The Linker Connecting the Two Kringles Plays a Key Role in Prothrombin Activation

The linker connecting the two kringles plays a key role in prothrombin activation

Nicola Pozzi, Zhiwei Chen, Leslie A. Pelc, Daniel B. Shropshire, and Enrico Di Cera1

Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO 63104

Edited by David W. Russell, University of Texas Southwestern Medical Center, Dallas, TX, and approved April 22, 2014 (received for review February 28, 2014) The zymogen prothrombin is proteolytically converted by factor between the autolysis loop (9) and exosite I (10). Factor Xa Xa to the active protease thrombin in a reaction that is accelerated interacts with kringle-2 (11) and residues near exosite II of the >3,000-fold by cofactor Va. This physiologically important effect is catalytic B chain (12), and with the Gla domain (13). These studies paradigmatic of analogous cofactor-dependent reactions in the have targeted regions of the zymogen also present in other vitamin coagulation and complement cascades, but its structural determi- K-dependent clotting factors and proteolytic enzymes (14). The nants remain poorly understood. Prothrombin has three linkers recent crystal structure of Gla-domainless prothrombin (GD-ProT; connecting the N-terminal Gla domain to kringle-1 (Lnk1), the residues 45–579) has revealed the architecture of the kringles two kringles (Lnk2), and kringle-2 to the C-terminal protease do- and protease domain arranged into an overall bent conformation main (Lnk3). Recent developments indicate that the linkers, and with the domains not vertically stacked (15). Importantly, none particularly Lnk2, confer on the zymogen significant flexibility in of the three linkers could be resolved in the electron density solution and enable prothrombin to sample alternative conforma- map. Complete disorder was detected in Lnk1, residues 144–168 tions. The role of this flexibility in the context of prothrombin of Lnk2, and residues 255–273 of Lnk3 containing the site of activation was tested with several deletions. Removal of Lnk2 in cleavage at R271. Disorder of Lnk3 was already documented in Δ – almost its entirety (ProT 146 167) drastically reduces the enhance- previous structures of meizothrombin desF1 (16) and pre- > ment of thrombin generation by cofactor Va from 3,000-fold to thrombin-1 (17), but the flexibility of Lnk1 and Lnk2 could not 60-fold because of a significant increase in the rate of activation in be anticipated before the structure of GD-ProT became avail- the absence of cofactor. Deletion of Lnk2 mimics the action of able. Luminescence resonance energy transfer measurements of cofactor Va and offers insights into how prothrombin is activated Δ – prothrombin in solution with probes attached to kringle-1 and at the molecular level. The crystal structure of ProT 146 167 reveals kringle-2 have shown that Lnk2 assumes a range of conformations a contorted architecture where the domains are not vertically stacked, and functions like a flexible spring connecting the kringle-2/protease kringle-1 comes within 9 Å of the protease domain, and the Gla- domain pair on the C-terminal side to the Gla domain/kringle-1 pair domain primed for membrane binding comes in contact with ontheN-terminalside(15).TheflexibilityofLnk2maybecritical kringle-2. These findings broaden our molecular understanding of to the function of prothrombin free in solution or bound to the a key reaction of the blood coagulation cascade where cofactor Va prothrombinase complex. enhances activation of prothrombin by factor Xa by compressing Here, we report the functional consequences of deleting Lnk1 and Lnk2 and morphing prothrombin into a conformation similar to the Lnk2 of prothrombin. Removal of these linkers does not eliminate structure of ProTΔ146–167. the site of cleavage at R271 residing on Lnk3 and affords an analysis clotting factor | zymogen activation | structural biology oftheperturbationwithbothR271 and R320 available for proteolytic attack by factor Xa. Deletion of the flexible Lnk2 greatly facilitates crystallization (Table S1) and reveals the architecture of all he clotting factor prothrombin is proteolytically converted by factor Xa to the active protease thrombin by cleavage at two T Significance sites, R271 and R320, along two mutually exclusive pathways producing the intermediates prethrombin-2 (cleavage at R271 first) or meizothrombin (cleavage at R320 first). The reaction is Deletion of the flexible linker connecting the two kringles of greatly accelerated through a drastic increase in k when cata- prothrombin reduces the drastic enhancement of thrombin cat > lyzed by the prothrombinase complex composed of factor Xa, generation by cofactor Va from 3,000-fold observed with + cofactor Va, Ca2 , and phospholipids (1, 2). Both the rate en- wild-type prothrombin to only 60-fold. The change proves that hancement and pathway of activation are under the control of deletion of the linker mimics the effect of cofactor Va on pro- cofactor Va and phospholipids (3, 4), although the cofactor has thrombin activation. The crystal structure of the deletion mutant reveals a contorted conformation where the domains are the greatest effect in enhancing kcat. The molecular determinants of this physiologically important effect are of substantial mech- not vertically stacked, kringle-1 comes close to the protease anistic interest because they bear on the cofactor-dependent domain, and the Gla-domain contacts kringle-2. These findings activation of zymogens in many proteolytic cascades (5, 6). broaden our understanding of a key reaction of the blood co- Prothrombin is composed of fragment 1 (residues 1–155), agulation cascade. Cofactor Va enhances activation of pro- fragment 2 (residues 156–271), and a protease domain (residues thrombin by altering the architecture of the linker and inducing 272–579). Fragment 1 contains the Gla domain (residues 1–46) a conformation similar to the structure of the deletion mutant. and kringle-1 (residues 65–143), fragment 2 contains kringle-2 – Author contributions: N.P. and E.D.C. designed research; N.P., Z.C., L.A.P., and D.B.S. (residues 170 248), and the protease domain contains the A chain performed research; N.P., L.A.P., and D.B.S. contributed new reagents/analytic tools; (residues 272–320) and the catalytic B chain (residues 321–579). N.P., Z.C., and E.D.C. analyzed data; and E.D.C. wrote the paper. Three linkers—to be referred to as Lnk1, Lnk2, and Lnk3—connect The authors declare no conflict of interest. kringle-1 to the Gla domain (residues 47–64), the two kringles This article is a PNAS Direct Submission. – – (residues 144 169), and kringle-2 to the A chain (residues 249 284), Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, respectively (Fig. 1). Previous mutagenesis studies have identi- www.pdb.org (PDB ID codes 4NZQ and 4O03). fied possible epitopes for the interaction of prothrombin with 1To whom correspondence should be addressed. E-mail: [email protected]. prothrombinase. Kringle-1 and kringle-2 interact with cofactor This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Va (7, 8) and so do residues within the catalytic B chain scattered 1073/pnas.1403779111/-/DCSupplemental.

7630–7635 | PNAS | May 27, 2014 | vol. 111 | no. 21 www.pnas.org/cgi/doi/10.1073/pnas.1403779111 cofactor Va is to optimize the factor Xa–prothrombin complex for catalysis by acting in concert with phospholipids (3). Deletion of the entire Lnk1 (ProTΔ47–64) impairs the rate of thrombin generation >20-fold by increasing Km (Table 1). Re- + moval of Lnk1 compromises Ca2 binding and results in a lower level of γ-carboxylation of the Gla domain (Table S2 and Fig. S1), two effects that reduce prothrombin binding to the membrane (21). Furthermore, the proximity of Lnk1 to the Gla domain may influence the optimal orientation of the adjacent ω-loop responsible for membrane binding (see below). The functional defect of ProTΔ47–64 is rescued by the addition of cofactor Va (Fig. 1) that brings both kcat and Km to values comparable to those of wild type. As a result, the overall enhancement of thrombin generation produced by the addition of cofactor Va becomes almost 20,000-fold (Table 1). Notably, the pathway of activation in the presence of cofactor Va switches from meizothrombin to prethrombin-2 (Fig. 2), as reported for perturbations of prothrombin affecting the Gla domain (18, 22, 23). The role of Lnk2 was studied with two mutations: one deleting six residues (ProTΔ154–159) bridging the site of prethrombin-1 cleavage at R155 and the other deleting 22 of the 26 residues Fig. 1. (A) Schematic representation of the structural domains of pro- Δ – thrombin and the deletion mutants characterized in this study. (B and C) of Lnk2 (ProT 146 167). The shorter deletion is largely in- Generation of activity in the absence (B) or presence (C) of cofactor Va for consequential on prothrombin function (Figs. 1 and 2), except wild-type and mutant prothrombins. Experimental data obey Michaelis– for a small but noticeable increase in the rate of activation in the Menten kinetics with best-fit parameter values reported in Table 1. Experi- absence of cofactor Va due to an increase in kcat. This small mental conditions are as follows: 150 mM NaCl, 5 mM CaCl2, 0.1% PEG 8000, effect presages the properties of ProTΔ146–167, whose activa- − and 20 mM Tris at pH 7.4 and 25 °C. Activity on the y axis is expressed in s 1 tion in the absence of cofactor Va proceeds with a rate nearly 20- units to facilitate visualization of kcat values. Colors refer to the constructs in fold faster compared with wild-type (Table 1 and Fig. 1). The A:wt(red),proTΔ47–64 (blue), ProTΔ154–159 (green), ProTΔ146–167 (purple). enhancement is due almost entirely to kcat with a small decrease in Km and reproduces on a smaller scale the effect of cofactor Va domains of prothrombin in what is likely the conformation of the and phospholipids (3, 4) on the rate of prothrombin activation. The addition of cofactor Va further increases the rate of acti- zymogen bound to the prothrombinase complex. vation along the meizothrombin pathway (Fig. 2) and brings the k K k Results values of cat/ m and cat close to those of wild type (Table 1 and + Fig. 1). As a result, the effect of cofactor Va on the rate of Activation of prothrombin in the presence of factor Xa, Ca2 , – thrombin generation is reduced from 3,200-fold in the wild type and synthetic phospholipids obeys Michaelis Menten kinetics to only 60-fold in ProTΔ146–167 because of a significant in- k K = ± μ −1· −1 k = ± with values of cat/ m 0.12 0.01 M s , cat 0.022 crease in the rate of activation in the absence of cofactor. −1 K = ± μ 0.002 s , and m 0.19 0.01 M (Table 1 and Fig. 1), in The intriguing functional properties of ProTΔ146–167 suggest agreement with previous studies (1, 18). The activation proceeds that its structural architecture may reveal how the conformation with accumulation of prethrombin-2 and fragment 1, although an of prothrombin changes in the prothrombinase complex under additional cleavage is detected at R155-generating prethrombin- the effect of cofactor Va and phospholipids. Removal of residues 1 (Fig. 2). The cleavage is due to factor Xa and not thrombin 146–167 from Lnk2 greatly facilitated crystallization from initial because the prethrombin-1 band is also observed when the pro- screenings compared with GD-ProT (15) and produced diffrac- Δ – 2+ 2+ thrombin mutant S525A is used as substrate to generate a catalyti- tion quality crystals of ProT 146 167 in the Ca -free and Ca - bound forms. The two structures differ mainly in the architecture cally inactive product. The addition of cofactor Va to form the + of the Gla domain, which is completely resolved in the Ca2 -bound prothrombinase complex produces a drastic (3,200-fold) enhance- form to be discussed below but misses the first 33 residues in the ment of prothrombin activation with values of kcat/Km = 380 ± 10 2+ − − − Ca -free form notwithstanding a higher resolution (Table S1). μ 1· 1 k = ± 1 K = ± μ + M s , cat 42 2s ,and m 0.11 0.01 M, and the Disorder in the Ca2 -free form was reported for a high resolu- reaction proceeds along the meizothrombin pathway, again consis- tion (2.2 Å) crystal structure of bovine fragment 1 in the absence tent with previous studies (1, 18–20). The effect of cofactor Va is of cation (24). The structure of ProTΔ146–167 spans a total directed mainly on kcat, which increases 1,900-fold. Because kcat length of 87 Å with the domains not vertically stacked and is a property of the enzyme–substrate complex, the function of kringle-1 positioned almost perpendicular to the main axis of

Table 1. Kinetic parameters for activation of prothrombin wild-type and mutants Without cofactor Va With cofactor Va

−1· −1 −1 −1· −1 −1 kcat/Km, μM s kcat,s Km, μM kcat/Km, μM s kcat,s Km, μM

WT 0.12 ± 0.01 0.022 ± 0.002 0.19 ± 0.01 380 ± 10 42 ± 2 0.11 ± 0.01 ProTΔ47–64 0.0049 ± 0.0005 0.020 ± 0.002 4.1 ± 0.8 95 ± 519± 2 0.20 ± 0.01 ProTΔ154–159 0.18 ± 0.01 0.030 ± 0.002 0.17 ± 0.01 350 ± 10 38 ± 1 0.11 ± 0.01 ProTΔ146–167 2.0 ± 0.2 0.26 ± 0.02 0.13 ± 0.01 120 ± 10 11 ± 1 0.092 ± 0.008

Experimental conditions are 25 μM phospholipids, 150 mM NaCl, 5 mM CaCl2, 0.1% PEG 8000, 20 mM Tris at pH 7.4 and 25 °C. The concentration of factor Xa in the absence of cofactor Va is 0.1–7.5 nM, depending on the BIOCHEMISTRY prothrombin construct used, and 10 pM in the presence of cofactor.

Pozzi et al. PNAS | May 27, 2014 | vol. 111 | no. 21 | 7631 Fig. 2. Activation of prothrombin wild-type and mutants in the absence (-FVa) or presence (+FVa) of cofactor Va. In the absence of cofactor Va, activation of prothrombin wild-type and mutants leads to accumulation of prethrombin-2 (Pre-2) and fragment 1.2. (F1.2) as main products. Notably, the band of the intact ProTΔ146–167 disappears at a rate considerably faster than wild-type prothrombin (see also Fig. 1). The inhibitor DAPA was added to inhibit thrombin and meizothrombin activity. Nonetheless, cleavage at R155 and formation of prethrombin-1 (Pre-1) was detected in prothrombin wild-type and ProTΔ47–64, but not for ProTΔ146–167 and ProTΔ154–159 where R155 is missing. The cleavage is due to factor Xa and not thrombin because the prethrombin-1 band is also observed when the prothrombin mutant S525A is used as substrate to generate a catalytically inactive product. The chemical identity of this band was verified by N-terminal sequencing (156SEGSS160). The faint bands around 30 kDa refer to fragment 1 because they are not detected when R155 is missing. In the presence of cofactor Va, prothrombin wild-type is cleaved at R320 along the meizothrombin pathway to generate fragment F1.2.A and the B chain. The same profile is observed for ProTΔ154–159 and ProTΔ146–167. However, activation of ProTΔ47–64 clearly differs from wild type and follows the prethrombin-2 pathway. Experimental conditions are 150 mM NaCl, 5 mM CaCl2, 0.1% PEG 8000, and 20 mM Tris at pH 7.4 and 25 °C. Time points for the SDS/PAGE experiments are in minutes. kringle-2 that remains coaxial to the protease domain (Fig. 3). At protease domain and the adjacent fragment 2 is very similar (rmsd = the artificial junction between residues Q145 and E168 created 0.77 Å) to that seen in GD-ProT (15). In the active site region, by the deletion (Fig. 4), kringle-1 swings almost 50 Å from the there is drastic shortening of the critical G548-G523 (G216-G193) position occupied in GD-ProT (15). An overlay of the two crystal Cα-Cα distance (chymotrypsin numbering is shown by parenthe- structures (Fig. 3) reveals a shorter overall length for ProTΔ146– ses) measuring the width of the catalytic pocket, from 14.3 Å in 167 caused by its contorted conformation in which kringle-1 GD-ProT (15) to 10.6 Å in ProTΔ146–167. The shift is due to the > comes within 9 Å of the protease domain instead of being 60 Å conformation of W468 (W148) that sits on a helical turn in the away, and the Gla domain primed for membrane binding brings usually disordered autolysis loop and penetrates the active site in E48 in interaction with R181 of kringle-2. Δ – a substrate-like orientation, making its way between a flipped All domains of ProT 146 167 are clearly resolved in the W547 (W215) and W370 (W60d), and pulling in the 547–549 electron density map, except for several residues of Lnk3. The (215–217) segment. The occluded conformation of the active site important cleavage site at R271 is contained in this segment and of ProTΔ146–167 is caused mainly by the repositioning of W468 has so far eluded X-ray detection in all published structures of (W148) and not by the collapse of W547 (W215) observed in the human GD-ProT (15), prethrombin-1 (17) or meizothrombin desF1 (16). Disorder in this region supports the conclusion that structures of other inactive thrombin precursors such as GD-ProT (15), prethrombin-1 (17), and prethrombin-2 (25). The width of R271 is constitutively exposed to solvent for proteolytic attack, Δ – regardless of the conformation of Lnk2. The site of cleavage at the active site of ProT 146 167 is similar to that reported for the R320, however, is clearly visible in the electron density map (Fig. zymogen of complement factor C1r (26) and trypsinogen (27) and 4), and the side chain is not caged as in prethrombin-2 (25), or in falls toward the lower end of the distribution of trypsin-like zym- contact with E323 as in GD-ProT (15) or prethrombin-1 (17), ogens (28) that ranges from 8.1 Å in the zymogen of complement but almost fully (86%) exposed to solvent. Although the two sites factor D (29) to 14.6 Å in prethrombin-1 (17). Another critical Cα- of cleavage are both exposed to solvent, they reside in segments Cα distance between the catalytic S525 (S195) and D519 (D189) of the zymogen that are intrinsically disordered (R271) or quite measures the depth of the active site pocket and increases slightly rigid (R320), which may have a bearing on the selection of the from 14.4 Å in GD-ProT to 15.6 Å in ProTΔ146–167 (Fig. 4). The pathway of prothrombin activation. The architecture of the entire distance is >4 Å longer than that measured for the mature enzyme

7632 | www.pnas.org/cgi/doi/10.1073/pnas.1403779111 Pozzi et al. Fig. 3. (A) X-ray crystal structure of ProTΔ146–167 + in the Ca2 -bound form showing the arrangement of the various domains (Ac, A chain; Bc, B chain; GD, Gla domain; K1, kringle-1; K2, kringle-2) that are not vertically stacked. The structure spans approxi- mately 87 Å in length, from the tip of F4 in the Gla domain to the site of cleavage R320 that is fully exposed to solvent (see also Fig. 4). The site of cleavage at R271 is in a disordered region in Lnk3. The active site region is circled. Details of the Gla domain containing five bound Ca2+ ions are shown in Fig. 4. (B) Overlay of the structures of ProTΔ146– 167 (ribbon colored as in A) and GD-ProT (wheat) reported recently (15). The two structures (rmsd = 1.1 Å) overlap significantly at the level of kringle-2 and the protease domain (rmsd = 0.77 Å), but differ sharply in the orientation of kringle-1 that shifts >50 ÅinProTΔ146–167 from its position in GD-ProT (K1 labeled in purple).

(30, 31) and underscores the compromised architecture of the S23-L31 (helix 2), T37-A50 (helix 3), P53-G63 (helix 4), and the primary specificity site in the zymogen. hydrophobic ω-loop (F4-L5-V8) primed for membrane binding. Prothrombin binds phospholipids by using the Gla domain The overall architecture of the Gla domain is comparable to that spanning residues 1–46 (Figs. 3 and 4), and the structure of detected by NMR (35), but helix 4 in Lnk1 creates a slightly different ProTΔ146–167 finally reveals the architecture of this domain environment compared with other clotting factors (36–38) where the in the context of the entire zymogen. There are nine conserved Gla domain attaches to EGF domains instead of kringles. The im- γ-carboxyglutamic acid residues, and the additional γ-carbox- portance of this helix pinned to helix 3 by the C47-C60 disulfide yglutamic acid at position 32 has been shown to be dispensable bond is directly supported by mutagenesis data (Figs. 1 and 2). + for prothrombin activation by the prothrombinase complex yet Deletion of Lnk1 in ProTΔ47–64 compromises Ca2 binding and required for binding to phospholipids (32). Residue 32 has been full γ-carboxylation (Table S2 and Fig. S1). Additionally, the de- implied for high-affinity binding and interspecies differences letion may compromise optimal orientation of the adjacent ω-loop among naturally occurring vitamin K-dependent proteins S, Z, responsible for membrane binding contributing to the increased and C (33) and explains the peculiar kinetics of prothrombin Km (Table 1 and Fig. 1) and the resulting shift in the pathway binding to membranes (34). The Gla domain of ProTΔ146–167 of thrombin generation (Fig. 2). Notwithstanding the low reso- and its linker connection to kringle-1 are clearly detected from lution of the structure, extra density is clearly detected in the A1 to N64 with four helices spanning residues N12-T21 (helix 1), area hosting cation binding sites in the structure of bovine fragment

Fig. 4. (A) Artificial connection between Q145 in kringle-1 and E168 in kringle-2 generated by deletion of Lnk2 in ProTΔ146–167. (B) Activation domain of ProTΔ146–167 showing the side chain of R320 at the site of cleavage fully exposed to solvent for proteolytic attack. (C) Active-site met- rics of ProTΔ146–167 showing the Cα-Cα distances G548–G523 (G216–G193) and S525–D519 (S195– D189) measuring the width and depth of the catalytic pocket (chymotrypsin numbering is shown by parentheses). W468 (W148) from the autolysis loop penetrates the active site pushing away the flipped W547 (W215) and W370 (W60d) in the 60- loop and pulling in the 547–549 (215–217) segment.

Electron densities in A–C are 2 F0–Fc maps contoured at 1 σ.(D) Architecture of the Gla domain of ProTΔ146–167 revealing four helical segments N12- T21 (H1), S23-L31 (H2), T37-A50 (H3), P53-G63 (H4) and five Ca2+ ions bound to sites 2–6 in the Tulin- sky’s numbering (24). (E) Detailed view of the five + Ca2 ions bound to γ-carboxyglutamic acid residues (labeled as E) of the Gla domain organizing the hydrophobic ω-loop (F4-L5-V8) for membrane + binding. Ca2 binding sites (numbered 2–6 from right to left, see also C) were validated with the program VALE (39) that returned valence units/ coordination of 1.7/5 (site 2), 1.1/4 (site 3), 1.4/8 (site4),1.0/6(site5),and0.64/2(site6).Thelow numbers for site 6 suggest weak binding affinity BIOCHEMISTRY for this site and contribution of water molecules to the coordination shell not detected because of the low resolution. Electron density is an F0–Fc + map contoured at 3.5 σ and calculated after removing the bound Ca2 ions.

Pozzi et al. PNAS | May 27, 2014 | vol. 111 | no. 21 | 7633 + 1 (24). Valence calculations (39) identify five Ca2 binding sites at exposed to solvent for proteolytic attack in the structure of positions 2–6 with high confidence, but no cations bound at posi- ProTΔ146–167 (Figs. 3 and 4), the directionality of the cleavage tions 1 and 7. Overall, kringle-1 assumes its expected fold and can be influenced long range by the Gla domain via Lnk1 and overlaps well with the published structures of bovine fragment 1 Lnk2 in response to the nature of the membrane (18, 19, 22, 23, (24) (rmsd = 0.46 Å) and GD-ProT (15) (rmsd = 0.60 Å). 45–47) or interaction with the Gla domain of factor Xa (13). Discussion Materials and Methods The results reported here unravel a completely unanticipated Reagents. Recombinant full-length prothrombin (residues 1–579) wild-type and structure-function link for a key reaction of the blood co- mutants S525A, proTΔ47–64, ProTΔ154–159, and ProTΔ146–167 were cloned in agulation cascade. Removal of the linker connecting the two a pNUT vector, expressed in baby hamster kidney cells and purified by affinity kringles causes substantial rearrangement in the structure of chromatography, ion exchange chromatography, and size exclusion chroma- prothrombin and produces a contorted architecture where the tography as described (15, 28, 48). Homogeneity and chemical identity of final preparations were verified by SDS/PAGE, N-terminal sequencing, and mass domains are not vertically stacked, kringle-1 comes within 9 Å of spectrometry. The level of γ-carboxylation (Table S2) was determined by alka- the protease domain, and the Gla domain primed for membrane line hydrolysis coupled to amino acid analysis (49, 50) performed by the protein binding comes in contact with kringle-2. This conformation is chemistry laboratory at Texas A&M University. Small unilamellar vesicles were associated functionally with an enhanced rate of prothrombin prepared by sonication (51). Phosphotidylcholine (PC) and phosphotidylser- activation in the absence, but not the presence, of cofactor Va, ine (PS) (Avanti Polar Lipids) were solubilized in chloroform in a 3:1 PC:PS thereby indicating that the structure of ProTΔ146–167 may molar ratio. After being dried under N2 gas, they were resuspended in 150 be a close representation of the conformation of prothrombin mM NaCl and 20 mM Tris at pH 7.4 and then sonicated in a water bath. bound to the prothrombinase complex. Dynamic light scattering showed 97.2% of the mass had a radius of 26 ± 3 Prothrombin activation is greatly accelerated by the pro- nm. Human purified factor Va, factor Xa, and the thrombin specific inhibitor thrombinase complex (1, 2), and both the rate enhancement and dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide (DAPA) were purchased from Hematologic Technologies. Protein concentrations were determined by pathway of activation are under the control of cofactor Va and reading at 280 nm with molar extinction coefficients reported in Table S2. phospholipids (3, 4). The molecular determinants of this physiologically important effect have been difficult to identify because Activation of Prothrombin. Prothrombin wild-type and mutants were buffer they are the result of contributions of direct and linked effects exchanged into 150 mM NaCl, 20 mM Tris, and 5 mM CaCl2 by using a Zeba 7K from the various components of the prothrombinase complex MWCO desalting columns. Prothrombin activation assays were performed as acting on prothrombin. Removal of the flexible Lnk2 connecting described (18, 20). Prothrombin (0–8 μM) was activated by the addition of factor the two kringles of the zymogen produces a prothrombin variant, Xa (0.1–7.5 nM) and phospholipids (25 μM) or factor Xa (10 pM), phospholipids ProTΔ146–167, that functionally mimics the action of pro- (25 μM), and cofactor Va (30 nM). Aliquots were quenched after 0.5, 1, 2, and 5 thrombinase on prothrombin without compromising the pro- min, and active site generation was quantified in terms of the hydrolysis of the thrombinase–prothrombin complex (Table 1). Efficient catalysis chromogenic substrate H-D-Phe-Pro-Arg-p-nitroanilide specific for thrombin of prothrombin by prothrombinase does not require the full and meizothrombin. Initial velocities were converted to concentrations by using a standard reference curve measured at the time of the experiment. length of Lnk2, contrary to the conclusions drawn from recent μ – Additionally, activation by factor Xa (10 nM) and phospholipids (25 M) or structural models of the prothrombinase prothrombin complex factor Xa (0.15 nM), phospholipids (25 μM), and cofactor Va (30 nM) was (40, 41). Factor Xa and prothrombin anchored to the membrane carried out with 1.4 μM prothrombin dissolved in 150 mM NaCl, 20 mM Tris, by their Gla domain need to align properly for efficient thrombin and 5 mM CaCl2 and incubated at 25 °C for 5 min in the presence 60 μMofthe generation. The flexibility of Lnk2, supported by the crystal specific thrombin inhibitor DAPA (except for mutant S525A), 25 μM phos- structure of GD-ProT and luminescence resonance energy transfer pholipids, and cofactor Va as indicated. The reaction was started by the ad- measurements in solution (15), enables prothrombin to sample dition of factor Xa and quenched at different time intervals with 10 μLof multiple conformations. This property could constitute an en- NuPAGE LDS buffer containing β-mercaptoethanol as the reducing agent and – tropic barrier to optimal interaction with factor Xa that may 20 mM EDTA. Samples were processed by NuPAGE Novex 4 12% (mass/vol) Bis-Tris protein gels run with Mes buffer, stained in SDS/PAGE with Coomassie itself undergo conformational transitions on the plane of the brilliant blue R-250, and analyzed by quantitative densitometry. membrane (42). We propose that binding of cofactor Va acting in concert with the membrane (3) relieves this entropy cost by X-Ray Studies. Crystallization of ProTΔ146–167 was achieved at 20 °C by the providing a scaffold that aligns both factor Xa and prothrombin vapor diffusion technique by using an Art Robbins Instruments Phoenix for optimal catalysis. A key component of this action is to liquid handling robot and mixing equal volumes (0.3 μL) of protein and “compress” prothrombin in a way that is mimicked by the reservoir solution (Table S1). ProTΔ146–167 (10 mg/mL) was dissolved in + structure of ProTΔ146–167 reported here. 50 mM ChCl, 50 mM Mes at pH 5.5 for the Ca2 -free crystals and 50 mM ChCl, 2+ A hotly debated issue in the field is how prothrombinase 5 mM CaCl2, 2 mM MgCl2, and 50 mM Mes at pH 5.5 for the Ca -bound selects the pathway of activation and imposes directionality of crystals. Optimization of crystal growth was obtained by the hanging drop vapor diffusion method mixing 3 μL of protein (10 mg/mL) with equal vol- cleavage at R271 and R320 depending on the context (18, 20, 43, + umes of reservoir solution. Crystals of Ca2 -free ProTΔ146–167 grew in less 44). Under conditions most relevant to physiology, i.e., on the + than 2 wk and were frozen directly. Crystals of Ca2 -bound ProTΔ146–167 surface of platelets (45, 46), prothrombinase activates pro- also grew in less than 2 wk and were frozen in a solution similar to the thrombin along the prethrombin-2 pathway. On nonplatelet mother liquor and containing 15% (vol/vol) glycerol. X-ray diffraction data surfaces such as red blood cells (47) or synthetic phospholipids were collected with a home source (Rigaku 1.2 kW MMX007 generator with (1, 18, 19), prothrombinase activates prothrombin along the al- Very High Flux optics) Rigaku Raxis IV++ detector and were indexed, in- ternative meizothrombin pathway. Deletion of Lnk1 switches the tegrated, and scaled with the HKL2000 software package (52). The structure pathway of activation from meizothrombin to prethrombin-2, of Ca2+-free ProTΔ146–167 was solved by molecular replacement using + and the effect may be due to the lack of Ca2 binding or com- PHASER from the CCP4 suite (53) and Protein Data Bank ID codes 3NXP for plete γ-carboxylation of this mutant (Table S2 and Fig. S1). prethrombin-1 (17) and 2PF1 for bovine prothrombin fragment 1 (54) as search 2+ Δ – However, it is worth pointing out that the region connecting the models. The structure of Ca -bound ProT 146 167 was solved by molecular replacement using MOLREP from the CCP4 suite (53). The structure of Ca2+-free Gla domain to kringle-1 revealed by the structure of ProTΔ146– + ProTΔ146–167 was used as search model, and residues 1–33 of the Ca2 -bound 167 comprises two helices connected by the C47-C60 disulfide Gla domain were then modeled by using Protein Data Bank ID code 1NL1 + bond. Through this hinge region, the Gla domain anchored to for bovine prothrombin fragment 1 bound to Ca2 (35). Refinement the membrane may influence the position of kringle-1 and and electron density generation were performed with REFMAC5 from the propagate conformational changes to the rest of the molecule CCP4 suite, and 5% of the reflections were randomly selected as a test by using the flexible Lnk2. Because both sites of cleavage are set for cross-validation. Model building and analysis of the structures

7634 | www.pnas.org/cgi/doi/10.1073/pnas.1403779111 Pozzi et al. were conducted with COOT (55). Ramachandran plots were calculated by ACKNOWLEDGMENTS. We thank Dr. David W. Gohara for carrying out the using PROCHECK (56). Statistics for data collection and refinement are valence calculations and building a model of the prothrombinase–prothrom- summarized in Table S1. Atomic coordinates and structure factors have bin complex, and Ms. Tracey Baird for help with illustrations. This work was been deposited in the Protein Data Bank (ID codes 4NZQ for Ca2+-free supported in part by the National Institutes of Health Research Grants + ProTΔ146–167 and 4O03 for Ca2 -bound ProTΔ146–167). HL49413, HL73813, and HL112303.

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