PDBsum entry 3ela

Hydrolase/hydrolase inhibitor

PDB id: 3ela

Contents

Protein chains

95 a.a. *

254 a.a. *

191 a.a. *

Ligands

GLC

FUC

0Z6

Metals

_CA

Waters ×214

* Residue conservation analysis

PDB id:

3ela

Name:

Hydrolase/hydrolase inhibitor

Title:

Crystal structure of active site inhibited coagulation factor viia mutant in complex with soluble tissue factor

Structure:

Coagulation factor viia light chain. Chain: l. Fragment: unp residues 61-212. Engineered: yes. Coagulation factor viia heavy chain. Chain: h. Fragment: unp residues 213-466. Engineered: yes. Mutation: yes.

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: f7. Expressed in: cricetulus griseus. Expression_system_taxid: 10029. Expression_system_cell: cho-k1. Gene: f3. Expressed in: escherichia coli.

Resolution:

2.20Å R-factor: 0.236 R-free: 0.294

Authors:

J.R.Bjelke,M.Fodje,L.A.Svensson

Key ref:

J.R.Bjelke et al. (2008). Mechanism of the Ca2+-induced enhancement of the intrinsic factor VIIa activity. J Biol Chem, 283, 25863-25870. PubMed id: 18640965 DOI: 10.1074/jbc.M800841200

Date:

21-Sep-08 Release date: 04-Nov-08

Protein chain

P08709  (FA7_HUMAN) -  Coagulation factor VII from Homo sapiens

Seq: 466 a.a.

Struc: 95 a.a.

Protein chain

P08709  (FA7_HUMAN) -  Coagulation factor VII from Homo sapiens

Seq: 466 a.a.

Struc: 254 a.a.*

Protein chain

P13726  (TF_HUMAN) -  Tissue factor from Homo sapiens

Seq: 295 a.a.

Struc: 191 a.a.

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

Enzyme reactions

• Enzyme class: Chains L, H: E.C.3.4.21.21 - coagulation factor VIIa.
• Reaction: Hydrolyzes one Arg-|-Ile bond in factor X to form factor Xa.

DOI no: 10.1074/jbc.M800841200

J Biol Chem 283:25863-25870 (2008)

PubMed id: 18640965

Mechanism of the Ca2+-induced enhancement of the intrinsic factor VIIa activity.

J.R.Bjelke, O.H.Olsen, M.Fodje, L.A.Svensson, S.Bang, G.Bolt, B.B.Kragelund, E.Persson.

ABSTRACT

The intrinsic activity of coagulation factor VIIa (FVIIa) is dependent on Ca(2+) binding to a loop (residues 210-220) in the protease domain. Structural analysis revealed that Ca(2+) may enhance the activity by attenuating electrostatic repulsion of Glu(296) and/or by facilitating interactions between the loop and Lys(161) in the N-terminal tail. In support of the first mechanism, the mutations E296V and D212N resulted in similar, about 2-fold, enhancements of the amidolytic activity. Moreover, mutation of the Lys(161)-interactive residue Asp(217) or Asp(219) to Ala reduced the amidolytic activity by 40-50%, whereas the K161A mutation resulted in 80% reduction. Hence one of these Asp residues in the Ca(2+)-binding loop appears to suffice for some residual interaction with Lys(161), whereas the more severe effect upon replacement of Lys(161) is due to abrogation of the interaction with the N-terminal tail. However, Ca(2+) attenuation of the repulsion between Asp(212) and Glu(296) keeps the activity above that of apoFVIIa. Altogether, our data suggest that repulsion involving Asp(212) in the Ca(2+)-binding loop suppresses FVIIa activity and that optimal activity requires a favorable interaction between the Ca(2+)-binding loop and the N-terminal tail. Crystal structures of tissue factor-bound FVIIa(D212N) and FVIIa(V158D/E296V/M298Q) revealed altered hydrogen bond networks, resembling those in factor Xa and thrombin, after introduction of the D212N and E296V mutations plausibly responsible for tethering the N-terminal tail to the activation domain. The charge repulsion between the Ca(2+)-binding loop and the activation domain appeared to be either relieved by charge removal and new hydrogen bonds (D212N) or abolished (E296V). We propose that Ca(2+) stimulates the intrinsic FVIIa activity by a combination of charge neutralization and loop stabilization.

Selected figure(s)

Figure 2.
Carbamylation inhibition assay using KOCN. At the indicated time points, aliquots of wild-type FVIIa (♦), FVIIa[D212N] (▴), and FVIIa[DVQ] (▪) were withdrawn and the residual activity measured. The curves show the result of a representative experiment. Similar results were obtained in two additional experiments in which samples were withdrawn at time points different from the experiment presented (data not shown).

Figure 4.
Highlight of residues in the Ca^2+-binding loop of the protease domain in the structures of FVIIa[D212N] (A and B; 2.7 Å) and wild-type FVIIa (C; 2.0 Å; PDB code 1DAN). The N terminus is indicated with an arrow and hydrogen bonds shown by dotted lines.2F[o] – F[c] electron density maps are shown at 1σ (blue) and 2σ (red). The mutant structure shows changes of hydrogen bond networks in the Ca^2+-binding loop (Ca^2+ shown as a green sphere), for example, surrounding mutated residue Asn^212 and Ser^214 and Glu^296. A hydrogen bond is abolished between Asn^212 and Ser^214 because of a side chain movement of Ser^214 in the mutant structure. A hydrogen bond network is introduced between Asn^212, Glu^296, and two strongly defined water molecules. In the wild-type structure, Asp^212 and Glu^296 are not in an electrostatically optimal configuration because of charge repulsion and the conformations of the two residues are slightly changed in the mutant structure. A distinct side chain movement of Asp^217 can be observed as well. In turn, a hydrogen bond between Lys^161 and Asp^217 is lost, whereas bonding to Asp^219 is strengthened: Asp^219 to Lys^161 is 2.6 Å in the mutant structure (see B) versus 3.9 Å in the wild-type structure (see C). Root mean square displacements (Cα) of the Ca^2+-binding loop of the mutant structure versus the wild structure were 0.74 Å compared with an overall of 0.65 Å for the heavy chains.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2008, 283, 25863-25870) copyright 2008.

Figures were selected by an automated process.