Báo cáo Y học: Assignment of molecular properties of a superactive coagulation factor VIIa variant to individual amino acid changes potx

Assignment of molecular properties of a superactive coagulation factor VIIa variant to individual amino acid changes Egon Persson 1 and Ole H. Olsen 2 1 Haemostasis Biology and 2 Medicinal Chemistry Research IV, Novo Nordisk A/S, Ma ˚ løv, Denmark The most active factor VIIa (FVIIa) variants identified to date carry concurrent substitutions at positions 158, 296 and 298 with the intention of generating a thrombin-mimicking motif, optionally combined with additional replacements within the protease domain [Persson, E., Kjalke, M. & Olsen, O. H. (2001) Proc. Natl Acad. Sci. USA 98, 13583– 13588]. Here we have characterized variants of FVIIa mutated at one or two of these positions to assess the relative importance of the individual replacements. The E296V and M298Q mutations gave an increased intrinsic amidolytic activity (about two- and 3.5-fold, respectively) compared with wild-type FVIIa. An additive effect was observed upon their combination, resulting in the amidolytic activity of E296V/M298Q-FVIIa being close to that of the triple mutant. The level of amidolytic activity of a variant was correlated with the rate of inhibition by antithrombin (AT). Compared with wild-type FVIIa, the Ca 2+ dependence of the intrinsic amidolytic activity was significantly attenuated upon introduction of the E296V mutation, but the effect was most pronounced in the triple mutant. Enhancement of the proteolytic activity requires substitution of Gln for Met298. The simultaneous presence of the V158D, E296V and M298Q mutations gives the highest intrinsic activity and is essential to achieve a dramatically higher relative increase in the proteolytic activity than that in the amidolytic activity. The N-terminal Ile153 is most efficiently buried in V158D/ E296V/M298Q-FVIIa, but is less available for chemical modification also in the presence of the E296V or M298Q mutation alone. In summary, E296V and M298Q enhance the amidolytic activity and facilitate salt bridge formation between the N-terminus and Asp343, E296V reduces the Ca 2+ dependence, M298Q is required for increased factor X (FX) activation, and the simultaneous presence of the V158D, E296V and M298Q mutations gives the most pro- found effect on all these parameters. Keywords: factor VIIa variant; factor X activation; intrinsic activity; superactivity; zymogenicity. Coagulation factor VIIa (FVIIa), in contrast to other, homologous serine proteases, possesses an active confor- mation that is energetically unfavourable. The consequence is a far from optimal enzymatic activity of free FVIIa, which is dramatically enhanced upon binding to the cognate, membrane-bound cofactor tissue factor (TF) or to its extracellular, soluble portion (sTF) [1]. In the natural environment, the zymogenicity of free FVIIa ensures timely triggering and appropriate location of FVIIa haemostatic activity upon vascular lesion and concomitant TF exposure. The three-dimensional structure of the protease domain of free FVIIa is, apart from certain loop regions, virtually identical to that of thrombin and other constitutively active and homologous serine proteases [2,3]. In addition, the structural differences between free [3–5] and TF-bound FVIIa [6,7] are subtle; thus the details in molecular architecture that restrict the activity of free FVIIa remain elusive. However, the high degree of similarity may be due to the presence of an active site inhibitor in the structure of the free FVIIa. The crystal (or solution) structure of noninhibited FVIIa is presumably needed to reveal the structural differences between ÔlatentÕ (zymogen-like) and ÔactiveÕ FVIIa. However, information possibly pertaining to the latent conformation of free FVIIa has been obtained from the crystal structure of zymogen FVII [8]. This structure suggests that relative b strand movements and a hydrogen bond involving Glu296{154} (chymotrypsinogen numbering is given in curly brackets to facilitate compar- isons with homologous enzymes) regulate the activity state of FVIIa. Recent advances in our understanding of the mechanisms regulating the activity of FVIIa have pinpointed side chains that function as zymogenicity determinants in the free enzyme. Replacements of these amino acid residues have resulted in FVIIa molecules with improved intrinsic (TF-independent) catalytic efficiency [9–11]. The relatively high intrinsic activity of some of these FVIIa variants suggests that the zymogen-like conformation of free factor VIIa is dictated by a limited number of key amino acid residues. We have previously shown that one of these superactive FVIIa variants, containing the mutations V158{21}D, E296{154}V and M298{156}Q, exhibits several properties resembling TF-bound rather than free FVIIa [9]. Apart from increased intrinsic enzymatic activity and inhibitor susceptibility as compared with wild-type FVIIa, this mutant has a diminished requirement for calcium ions and a more deeply buried protease domain N-terminus Correspondence to E. Persson, Haemostasis Biology, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Ma ˚ løv, Denmark. Fax: + 45 44434417, Tel.: + 45 44434351, E-mail: egpe@novonordisk.com Abbreviations: FVIIa, coagulation factor VIIa; FX, coagulation factor X; sTF, soluble tissue factor (residues 1–219); TF, tissue factor; AT, antithrombin (III). (Received 20 June 2002, revised 2 October 2002, accepted 22 October 2002) Eur. J. Biochem. 269, 5950–5955 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03323.x (Ile153{16}) indicating salt bridge formation of this residue with Asp343 {194}. To pinpoint the underlying mutation(s) responsible for these molecular and functional properties of the triple mutant, and thereby gain more knowledge about the zymogenicity determinants of FVIIa, mutants with substitutions at one or two of positions 158{21}, 296{154} and 298{156} were expressed and characterized. MATERIALS AND METHODS Proteins, chemical reagents and standard methods Wild-type FVIIa [12] and sTF [13] were prepared according to published procedures. The concentrations of FVIIa and sTF were determined by ELISA and spectrophotometry, respectively, as described [9]. SDS/PAGE was run on 8–25% gradient gels using the PhastSystem (Amersham Pharmacia) and followed by silver staining to check the purity of the FVIIa mutants and to verify their conversion to the activated, two-chain form. Factor X (FX) and factor Xa were from Enzyme Research Laboratories (South Bend, IN, USA) and antithrombin (AT) from Hematologic Technologies (Essex Junction, VT, USA). Unfractionated heparin was from Leo Pharmaceutical Products (Ballerup, Denmark), potassium cyanate (KOCN) from Merck and the chromogenic substrates S-2288 ( D -Ile-Pro-Arg-p-nitro- anilide) and S-2765 (benzyloxycarbonyl- D -Arg-Gly-Arg- p-nitroanilide) from Chromogenix (Mo ¨ lndal, Sweden). Mutagenesis and preparation of FVIIa mutants The FVII expression plasmid pLN174 [14] was used as the template for site-directed mutagenesis using the Quik- Change kit (Stratagene, La Jolla, CA, USA). The primer (only sense primer is given) used to introduce the E296V mutation, with base substitution in italic and the affected codon underlined, was GCC ACG GCC CTG GTGCTC ATG GTC CTC. The primers used to introduce the V158D, E296V/M298Q and M298Q mutations have been described [9]. Plasmid preparation, baby hamster kidney cell trans- fection and selection, and the expression, purification and autoactivation of FVII mutants were carried out as described [9,15]. The presence of none but the desired mutation(s) was verified by sequencing the portion of the cDNA encoding residues  80–406, encompassing the second epidermal growth factor-like and serine protease domains, on a MegaBACE 1000 (Amersham Pharmacia Biotech). Activity and inhibition assays The enzymatic activity and inhibition rates of the FVIIa variants were measured as described [9] using an assay buffer containing 50 m M Hepes, 0.1 M NaCl, 5 m M CaCl 2 and 1 mgÆmL )1 bovine serum albumin (pH 7.4). Briefly, the amidolytic activity was measured by mixing 180 lL wild- type or mutant FVIIa alone (final concentration 100 n M )or 180 lL FVIIa together with sTF (final concentrations 10 n M FVIIa and 50 n M sTF) with 20 lL10m M S-2288 at 25 °C. The measurement with free FVIIa was also per- formed in an assay buffer where CaCl 2 was replaced by EDTA. The ability of the FVIIa variants to activate FX (the proteolytic activity) was studied by incubating 10 n M (M298Q-, V158D/M298Q-, E296V/M298Q- and V158D/ E296V/M298Q-FVIIa) or 50 n M (wild-type, V158D-, E296V-, and V158D/E296V-FVIIa) FVIIa variant alone or 5 n M FVIIa variant plus 100 n M sTF with various concentrations of FX (0.1–4.8 l M )for20minatambient temperature (22 ± 1 °C). Buffer containing S-2765 was then added to give a chromogenic substrate concentration of 0.5 m M , whereafter the factor Xa-catalyzed hydrolysis was measured for 2 min. The inherent activity of the FX substrateandoftheFVIIa/sTFmixtureweresubtracted and the net amount of factor Xa generated was derived from a standard curve. The kinetic parameters of FX activation were calculated using GRAFIT 4.06 (Erithacus Software, Ltd). The rates of inhibition by AT/heparin and potassium cyanate were studied according to published methods [9] by incubation of the FVIIa variants (100 n M with AT and 1 l M with cyanate) with the inhibitor for 15 and 60 min, respectively, followed by measurements of the residual amidolytic activity. Bovine serum albumin was omitted from the buffer during the incubation with cyanate. RESULTS Enzymatic activity of FVIIa variants Two forms of enzymatic activity are analyzed. The amido- lytic activity is measured to assess the functional status of the active site, whereas the proteolytic activity reflects both this and exosite alterations resulting in further increased macromolecular substrate turnover. In the absence of TF, V158D/E296V/M298Q-FVIIa has seven- to eight-fold higher intrinsic amidolytic activity compared with wild-type FVIIa as measured by the rate of hydrolysis of the chromogenic substrate S-2288 [9]. When the mutations were introduced individually into FVIIa, V158D had no significant effect on the amidolytic activity, whereas E296V and M298Q yielded approximately two- and 3.5-fold enhancement, respectively (Table 1). The result with M298Q-FVIIa agrees with earlier reports [9,10]. The double mutant E296V/M298Q-FVIIa had an amidolytic activity six times higher than that of wild-type FVIIa and close to that of V158D/E296V/M298Q-FVIIa. In addition, V158D/ E296V-FVIIa had significantly lower amidolytic than E296V-FVIIa and V158D/M298Q-FVIIa had similar or slightly lower activity than M298Q-FVIIa. This shows that the simultaneous presence of the E296V and M298Q mutations suffices to achieve an amidolytic activity similar Table 1. Enzymatic activity of free FVIIa variants. All values are means ± SD (n ¼ 3). The amidolytic activity is given as the ratio between the activity of mutant and wild-type FVIIa. FVIIa variant Amidolytic activity (mutant/wt) FX activation (k cat , · 10 )3 s )1 ) Wild-type 0.088 ± 0.006 V158D 1.0 ± 0.1 0.069 ± 0.008 E296V 2.1 ± 0.3 0.083 ± 0.010 M298Q 3.4 ± 0.4 0.62 ± 0.04 V158D/E296V 0.6 ± 0.1 0.081 ± 0.009 V158D/M298Q 3.0 ± 0.5 0.60 ± 0.05 E296V/M298Q 6.0 ± 0.5 0.47 ± 0.07 V158D/E296V/M298Q 7.6 ± 0.5 2.2 ± 0.3 Ó FEBS 2002 Dissection of a superactive FVIIa variant (Eur. J. Biochem. 269) 5951 to that of the triple mutant and that the substitution of Asp for Val at position 158 has an insignificant or negative impact on the amidolytic activity. In complex with sTF, none of the investigated mutants had an amidolytic activity significantly different from that of wild-type FVIIa (not shown). The amidolytic activity of free FVIIa has been shown to be at least about 10 times higher in the presence of 5 m M Ca 2+ than in the absence of the metal ion [16,17]. As expected, in the absence of Ca 2+ wild-type FVIIa exhibited an amidolytic activity corresponding to about one tenth of its activity in the presence of 5 m M Ca 2+ . This was also the case for V158D-FVIIa, M298Q-FVIIa and V158D/M298Q- FVIIa. In contrast, all FVIIa variants containing the E296V mutation retained a larger fraction of their activity when Ca 2+ was omitted from the assay buffer; E296V-FVIIa (retained 24% of the activity), V158D/E296V-FVIIa (29%), E296V/M298Q-FVIIa (39%) and, in particular, V158D/ E296V/M298Q-FVIIa (64%). This shows that the substitu- tion of Val for Glu296{154}, which contacts the acidic Ca 2+ binding loop in the protease domain, attenuates the Ca 2+ dependence of FVIIa and confirms that this replacement is responsible for the diminished Ca 2+ requirement observed for V158D/E296V/M298Q-FVIIa [9]. The additional sub- stitution of Gln for Met298{156}, especially in combination with the replacement of Val158{21} by Asp, further attenuates the Ca 2+ dependence. We have previously shown that, in the absence of TF, the k cat values for FX activation by M298Q-FVIIa and V158D/ E296V/M298Q-FVIIa were increased 5.5- and 28-fold compared with that of wild-type FVIIa, respectively [9]. In agreement with these results, the new batches of the two variants displayed seven- and 25-fold higher values, respect- ively (Table 1). E296V/M298Q-FVIIa and V158D/M298Q- FVIIa activated FX five to seven times more rapidly than did wild-type FVIIa, rates similar to that seen with M298Q- FVIIa. This indicates that the introduction of the V158D or E296V mutation on the M298Q-FVIIa background does not contribute to an increased proteolytic activity. Indeed, an increased rate of FX activation was only observed for the FVIIa variants containing the M298Q mutation, and V158D-FVIIa, E296V-FVIIa and V158D/E296V-FVIIa exhibited a rate of catalysis of FX activation similar to or slightly below that of wild-type FVIIa. It is noteworthy that FX activation occurs much faster when catalyzed by V158D/E296V/M298Q-FVIIa than when catalyzed by V158D/E296V-FVIIa, V158D/M298Q-FVIIa or E296V/ M298Q-FVIIa. This suggests that the triad composed of residues 158{21}, 296{154} and 298{156} works as a unit regulating macromolecular substrate processing by free FVIIa. All FVIIa variants (including the wild-type enzyme) gave K m values for FX between 2.2 and 2.8 l M indicating that the mutations did not affect substrate affinity to a detectable extent (data not shown). When bound to sTF, none of the studied FVIIa variants exhibited an increased ability to activate FX as compared with wild-type FVIIa (not shown). Inhibition of FVIIa variants by antithrombin and potassium cyanate The susceptibility of the FVIIa variants to inhibition by two mechanistically different agents, antithrombin (active site-directed) and potassium cyanate (N-terminal carbamy- lation), was investigated. The rate of inhibition by anti- thrombin reflects the reactivity of the active site and has previously been found to nicely correlate to the level of amidolytic activity of FVIIa variants [9]. The results herein show that the new variants also obey this rule, with a strong relationship between amidolytic activity enhancement and increased inhibition rate (Table 2). The inhibition resulting from potassium cyanate-mediated, N-terminal carbamyla- tion reflects the degree of exposure of the protease domain N-terminus. When compared with that of wild-type FVIIa, the susceptibility to carbamylation was found to be strikingly reduced for V158D/E296V/M298Q-FVIIa (and reduced to some extent also for M298Q-FVIIa) indicative of a more buried N-terminal amino group [9]. A majority of the present FVIIa variants exhibits an intermediate level of protection from carbamylation (Table 2). E296V-FVIIa retains about half of its activity after incubation with potassium cyanate, which is slightly more than wild-type FVIIa. V158D/E296V-FVIIa, V158D/M298Q-FVIIa and E296V/M298Q-FVIIa all retain about 60% of their activity, which is similar to the residual activity of M298Q-FVIIa but considerably less than that of V158D/E296V/M298Q- FVIIa. This indicates that no single mutation is particularly efficient in terms of promoting the insertion of the N-terminus and, importantly, demonstrates that all three mutations are needed for stable burial of the N-terminus, most likely through salt bridge formation with Asp343{194}). DISCUSSION V158D/E296V/M298Q-FVIIa has been found to possess unique properties that differ dramatically from those of wild-type FVIIa (Fig. 1). This includes an increased intrinsic activity, a reduced activity dependence on Ca 2+ and a buried protease domain N-terminus [9]. The characteriza- tion of FVIIa variants containing one or two of the mutations at positions 158{21}, 296{154} and 298{156} has enabled the identification of the amino acid changes mainly responsible for the unique profile of the triple mutant. M298Q is the single mutation that enhances the amidolytic activity to the largest extent. The E296V mutation, on the other hand, appears to be responsible for the decreased calcium ion dependence of the amidolytic activity, with the other two mutations functioning as modulators of this Table 2. Inhibitor susceptibility of free FVIIa variants. The residual activity (%) after incubation with the inhibitor for 15 min (anti- thrombin) and 60 min (KNCO) is given (means ± SD, n ¼ 3). FVIIa variant Inhibitor Antithrombin KNCO Wild-type 71 ± 4 38 ± 4 V158D 75 ± 6 44 ± 7 E296V 67 ± 6 48 ± 6 M298Q 48 ± 4 66 ± 5 V158D/E296V 74 ± 8 60 ± 6 V158D/M298Q 41 ± 5 58 ± 8 E296V/M298Q 24 ± 4 64 ± 3 V158D/E296V/M298Q 19 ± 2 87 ± 4 5952 E. Persson and O. H. Olsen (Eur. J. Biochem. 269) Ó FEBS 2002 property in the presence of valine at position 296. It is possible that the increased activity of FVIIa after Ca 2+ binding to the acidic 210–220 {70–80} loop [18] is induced, at least to some extent, by a conformational change resulting from charge neutralization. The altered local charge distribution upon replacement of Glu296 by Val might in part mimic this effect and contribute to an increased activity and reduce the positive effect of Ca 2+ binding (Fig. 1A). The relative intrinsic proteolytic activity, i.e. the degree of enhanced catalysis of FX activation, follows a pattern slightly different from that of the amidolytic activity. The M298Q mutation, in contrast to V158D and E296V, enhances the proteolytic activity and is indeed present in all members of this family of FVIIa variants with increased proteolytic activity. Moreover, V158D/E296V/M298Q-FVIIa is far superior to the other variants, indicating that the three mutations work together in a concerted fashion to dramatically boost the proteolytic activity. The fact that FX itself binds Ca 2+ precludes direct studies of the influence of Ca 2+ on the proteolytic activity. An intriguing property of V158D/E296V/M298Q-FVIIa is the nonparallel increase in amidolytic and proteolytic activity compared with wild-type FVIIa. Such a behaviour, but less pronounced, is also observed for M298Q- and V158D/M298Q-FVIIa. This demonstrates that the three mutations together, and to some extent the M298Q mutation alone, somehow result in an additional facilitation of macromolecular substrate processing on top of the activity increase detected with a low-molecular-mass, chromogenic substrate. The V158D and E296V mutations need to be present simultaneously to achieve a proteolytic activity higher than that observed with M298Q-FVIIa. This indicates that local net charge and charge distribution are critical, presumably in order to allow for a stable local conformation to involve in an exosite interaction with FX. A recent study has clearly shown that the charge on residue 158 is pivotal for enhanced FX activation [19]. The relative rate of hydrolysis of peptides of various lengths (from Fig. 1. Activation pocket region of FVIIa. (A) The carbon atoms of the N-terminal residues 153{16} to 158{21} are shown in green, those of b strand B2 in the active FVIIa conformation (residues 296{154} to 305{163}) in gray and in the zymogen or inactive conformation (residues 296{154} to 302{160}) in magenta, and the Ca 2+ binding loop (residues 210{70} to 220{80}) is shown as a gray ribbon with the Ca 2+ ion represented by a magenta sphere. The residues in position 158{21} and in positions 296{154} and 298{156} in the active B2 conformation are those found in V158D/ E296V/M298Q-FVIIa. The water molecule interacting with the backbone carbonyl of Ile153{16}, the backbone amides of residues 155{18} and 156{19} and the side chain of Gln298{156} is shown as a red sphere. (B and C) Detail of the activation pocket in V158D/E296V/M298Q-FVIIa and wild-type FVIIa, respectively. The structure is from the FVIIa-TF complex (6, PDB entry code 1dan), except for the zymogen conformation of strand B2 which is from the structure of FVII (8, PDB entry code 1jbu). The drawings were made using QUANTA 2000 (Accelrys Inc.). Ó FEBS 2002 Dissection of a superactive FVIIa variant (Eur. J. Biochem. 269) 5953 P4-P1¢ to P4-P7¢ of FX) by V158D/E296V/M298Q-FVIIa as compared with that of wild-type FVIIa was constant, indicative of that the three mutations do not simply increase the accessibility of the substrate binding cleft to longer substrates (E. Persson, A. M. Hansen, K. Madsen and O. H. Olsen, unpublished observation). However, the peptides may not correctly mimic the corresponding sequences when part of FX. Finally, the high proteolytic activity of V158D/ E296V/M298Q-FVIIa appears to be accompanied by a stabilized salt bridge between Ile153{16} and Asp343{194}. Crystallographic data and molecular dynamics simula- tions of FVII suggest that the purpose of the activation to FVIIa is to maturate and open the substrate binding site, in particular the S1 pocket, whereas an appropriate catalytic triad geometry appears to be preformed in the zymogen [8,20]. However, even after conversion to FVIIa the conformational equilibrium appears to be shifted toward an enzymatically latent form. Thus, the role of TF, apart from localizing FVIIa to the site of vascular injury, optimally positioning the active site [21] and contributing to an extended, specificity-determining, factor IX/X binding surface [22–24], is to stabilize the active FVIIa conforma- tion. Strong evidence supports that Met306 in FVIIa is the starting point for the TF-mediated effect on the FVIIa conformation leading to allosteric stimulation of the enzymatic activity [6,15,25,26]. Recently, site-directed muta- genesis on FVIIa has been able to mimic the effect of TF binding, at least in part, resulting in FVIIa molecules with enhanced intrinsic activity [9–11,19]. Two published hypo- theses accommodate an instrumental role of the activation region in the regulation of the activity of free FVIIa, the first dealing with the structural requirements for FVIIa to be in an active conformation [8], whereas the other tries to explain the effects of the activity-enhancing mutations [9]. They are complementary and contribute to our understanding of how the activity-enhancing mutations in this region of FVIIa might exert their influence on the enzymatic activity. Replacement of Met298{156} by Gln could prevent relative b strand movement and stabilize strand B2 in a position compatible with an active FVIIa conformation (Fig. 1A). This is accomplished by introducing an extra hydrogen bond to the water molecule that interacts only with residues 155{18} and 156{19} in wild-type FVIIa (Fig. 1B,C). Substitution of Asp for Val at position 158{21} would not be expected to affect the activity of FVIIa unless Gln is simultaneously present at position 298{156} to allow the establishment of a hydrogen bond between the two introduced side chains that in turn stabilizes the inserted N-terminus (Fig. 1B). An effect of the V158D mutation becomes evident only after simultaneous replacement of Glu296 by Val, presumably due to electrostatic repulsion between Asp at position 158 and Glu at position 296. In addition, according to the structure of zymogen FVII [8], the change at position 296{154} (Glu to Val) eliminates hydrogen bonds between the Glu side chain and the backbone carbonyls of residues 158{21} and 159{22}. This would remove an obstacle for the formation of a salt bridge between the N-terminal residue 153{16} and Asp343{194} as well as for the b strand reregistration that appears to be required for FVIIa to attain its active conformation. Together, the three mutations result in a highly active FVIIa molecule that is more comfortable in the ÔactiveÕ b strand registration and with a buried N-terminus. Moreover, the mentioned b strand B2 and the preceding loop contain Glu296{154} and Arg290{147} which have been shown to be important for FX activation [25,27]. This might explain why an ordering of this region selectively increases the proteolytic activity more than the amidolytic activity. In accordance with this, displacement of this region by a peptide exosite inhibitor causes a larger effect on the proteolytic activity of FVIIa than on its amidolytic activity [5]. ACKNOWLEDGEMENT We thank Anette Østergaard and Helle Bak for excellent technical assistance. REFERENCES 1. 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(1994) Factor VIIa residue Arg 290 is required for efficient activation of the macromolecular substrate factor X. Biochemistry 33, 11631–11636. Ó FEBS 2002 Dissection of a superactive FVIIa variant (Eur. J. Biochem. 269) 5955 . on all these parameters. Keywords: factor VIIa variant; factor X activation; intrinsic activity; superactivity; zymogenicity. Coagulation factor VIIa (FVIIa),. Assignment of molecular properties of a superactive coagulation factor VIIa variant to individual amino acid changes Egon Persson 1 and Ole H.