beh

Contents

Protein chain

413 a.a.

Ligands

NAG-NAG ×2

NAG-NAG-MAN

NAG ×2

GOL

Waters ×17

References listed in PDB file

Key reference

Title

Crystal structure of monomeric native antithrombin reveals a novel reactive center loop conformation.

Authors

D.J.Johnson, J.Langdown, W.Li, S.A.Luis, T.P.Baglin, J.A.Huntington.

Ref.

J Biol Chem, 2006, 281, 35478-35486. [DOI no: 10.1074/jbc.M607204200]

PubMed id

16973611

Abstract

The poor inhibitory activity of circulating antithrombin (AT) is critical to the formation of blood clots at sites of vascular damage. AT becomes an efficient inhibitor of the coagulation proteases only after binding to a specific heparin pentasaccharide, which alters the conformation of the reactive center loop (RCL). The molecular basis of this activation event lies at the heart of the regulation of hemostasis and accounts for the anticoagulant properties of the low molecular weight heparins. Although several structures of AT have been solved, the conformation of the RCL in native AT remains unknown because of the obligate crystal contact between the RCL of native AT and its latent counterpart. Here we report the crystallographic structure of a variant of AT in its monomeric native state. The RCL shifted approximately 20 A, and a salt bridge was observed between the P1 residue (Arg-393) and Glu-237. This contact explains the effect of mutations at the P1 position on the affinity of AT for heparin and also the properties of AT-Truro (E237K). The relevance of the observed conformation was verified through mutagenesis studies and by solving structures of the same variant in different crystal forms. We conclude that the poor inhibitory activity of the circulating form of AT is partially conferred by intramolecular contacts that restrain the RCL, orient the P1 residue away from attacking proteases, and additionally block the exosite utilized in protease recognition.

Figure 3.
FIGURE 3. Stereo views of the structure of monomeric AT reveals a novel RCL conformation and contacts. A, superimposed C traces of native AT from crystals of the heterodimer (gray) and of monomeric AT (oriented as in Fig. 1A) reveal regions of conformational difference. Monomeric AT is colored according to C root mean squared deviation (yellow to red, from 1 to 6 Å) with the active component of 1E04 ( -glycoform). Disulfide bonds are shown as green rods, and the P1 residue is indicated by a blue ball. Regions that differ most significantly are the RCL (top) and the heparin binding site (lower right). B, the RCL of monomeric AT (from P5 to P3') is shown with corresponding electron density (contoured at 1 ). C, extensive intramolecular contacts are observed between the RCL and the body of AT; those involving the P1 Arg-393 are indicated by dashed lines.

Figure 5.
FIGURE 5. Equilibrium model for the conformational states of AT. Similar to the revised model proposed by Chuang et al. (9), our data support a two-state conformational equilibrium for AT in the absence of heparin. Ribbon diagrams are given to represent the one activated and two native states. State N is the monomeric AT structure presented here with its RCL held close against the body of AT and the P1 side chain sequestered in the acidic pocked used as an exosite for factor Xa binding. This state would be nonreactive toward proteases but is in rapid equilibrium with state N' (based on the structure of heterodimeric native AT), where there are fewer contacts to constrain the RCL and the P1 residue is free to interact with proteases. Activation of AT by the pentasaccharide results ultimately in the expulsion of the hinge and the liberation of the entire RCL. Monomer A is based on the structure of AT in complex with the pentasaccharide and S195A factor Xa (23).

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2006, 281, 35478-35486) copyright 2006.