Structural architecture of the acidic region of the B domain of coagulation factor V

Summary

Background:

Coagulation factor V (fV) features an A1-A2-B-A3-C1-C2 domain organization and functions as the inactive precursor of fVa, a component of the prothrombinase complex required for rapid thrombin generation in the penultimate step of the coagulation cascade. An intramolecular interaction within the large B domain (residues 710–1545) involves the basic region (BR, residues 963–1008) and acidic region (AR, residues 1493–1537) and locks fV in its inactive state. However, structural information on this important regulatory interaction, or on the separate architecture of the AR and BR, remain elusive due to conformational disorder of the B domain.

Objective:

To reveal the structure of the BR-AR interaction or of its separate components.

Methods:

The structure of fV is solved by cryogenic electron microscopy (cryo-EM).

Results:

A new 3.05 Å resolution cryo-EM structure of fV confirms the overall organization of the A and C domains but resolves the segment 1507–1545 within a largely disordered B domain. The segment contains most of the AR and is organized as recently reported in fV short, a spliced variant of fV with a significantly shorter and less disordered B domain.

Conclusions:

The similar architecture of the AR in fV and fV short provides structural context for physiologically important interactions of this region with the BR in fV and with the basic C-terminal end of tissue factor pathway inhibitor α fV short.

Introduction

Coagulation factor V (fV) is a large (2196 residues, MW 330 kDa) protein with an A1-A2-B-A3-C1-C2 domain organization. The protein has a procoagulant role as the inactive precursor of fVa that, together with the protease fXa, assembles in the prothrombinase complex and enables rapid conversion of prothrombin to thrombin in the penultimate step of the coagulation cascade [1]. In addition, fV features an anticoagulant role as a component of the tissue factor pathway inhibitor α (TFPIα) and protein C pathways that down-regulate the coagulation response [2]. The structure of fV [Protein Data Bank (PDB) 7KVE] was solved recently by cryogenic electron microscopy (cryo-EM) [3] and contains well organized A and C domains but a large disordered B domain (residues 710–1545). The disorder precludes assignment of the basic region (BR, residues 963–1008) and acidic region (AR, residues 1493–1537) that are assumed to interact intramolecularly to lock fV in its inactive state [4, 5]. Hence, a potentially important regulatory mechanism of the function of fV lacks structural validation and the molecular organization of the AR and BR remains unknown.

The spliced variant fV short is associated with the East Texas bleeding disorder [6, 7] and lacks a large portion of the B domain, including the BR [8]. This structural perturbation produces constitutive fVa-like activity and unmasks a high affinity binding site in the AR for tissue factor pathway inhibitor α (TFPIα) [2], a key regulator of blood coagulation [9, 10]. Importantly, the BR of fV and the C-terminal basic segment of TFPIα share significant sequence homology [11], which underscores their similar propensity to interact with the AR [2, 12, 13] and their similar functional interference with the site of thrombin activation at R1545 located at the C-terminal end of the B domain [14]. A recent cryo-EM structure of fV short (PDB 8FDG) [15] has revealed details of the shorter B domain with the AR organized in acidic and hydrophobic clusters. The architecture of the AR unraveled for fV short supports possible interaction with TFPIα and in a way that would interfere with thrombin cleavage at R1545, which is consistent with the functional properties of this variant [8, 11, 16]. Therefore, it is important to establish if this structural organization of the AR is the result of the splicing reaction of fV short or extends to fV, where it can provide a locale for intramolecular interaction with the BR.

Methods

In the original report of the cryo-EM structure of fV [3], human plasma fV (Sigma-Aldrich, St. Louis, MO) was buffer exchanged at pH 7.4 into 20 mM HEPES, 150 mM NaCl, 5 mM CaCl₂ by size exclusion chromatography using a Superdex 200 increase 10/300 GL column and tested for purity by SDS-PAGE (see Figure 1 of Supplemental Information of [3]). Two sets of grids containing fV at 0.1 mg/mL and 0.2 mg/mL, under the same experimental conditions, were frozen for data collection that was carried out over an interval of almost one year and generated a total of 3,982 (0.1 mg/mL sample) and 4,447 (0.2 mg/mL sample) micrographs. The grids were plunge frozen on Quantifoil R 2/2, Cu 300 mesh grids (EMS, Hatfield, PA) which had been plasma cleaned in an H₂O₂ plasma for 1 min using a Solarus 950 (Gatan, Warrendale, PA). Screening and data collection was performed at a pixel size of 1.1 Å, using a dose of 66 e⁻/Ų across 40 frames with a set defocus range of −1 to −2.5 μm on Titan Krios G3 cryo-TEM operating at 300 kV, as described [3]. The original structure of fV (PDB ID 7KVE, EMD-23048) was solved from analysis of a curated set of 2,817 micrographs obtained from the 0.2 mg/mL grids [3]. Recent algorithms for single particle averaging and introduction of non-uniform refinement [17] enabled a comprehensive analysis of the entire data set of 8,429 micrographs from the two grids. Furthermore, this analysis was deemed important after solving the structure of fV short [15] and yielded a new 3.05 Å structure of fV containing more details of the AR. Single particle analysis was performed in cryoSPARC v4.2.1 [18], as described [3]. Masked local refinement was carried out for each fV domain and a composite map was built and sharpened in Phenix [19]. Initial models using 7KVE and 8FDG were docked and fit as rigid bodies using USCF Chimera [20]. ModelAngelo [21] was used to produce an automatic atomic model for fV to guide model building in the B domain. All atoms refinement with restraint and model building were completed in COOT [22] using the composite map. The final model was real space refined against the composite map with AlphaFold AF-P12259 and 8FDG as reference models for secondary structure restraint using Phenix. Relevant parameters of the cryo-EM structure are summarized in Table 1. The structure and maps were deposited in the PDB with accession code 8TN9 and in the Electron Microscopy Data Bank with accession codes EMDB-41411 (composite map), EMDB-41400 (consensus map), EMDB-41401 (A1 domain focused map), EMDB-41402 (A2 domain focused map), EMDB-41403 (A3 domain focused map), EMDB-41407(C1 domain focused map) and EMDB-41408 (C2 domain focused map). The entire set of micrographs from the 0.1 mg/mL and 0.2 mg/mL grids was deposited into the EMPIAR with accession code 11761.

Figure 1ABCDEF. Cryo-EM structure of fV.

(A,B) Surface representation fV (PDB 8TN9) showing the front (A) and side (B) of fV with its constitutive domains: A1 (wheat), A2 (pale green), B (light blue), A3 (pale yellow), C1 (light pink) and C2 (grey). The B domain is largely disordered, except for the segment 1507–1545 (cyan) that contains most of the AR (residues 1493–1537). In the A2 domain, residues 660–678 (dark green) could be assigned but residues 679–709 appeared as discontinuous blobs of density that could not be modeled unequivocally. Shown in red are the site of thrombin activation at R1545 and the sites of APC cleavage at R306 and R506. (C) Composite map with fV cartoon documents the quality of the final model. (D) Representative micrograph. (E) Representative model free 2D classification. (F) Gold standard Fourier shell correlation (GSFSC) for the consensus map EMDB-41400.

Table 1.

Structural parameters for the cryo-EM structure of fV

	PDB ID 8TN9 EMD-41411
Data collection and processing
Nominal Magnification	105,000x
Voltage (kV)	300
Electron exposure (e-/Å2)	66
Defocus range (μm)	−1 to −2.5
Pixel size (Å)	1.1
Number of images	8,429
Final particle images	680,564
Symmetry imposed	C1
Resolution (unmasked, FSC threshold 0.143)	3.05
Model Composition
Refinement program	Phenix (real space)
Number of protein atoms (non-H)	11144
Protein residues	1368
Ligand molecules	0
R.m.s.d. deviations
Bond lengths (Å)	0.003
Bond angles (°)	0.656
Validation
All-atom clash score	10
Poor Rotamers (%)	0
Ramachandran disallowed (%)	0.0
Ramachandran allowed (%)	10
Ramachandran favored (%)	90
MolProbity score	2.1

Results and Discussion

The new, 3.05 Å resolution cryo-EM structure of fV confirms the overall arrangement reported previously (rmsd=0.68 Å over 955 Cα atoms) [3], with the C domains defining a membrane binding module that supports the A1 and A3 domains arranged side-by-side and the A2 domain wedged between them (Figure 1AB). The site of thrombin activation at R1545 is 64% exposed to solvent [23] for proteolytic attack and the sites of activated protein C (APC) cleavage at R306 and R506 are 39% and 53% exposed to solvent, respectively. Clear density is also detected for N-glycans attached to N211, N269, N1675 and N1982. The new structure retains disorder in the large B domain but enables assignment of the 1507–1545 region comprising a large portion of the AR (residues 1493–1537). The segment 1507–1545 stretches 40 Å across, from the site of thrombin cleavage at R1545 to the site of APC cleavage at R506 in the A2 domain (Figure 2AB). The ¹⁵⁰⁷EDDY¹⁵¹⁰ wedge at the top of this segment shifts up to 21 Å at the level of D1508 from its position in fV short (Figure 2C) and separates from the segment 660–678 in the A2 domain. Immediately downstream, the segment 679–709 of the A2 domain (31 residues total) appears as disordered blobs of density lacking continuity in the sharpened map. As a result, there is little or no information on regions like the lid (⁶⁷²ESTVMATRKMHDRLEPEDEE⁶⁹¹) and the gate (⁶⁹⁶YDYQNRL⁷⁰²) that were resolved in the recent structure of fV (PDB 7KVE) [3] and play an important role in fVa assembled in the prothrombinase complex [24]. Hydrophobic contacts connect Y1510 to F668 and the Y1515-V1516 pair to P670. Both F668 and P670 are contained in the segment ⁶⁶⁶EIFEPPE⁶⁷² that may interact with exosite I of thrombin [25] to promote activation of fV through cleavage at R709 and R1545 [26]. Notably, the 660–678 segment shows no density in fVa [3] and is weakly defined in fV short [15]. In the new structure of fV reported here (PDB 8TN9), the segment assumes a conformation that is distinct and better defined from that reported previously for fV (PDB 7KVE) [3] or fVa in the prothrombin-prothrombinase complex (PDB 7TPP) [24]. This conformational flexibility is expected of a domain that lacks a well-defined secondary structure (Figure 3).

Figure 2ABC. Architecture of the AR in fV.

(A) Surface representation of the entire segment ¹⁵⁰⁷EDDYAEIDYVPYDDPYKTDVRTNINSSRDPD¹⁵³⁷ in the B domain of fV, folded on the surface of the A2 (light green) and A3 (light yellow) domains. The segment contains 10 of the 14 acidic (cyan) residues of the AR (residues 1493–1537), as well as 11 of the 17 non-polar (grey) residues and terminates with the site of thrombin cleavage at R1545 (red). Basic (red) and polar (orange) residues are also shown for completeness. The acidic cluster (AC) defines the core of the AR and includes a strip of acidic residues (E1507, D1508, D1509, E1512, D1514) connected to an equally long patch of such residues from the A2 domain (D659, D660, D661, E662, D663). The AC borders the hydrophobic cluster (HC) 3 (A1511, I1513, Y1515, P1517, Y1518) that includes I657 and P658 from the A2 domain. Residues D1519 and D1520 are also part of the AC and border the HC3 from below. Residues of the HC4 (V1526, A1541, W1542, L1544) cluster near the site of thrombin cleavage at R1545 and the indole of W1542 comes close to the Arg and partially shields it from proteolytic attack. Next to the HC4, a cluster of polar (N1529, N1531, S1532, S1533, N1538) and acidic (D1535, D1537) residues define the polar cluster (PC), with K1523 prominently exposed immediately above it. (B) Map (grey) with the segment 1507–1545, rendered as a purple ribbon with residues shown as sticks, documents the quality of the final model of this region of the B domain. (C) Superposition of the segment 1507–1545 between fV (PDB 8TN9, cyan) and fV short (PDB 8FDG, brown). Residues R1545, Y1515 and E1507 are shown as sticks on the ribbon renderings. The alignment is particularly good in the 1515–1545 segment and the backbones starts to diverge upstream of Y1515.

Figure 3ABCD. Architecture of the 656–709 segment in different fV structures.

Shown are tile arranged and aligned 656–709 segments from: (A) fV short (PDB 8FDG), (B) fV (PDB 7KVE), (C) fVa in the prothrombinase complex (PDB 7TPP) and (D) fV in the new structure presented in this study (PDB 8TN9). The lid (residues ⁶⁷²ESTVMATRKMHDRLEPEDEE⁶⁹¹) and the gate (residues ⁶⁹⁶YDYQNRL⁷⁰²) are highlighted in red and magenta, respectively, and the side chains of I657, T678, and R709 are displayed as sticks. The PDB entry for the structure is indicated in each panel.

The architecture of the AR in the new structure of fV (Figure 2AB) is elucidated for the first time and is similar to that recently uncovered for the same region in fV short [15], which was the best defined segment in the density map of the shorter B domain. A superposition of the 1507–1545 segment in the two proteins shows excellent alignment in the 1515–1545 region (Figure 2C) and the presence of acidic and hydrophobic clusters (Figure 2A) that may work synergistically to engage the BR in an intramolecular interaction that locks fV in its inactive state. We speculate that interaction of the BR with the proximal portion of the AR (residues 1493–1520) may protect fV from cleavage by APC at R506, whilst interaction with the distal portion of the AR (residues 1521–1537) may protect from cleavage by thrombin at R1545. Validation of this model will await structural identification of the intramolecular interaction of the BR with the AR through higher resolution cryo-EM structures of fV or other biophysical approaches.

Disorder of the B domain has so far precluded resolution of the AR in fV [3]. On the other hand, the architecture of the AR in fV short may have been biased by the splice site at L1459 [15]. The new structure of fV indicates that the splice site at L1459 in fV short does not significantly alter the conformation and position of the 1515–1545 segment of the AR, which is similar in the two proteins. This feature offers a structural basis for the functional effects caused by binding of the BR in fV and of the homologous C-terminal basic segment of TFPIα in fV short. In both cases, binding protects R1545 from catalytic attack by thrombin, whilst cleavage at R1545 by thrombin converts fV to fVa and abolishes TFPIα binding to fV short [1, 2, 12–14]. On the other hand, the splice site at L1459 is close enough to the preAR (residues 1458–1492) to change the direction of the peptide backbone upstream of Y1515 (Figure 2C). As a result, the entire preAR of fV may occupy a different position compared to fV short, which should be clarified by future structural studies. The new structure of fV also draws attention to the intramolecular interaction between the BR and AR that locks fV in its inactive state [4, 5]. The structure detects the AR with no interactions with the BR or its fragments and in a conformation similar to that of fV short, where the BR is not present. An electrostatically-driven intramolecular interaction like the one involving the BR and AR is expected to take place with high-affinity and stability [27]. A weak or transient interaction would be difficult to detect by cryo-EM, but would also be unlikely to lock fV in its inactive state. Therefore, the molecular mechanism that keeps fV in its inactive state remains unresolved and should await resolution by future structural studies or alternative biophysical approaches.