Analysis of the factor XI variant Arg184Gly suggests a structural basis for factor IX binding to factor XIa

Abstract

Background: A patient with factor XI (FXI) deficiency was reported with an Arg184Gly substitution in the FXI A3 domain. The A3 domain contains an exosite required for binding of FIX to activated FXI (FXIa).

Objective: To test the effects of the Arg184Gly substitution on FIX activation, and to characterize the FIX-binding site on FXIa.

Methods: Recombinant FXIa and FIX variants were used to identify residues involved in FIX activation by FXIa. Analysis of the FXI structure was used to identify potential FIX-binding sites.

Results: The Km for FIX activation by FXIa-Gly184 was approximately three-fold higher than for FXIa, suggesting that Arg184 is part of the exosite. Arg184 and the adjacent residues, Ile183 and Asp185, contribute to charged and hydrophobic areas that are not present in the FXI homolog prekallikrein (PK). Replacing residues 183-185 with alanine abolished exosite activity, similarly to replacement of the entire A3 domain with the A3 domain from PK (FXIa/PKA3). Reintroducing FXI residues 183-185 into FXIa/PKA3 partially restored the exosite, and replacing residues 183-185 and 260-264 completely restored exosite function. FIX in which the Ω-loop (residues 4-11) was replaced with the FVII Ω-loop was activated poorly by FXIa, suggesting that the FIX Ω-loop binds to FXIa.

Conclusions: The results support a model in which the Ω-loop of FIX binds to an area on FXIa composed of residues from the N-terminus and C-terminus of the A3 domain. These residues are buried in zymogen FXI, and must be exposed upon conversion to FXIa to permit FIX binding.

Keywords: blood coagulation; factor IX; factor XI; factor XIa; proteolysis.

Figure 1. FXI and fXIa

Left panel - Non-reducing SDS-PAGE stained with GelCode blue of recombinant fXIWT (WT) and fXI-Gly184. Right panel – Reducing SDS–PAGE of the zymogen (XI) and protease (XIa) forms of the proteins in the left hand panel. Positions of fXI zymogen (XI) and the heavy chain (XIa-HC) and catalytic domain (XIa-CD) are shown to the right of the panel. Positions of molecular mass standards for both gels are indicated on the left in kilodaltons.

Figure 2. S-2366 cleavage

(A & B) Shown are the rates of cleavage of the tripeptide substrate S-2366 by 6 nM (A) fXIaWT or (B) fXI-Gly184. (C & D) Cleavage of S-2366 (500 nM) by 6 nM (C) fXIa fXIWT or (D) fXI-Gly184 in the presence of varying concentrations of fIXaβ. For all panels, data points represent averages of duplicate runs.

Figure 3. FIX cleavage by fXIa

(A) Non-reducing 17% polyacrylamide-SDS gels of 100 nM fIX in Assay Buffer with Ca2+ incubated at RT with 3 nM active sites of fXIaWT (top), fXIa/PKA3 (middle) or, fXIa-Gly184 (bottom). Positions of standards for fIX, fIXα and fIXaβ are indicated at the right of each panel. (B & C) Progress curves of fIX disappearance (●), and fIXα (○) and fIXaβ (Δ) generation for (B) fXIaWT or (C) fXI-Gly184 from panel A. Lines represent least-squares fits to the data. (D) Initial rates of fIX disappearance for fXIaWT (●), fXIa-Gly184 (○) and fXIa/PKA3 (Δ) determined over the first 10 min of activation from panel A. (E) Initial rates of fIXaβ formation for fXIaWT (●), fXIa-Gly184 (○) and fXIa/PKA3 (Δ) determined over the first 10 min of activation from panel A.

Figure 4. The FXI A3 Domain and fIX Gla-domain

(A) Topological diagram showing the A3 domain of human fXI with the β-sheet indicated in yellow and α-helix in grey. Side chains of residues in the locality of Arg184 are shown as sticks. (B) Surface representations of the fXI A3 domain are shown with partial transparency to highlight side chains of specific residues that are colored by atom type. Two rotations are shown related by 180 degrees. The software program Metapocket identified three pockets on the surface of A3 (shown as red balls) (C) Topological diagram of the human PK A3 domain model with side chains of non-conserved residues in the area of the pocket in fXI shown as sticks. Note that the side chain of Arg209 in PK occupies the site of the hydrophobic pocket in fXI. (D) Charged surface representations of fXI (left) and PK (right) A3 domains. Blue indicates positive charge and red negative charge. Note the absence of pocket 1 in the PK A3 domain model due to the Arg²⁰⁹ side chain. (E) Structure of the zymogen fXI monomer showing the A3 domain as a charged surface representation, and the A4 and catalytic domains as ribbon drawings. Note that Arg184 and the adjacent hydrophobic pocket 1 are covered by the catalytic domain. The inset shows specific interactions between side chains of the protease domain and A3. (F) Topological diagram of the human fIX Gla-domain with residues in the Ω-loop (4 to 11) that differ from the corresponding region of the human fVII Gla-domain highlighted in magenta. Positions of certain γ-carboxyglutamic acid residues are indicated by the symbol “γ”. Calcium ions in the vicinity of the Ω-loop are represented by green spheres. The image is derived from a structure for a complex between the human fIX Gla-domain and the antibody 10C12.²⁹ Figures prepared with Pymol (The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC. http://www.pymol.org/citing).

Figure 4. The FXI A3 Domain and fIX Gla-domain

(A) Topological diagram showing the A3 domain of human fXI with the β-sheet indicated in yellow and α-helix in grey. Side chains of residues in the locality of Arg184 are shown as sticks. (B) Surface representations of the fXI A3 domain are shown with partial transparency to highlight side chains of specific residues that are colored by atom type. Two rotations are shown related by 180 degrees. The software program Metapocket identified three pockets on the surface of A3 (shown as red balls) (C) Topological diagram of the human PK A3 domain model with side chains of non-conserved residues in the area of the pocket in fXI shown as sticks. Note that the side chain of Arg209 in PK occupies the site of the hydrophobic pocket in fXI. (D) Charged surface representations of fXI (left) and PK (right) A3 domains. Blue indicates positive charge and red negative charge. Note the absence of pocket 1 in the PK A3 domain model due to the Arg²⁰⁹ side chain. (E) Structure of the zymogen fXI monomer showing the A3 domain as a charged surface representation, and the A4 and catalytic domains as ribbon drawings. Note that Arg184 and the adjacent hydrophobic pocket 1 are covered by the catalytic domain. The inset shows specific interactions between side chains of the protease domain and A3. (F) Topological diagram of the human fIX Gla-domain with residues in the Ω-loop (4 to 11) that differ from the corresponding region of the human fVII Gla-domain highlighted in magenta. Positions of certain γ-carboxyglutamic acid residues are indicated by the symbol “γ”. Calcium ions in the vicinity of the Ω-loop are represented by green spheres. The image is derived from a structure for a complex between the human fIX Gla-domain and the antibody 10C12.²⁹ Figures prepared with Pymol (The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC. http://www.pymol.org/citing).

Figure 5. Progress curves of fIX activation by fXIa183-185 and fXIa/PKA3 gain-of-function variants

Shown are progress curves of fIX disappearance (●), and fIXα (○) and fIXaβ (Δ) generation for reactions containing 100 nM fIX and (A) 30 nM fXIa183-185, (B) 3 nM fXIa/PKA3-A, and (C) 3 nM fXIa/PKA3-B. Note the different fXIa concentrations used in the three reactions. FXIa concentrations represent the concentration of active subunits. (D) Initial velocities of cleavage of fIX after Arg145 (conversion of fIX to fIXα) by fXIa183-185 (Δ), fXIa/PKA3-A (●), and fXIa/PKA3-B (○) as a function of fIX concentration.

Figure 6. Factor IX with fVII sequence in the Gla-domain

(A) Primary sequences of the human factor IX (fIX) and factor VII (fVII) Gla-domains. The numbering system shown is for fIX. The symbol γ indicates the position of γ-carboxyglutamic acid residues. Underlined sequences were changed from fIX sequence to fVII sequence to generate chimeras C1, C2, C3 and C4. The amino acids changed in each chimera are highlighted by the gray boxes. (B) Stained SDS-polyacrylamide gel of purified plasma fIX (pIX), recombinant wild type fIX (WT), fIX/fVII chimeras (C1, C2, C3, and C4) and fIX in which the Gla-domain from residues 1 through 46 are changed to fVII sequence (fIX/VII-Gla - abbreviated VIIGla). The weaker band running underneath fIX C3 is contaminating bovine serum albumin from conditioned media. (C and D) FXIa (2 nM) was incubated with 250 nM fIXWT (○), C1 (●), C2 (□), C3 (■), C4 (Δ) and fIX/VII-Gla (▲) (250 nM) in TBS with calcium, as described under methods. Shown are concentrations of (C) fIXaβ and (D) fIXα at various times as determined by densitometry of SDS-polyacrylamide gels.