The natural mutation by deletion of Lys9 in the thrombin A-chain affects the pKa value of catalytic residues, the overall enzyme’s stability and conformational transitions linked to Na+ binding Raimondo De Cristofaro1, Andrea Carotti2, Sepideh Akhavan3,*, Roberta Palla3, Flora Peyvandi3, Cosimo Altomare2 and Pier Mannuccio Mannucci3 Haemostasis Research Centre, Institute of Internal Medicine and Geriatrics, Catholic University School of Medicine, Rome, Italy Department of Pharmaceutical Chemistry, University of Bari, Italy Angelo Bianchi Bonomi Hemophilia and Thrombosis Center and Fondazione Luigi Villa, IRCCS Maggiore Hospital University of Milan, Italy Keywords allostery; molecular dynamics; pKa values; stability; thrombin Correspondence R De Cristofaro, Haemostasis Research Centre, Institute of Internal Medicine and Geriatrics, Catholic University School of Medicine, Largo F Vito 1, 00168 Rome, Italy Fax: +39 30 155 915 Tel: +39 30 154 438 E-mail: rdecristofaro@rm.unicatt.it C Altomare, Department of Pharmaceutical Chemistry, University of Bari, Via E Orabona 4, 70125 Bari, Italy Fax: +39 80 544 2230 Tel: +39 80 544 2781 E-mail: altomare@farmchim.uniba.it *Present address ´ INSERM E0348, Faculte Xavier Bichat, University Paris 7, France (Received September 2005, revised 12 October 2005, accepted November 2005) doi:10.1111/j.1742-4658.2005.05052.x The catalytic competence of the natural thrombin mutant with deletion of the Lys9 residue in the A-chain (DK9) was found to be severely impaired, most likely due to modification of the 60-loop conformation and catalytic triad geometry, as supported by long molecular dynamics (MD) simulations in explicit water solvent In this study, the pH dependence of the catalytic activity and binding of the low-molecular mass inhibitor N-a-(2naphthylsulfonyl-glycyl)-4-amidinophenylalanine-piperidine (a-NAPAP) to the wild-type (WT) and DK9 thrombin forms were investigated, along with their overall structural stabilities and conformational properties Two ionizable groups were found to similarly affect the activity of both thrombins The pKa value of the first ionizable group, assigned to the catalytic His57 residue, was found to be 7.5 and 6.9 in ligand-free DK9 and WT thrombin, respectively Urea-induced denaturation studies showed higher instability of the DK9 mutant compared with WT thrombin, and disulfide scrambling experiments proved weakening of the interchain interactions, causing faster release of the reduced A-chain in the mutant enzyme The sodium ion binding affinity was not significantly perturbed by Lys9 deletion, although the linked increase in intrinsic fluorescence was lower in the mutant Essential dynamics (ED) analysis highlighted different conformational properties of the two thrombins in agreement with the experimental conformational stability data Globally, these findings enhanced our understanding of the perturbations triggered by Lys9 deletion, which reduces the overall stability of the molecule, weakens the A–B interchain interactions, and allosterically perturbs the geometry and protonation state of catalytic residues of the enzyme Recently, a homozygous deletion mutation of one of the two contiguous Lys9 ⁄ Lys10 residues in the A-chain of a-thrombin (DK9) was identified in patients with severe prothrombin deficiency and hemorrhagic diathesis [1,2] Compared with the wild-type (WT) form, the specificity constants of hydrolysis by DK9 of the synthetic substrate d-Phe-Pip-Arg-pNA and fibrinopeptide A were found to be 18- and 60-fold lower, respectively Abbreviations a-NAPAP, N-a-(2-naphthylsulfonyl-glycyl)-4-amidinophenylalanine-piperidine; Bis-Tris, (2-hydroxyethyl)iminotris(hydroxymethyl)methane; CHES, 2-(N-cyclohexylamino)ethanesulfonic acid; ED, essential dynamics; DK9, Lys9 deleted mutant; MD, molecular dynamics; Pip, pipecolyl; pNA, para-nitroanilide; SAS, solvent-accessible surface; WT, wild-type FEBS Journal 273 (2006) 159–169 ª 2005 FEBS No claim to original US government works 159 Functions of the thrombin A-chain R De Cristofaro et al Interaction with antithrombin was also reduced in the mutant, the association rate being % 20-fold lower than in WT thrombin Moreover, DK9 showed very weak platelet-activating capacity, whereas binding to the platelet glycoprotein Iba and thrombomodulin was unaffected At variance with these findings, inhibitors showed better binding to DK9 than to the WT form A long-term molecular dynamics (MD) simulation of DK9 thrombin in explicit water solvent supports the role of the A-chain in affecting the conformation and catalytic properties of the B-chain, particularly in some insertion loops of the enzyme, such as the 60-loop, as well as in the geometry of the catalytic triad residues Our MD analysis highlighted relevant modifications within the so-called ‘aryl-binding site’, in particular, expulsion ⁄ rearrangement of the W60d side chain (S2 site) and shifting of W215 (S3) Functional and computational data show that the catalytic cycle and efficient interaction with substrates and natural inhibitors by DK9 undergo a severe impairment, likely due to propagation to the active site residues of structural and conformational perturbations caused by Lys9 deletion in the A-chain [2] These findings prompted us to further investigate the pH dependence of the catalytic activity and stability of the DK9 mutant in comparison with WT thrombin, using experimental techniques in conjunction with computational approaches to prove the effects of the K9 deletion on the ionization of catalytic residues and the overall stability of the enzyme This investigation contributes to the unraveling of the mechanisms responsible for both the impaired catalytic activity of the K9-deleted natural mutant of thrombin in vitro and its hemorrhagic phenotype in vivo Results and Discussion Effect of pH on thrombin catalytic activity and inhibition The pH-dependent steady-state amidase activities of WT and K9-deleted mutant thrombins were studied in the pH range 5.5–10, using previously reported experimental and theoretical approaches [3,4] Kinetic schemes and equations (Eqns 2–4), allowing the effects of pH on the Michaelis–Menten parameters kcat and Km to be assessed, are reported in Experimental Procedures Although protons globally affected the amidase activity of both WT and DK9 thrombin forms in a similar way (Fig 1A–C), the pKa values of the ionizable groups involved in the catalytic cycle were found to be significantly different As reported in Table 1, we found an appreciable increase in the pKa value of the first ionizable group, which in the free enzyme showed a pKa value of 7.53 in the DK9 mutant and 6.86 in WT thrombin Because previous studies have assigned this group to the active site His57 side chain [3–5], this finding suggests that Lys9 deletion allosterically affects the protonation equilibrium of the active site His57, enhancing its affinity for protons both in free and substrate-bound forms of the enzymes The second pKa value was assigned to the N-terminal group of Ile16 (NTIle), which holds Asp194 in a salt bridge as a result of zymogen activation [3,5,6] The NTIle pKa Fig Analysis of pH dependence of Michaelis–Menten constants of D-Phe-PipArg-pNA hydrolysis (A–C) by WT (d) and DK9 thrombin (s), along with the Ki values of NAPAP binding (D) at 25 °C and 0.15 M NaCl Continuous lines were drawn according to the best-fit parameters values of Eqns (2–4) and listed in Table The vertical bars are the standard errors of the determinations 160 FEBS Journal 273 (2006) 159–169 ª 2005 FEBS No claim to original US government works R De Cristofaro et al Functions of the thrombin A-chain Table Best-fit pKa values of the ionizable groups of both WT and DK9 mutant thrombins (A), along with the best-fit kinetic parameters contained in Eqns (2–4) involved in the hydrolysis of the synthetic substrate D-Phe-Pip-Arg-pNA (B) at 25 °C in the presence of 0.15 M NaCl The best-fit pKa values of the ionizable groups of both WT and DK9 mutant thrombins, calculated using Eqn (2), are reported in C (A) Enzymes Group Group Free kcat s)1 Free Bound 6.86 ± 0.06 7.53 ± 0.12 WT DK9 Bound 6.30 ± 0.06 7.13 ± 0.09 8.45 ± 0.06 8.87 ± 0.09 9.04 ± 0.05 9.36 ± 0.12 kcat s)1 (B) Enzymes WT DK9 31.9 ± 1.02 ± 0.4 76.3 ± 3.82 ± 0.3 (C) Enzymes kcat s)1 32 ± 2.01 ± 0.2 °Km lM r0 · 106 (M)1Ỉs)1) r1 · 106 (M)1Ỉs)1) r2 · 106 (M)1Ỉs)1) 6.6 ± 0.3 3.6 ± 0.3 ± 0.6 0.31 ± 0.04 42 ± 3.1 ± 0.3 5.3 ± 0.6 0.69 ± 0.07 Group Group Free a Ki° ¼ 3.9 ± 0.10 nM b Free Bound 6.97 ± 0.05 7.53 ± 0.05 WTa DK9b Bound 6.52 ± 0.05 6.59 ± 0.05 8.63 ± 0.05 8.99 ± 0.06 9.00 ± 0.05 9.85 ± 0.07 Ki° ¼ 2.7 ± 0.13 nM value in the mutant thrombin undergoes a moderate increase from 8.45 to 8.87 in the free enzyme and from 9.04 to 9.36 in the substrate-bound form, for WT thrombin and DK9 mutant, respectively Table 1B reports changes in the kcat and kcat ⁄ Km values at the three protonation levels of WT and DK9 thrombins The kcat and kcat ⁄ Km values show a drastic decrease, mostly due to a net fall in the kcat value, which expresses the acylation rate By contrast, the decrease in the DK9 Km value is consistent with better accommodation of the substrate into the catalytic pocket of the unprotonated form of the mutant, as shown recently [2] Interestingly, the log kcat ⁄ Km values pertaining to WT and DK9 forms with protonated His57 (r1 of Eqn 4) in the presence of 0.15 m NaCl were inversely related to the respective His57 pKa values Based on a Brønsted mechanism [7], this observed relation is consistent with a transition state for breakdown of the tetrahedral intermediate involving partial carbon–nitrogen (C–N) bond cleavage, which is stabilized by H-bonding to the His57 imidazolium Ne nitrogen (see Experimental procedures Scheme 2) The imidazolium form would act as a general acid to facilitate amine expulsion (kA) from the tetrahedral intermediate (TI) Such a conclusion is in agreement with recent findings obtained with proton inventory studies of a-thrombin-catalyzed hydrolysis of amide substrates [6] Thus, in human thrombin the ratelimiting step in the acylation reaction is the breakdown of tetrahedral intermediate, being the different affinity for protons of His57 imidazole Ne nitrogen inversely related to the specificity constant of the amidase activity of the two thrombin forms According to our recent report, DK9 compared with WT thrombin achieves a better interaction with the low-molecular mass inhibitor N-a-(2-naphthylsulfonylglycyl)-4-amidinophenylalanine-piperidine (a-NAPAP) [2,8] As shown in Fig 1D, the pH dependence of a-NAPAP binding was characterized by a bell-shaped curve The best-fit pKa values calculated from this data set (Table 1C) were very close to those calculated from the pH-dependent enzyme activity profiles (Table 1A) The pKa values of the DK9 residues obtained in the two data sets showed almost the same experimental error This may be because the synthetic substrate used in the experiments is not a ‘sticky’ substrate for DK9 thrombin, as shown recently [2] In fact, according to the known following relation [3,5] pKðobsÞ ẳ pK log1 ỵ k2 =k1 ị 1ị in case of the interaction between thrombin and a < nonsticky substrate, where k2 < k1, pK(obs) should be equal to the true pK value, as found experimentally Slightly higher pKa values were obtained in a-NAPAP than in the substrate data set for WT thrombin (6.97 vs 6.86 and 8.63 vs 8.45, for the first and second ionizable group, respectively) This is likely because d-Phe-Pip-Arg-pNA is a ‘sticky’ substrate for WT thrombin [2] and thus, according to Eqn (1), the observed pKa value from steady-state kinetic experiments should be lower than the true pKa value by a factor equal to log(1 ) k2 ⁄ k1), as seen experimentally In analogy with the values calculated in enzymatic experiments, an increase of % 0.5 pK units of the His57 in DK9 mutant was also calculated analyzing the NAPAP data set (Table 1C) In fact, His57 undergoes FEBS Journal 273 (2006) 159–169 ª 2005 FEBS No claim to original US government works 161 Functions of the thrombin A-chain R De Cristofaro et al a decrease in pKa value upon NAPAP binding in the reversible complex formation, both in WT and DK9 thrombins The change in pKa value of the catalytic His residue in the mutant thrombin implies that the deletion of Lys9 allosterically affects the conformational state of relevant domains of the catalytic B-chain Thus, in an attempt to unravel the mechanisms responsible for the observed effects, we investigated the sodium-binding properties and conformational stability of the mutant enzyme in comparison with the WT thrombin form Binding of Na+ to thrombin Sodium ion binding to WT thrombin was characterized by a saturable increase in fluorescence at 342 nm (Fig 2) The apparent equilibrium dissociation constant of Na+ binding was calculated using a single site binding equation and was 22.0 ± 1.5 and 24 ± 2.6 mm for WT and DK9 thrombins, respectively, in reasonable agreement with previous results [9] However, the intrinsic fluorescence of the mutant was % 20% lower than that pertaining to WT thrombin and the magnitudes of the fluorescence change differed significantly, being equal to approximately +18 and +9% for WT and DK9 mutant thrombin, respectively These results suggest that the Na+-binding loop in the K9-deleted mutant retains the intrinsic affinity for the cation, although the conformational transitions linked to its binding are of more limited extension Fig Titration by steady-state fluorescence of Na+ binding to 75 nM WT (d) and DK9 thrombin (s) Na+ binding was investigated at 25 °C at ionic strength of 0.2, pH 8.00 Excitation wavelength was 280 nm Continuous lines were drawn according to single-site binding isotherms with best-fit Kd values of 22 ± 1.5 and 24 ± 2.6 mM for WT and DK9 thrombin, respectively The vertical bars are the standard errors of the measurements 162 compared with those of the WT form In other words, in the mutant thrombin the binding of sodium is not intrinsically perturbed, but should be uncoupled from the specific conformational transitions occurring in the WT form [9] This implies that the conformational transitions induced in the B-chain by Lys9 deletion are unique, being different from that of either the ‘fast’ or ‘slow’ form of WT thrombin [10] Essential dynamics (ED) analysis [11] was applied to key regions of WT and DK9 thrombin forms, with the aim of identifying motions relevant for their folding, separating them from those describing irrelevant local fluctuations ED analysis has proven to be a valid method allowing the correlation between motions of different parts of the protein to be assessed, overcoming possible artifacts which could derive from a simple rmsd analysis As shown in Fig 3, the A-chain, as well as the Na+-binding site, proved more flexible in WT than in the K9-deleted mutant The Na+-binding site undergoes a significant conformational transition after % 10 ns in DK9 thrombin, whereas this phenomenon occurs after % ns in WT thrombin The motions of the Na+-binding site were found to be correlated with those of regions of particular importance, such as the S3 specificity site (Trp215–Ile174), the Trp60 insertion loop, the Cys168–Cys182 disulfide bond, and the fibrinogen secondary binding exosite, all occurring after % 10 ns (results not shown) A previous X-ray diffraction study demonstrated that the Cys168– Cys182 disulfide bond undergoes a re-registration upon sodium binding [10] In particular, the distance between the sulfur atom of Cys182 and the Cb of ˚ Tyr225 was reduced by % A in WT thrombin by + Na binding and mediated the conformational transitions in the catalytic pocket of the enzyme responsible for the enhanced catalytic activity [10] The results of our calculations were in qualitative agreement with this behavior of the WT form, whereas the reduction of that distance in DK9 thrombin upon Na+ binding, along the whole productive MD, was found about half of that of WT (data not shown), highlighting reduced conformational mobility of the Cys168–Cys182 disulfide bond linked to Na+ binding It had been shown by others that there is an inverse relation between fluorescence intensity and exposure to solvent in a number of Trp residues (60d, 96, 148, 207, and 215) [12] In particular, Trp207 and Trp29 residues, located at the boundary between the A- and B-chains and interacting with Arg137 via three water structural molecules having low mobility (w321, w325, and w454) [12], have been shown to contribute about 35 and 9%, respectively, to the total intrinsic fluorescence of WT thrombin [12] In an attempt to understand the FEBS Journal 273 (2006) 159–169 ª 2005 FEBS No claim to original US government works R De Cristofaro et al Functions of the thrombin A-chain A B Fig Essential dynamics analysis of (A) A-chain and (B) Na+-binding site of WT ( ) and DK9 thrombin (—) Motions along the first eigenvector of the selected protein regions in the time frame of the MD simulation are reported observed difference in the fluorescence properties of the WT and DK9 thrombins, we calculated the average solvent (water)-accessible surface areas (SAS) of the above residues along the whole MD simulations and found that Trp207, whose predominant contribution to the total fluorescence is well established [12], exposes its surface to solvent about three times more in the DK9 ˚ mutant (average SAS ¼ 24.9 A2) than in the WT form ˚ 2) A similar trend was observed (average SAS ¼ 9.3 A ˚ for Trp29 (average SAS ¼ 9.6 and 14.1 A2 in WT and DK9, respectively), whereas the other Trp residues (60d, 96, 148 and 215), contributing < 11% to the total fluorescence [12], vary in their average SASs by < 25% in DK9 compared with WT thrombin Furthermore, it is likely that as a consequence of Lys14 deletion, the hydrogen bond between Glu8 and Trp207 is lost, as our MD calculations proved, with a consequent decrease in tryptophan fluorescence, in agreement with the known inverse relationship between the intrinsic fluorescence of a molecule and its conformational mobility [13] These computational results suggest that perturbation in the polarity and ⁄ or flexibility of the environment of Trp207 and 29 may significantly affect the intrinsic fluorescence of the DK9 variant, the higher the surface exposure and flexibility of their side-chains to the solvent the smaller the intrinsic fluorescence of the enzyme The smaller gain in the intrinsic fluorescence of DK9 upon Na+ concentration (9 vs 18% in WT) may reflect not only the lower flexibility of the Na+-binding loop, but also a different conformational rearrangement of Trp215 (as indicated by the ED results), which contributes solely to the gain in fluorescence observed in the Na+-bound conformer of thrombin [10,14] Thrombin stability studies Urea at m concentration induced complete denaturation of both WT and DK9 thrombin (Fig 4) The denaturating process at pH 6.80 was monitored by the Fig Urea-induced denaturation of WT (d) and DK9 thrombin (s) Measurements were performed at 25 °C in 10 mM Bis ⁄ Tris, 0.15 M NaCl, pH 6.80 Continuous lines were drawn according to the bestfit EC50 values of urea-induced denaturation: 2.94 ± 0.05 and 2.49 ± 0.04 M for WT and DK9 thrombin, respectively FEBS Journal 273 (2006) 159–169 ª 2005 FEBS No claim to original US government works 163 Functions of the thrombin A-chain R De Cristofaro et al decrease in fluorescence of the intrinsic enzyme and was almost perfectly reversible, because a 10-fold dilution of the thrombin samples in the same buffer without urea resulted in recovery (after correction for the dilution factor) of the fluorescence in the absence of urea The process was highly cooperative for both thrombin species (slope factor % in both cases), and the concentration of urea inducing the 50% effect on the fluorescence signal, [urea]1 ⁄ 2, was determined as 2.94 ± 0.05 m for WT and 2.49 ± 0.04 m for DK9 thrombin These values suggested that, in the presence of a saturating Na+ concentration, the mutant species was less stable than WT thrombin, which in turn showed a behavior similar to that reported previously [15] The higher sensitivity to urea denaturation shown by the mutant may explain why DK9 thrombin is clinically characterized by a much lower phenotypic expression in vivo, likely as a consequence of intracellular precipitation or enhanced degradation [2] The stability of the two thrombin variants was further investigated using a more complex denaturation procedure, referred to as disulfide scrambling [16] One disulfide bond connects covalently the A- and B-chain (Cys1–Cys122) in the thrombin molecule, whereas the B-chain is stabilized by three intrachain disulfide bonds (Cys42–Cys58, Cys168–Cys182 and Cys191–Cys220) [17] In disulfide scrambling, urea breaks noncovalent interactions between the two chains, subsequently enhancing the susceptibility of the connecting disulfide bonds to the reductive action of b-mercaptoethanol However, the low reducing agent concentration allows the disulfide bonds to scramble and rearrange according to conformational changes induced by the denaturant This allowed us to assess, better than by the simple urea-induced denaturation, whether the deletion of the K9 residue in the A-chain could affect the conformation stability of the whole thrombin molecule, as a consequence of perturbed intra- and interchain bonds After 180 denaturation with m urea and 0.2 mm b-mercaptoethanol, the A-chain was released and the the native enzyme form disappeared for both WT and mutant thrombin (Fig 5) The kinetic rate constant of free A-chain release was 1.69 ± 0.03 · 10)2Ỉmin)1 in the WT form and 2.96 ± 0.05 · 10)2Ỉmin)1 in DK9 thrombin (Fig 6) The disappearance of the intact enzyme (A- + B-chains) was characterized by a first-order rate constant equal to 1.69 ± 0.06 and 3.01 ± 0.02 · 10)2Ỉmin)1 for WT and DK9 thrombins, respectively Furthermore, the lag time for the early appearance of the stable isomer ‘3’ shown in Fig is shorter in DK9 (20 min) than in the WT form (33 min) 164 Fig HPLC chromatograms of disulfide scrambling of WT and DK9 thrombin Disulfide scrambling was obtained under M urea and 0.2 mM b-mercaptoethanol at 0.5 (upper) and 180 (remaining chromatograms) The HPLC chromatogram pertaining to WT thrombin after 180 of treatment is reported in the middle of the figure, whereas the chromatogram of DK9 form is given at the bottom The primed numbers refer to the stable B-chain isomers of DK9 thrombin Characterization of stabilizing interactions between A- and B-chains In WT thrombin, the A-chain assumes an overall boomerang-like shape interacting with the B-chain surface opposite to the active site [17] Stabilization within the A-chain and between the A- and B-chains occurs mainly through salt bridges and H bonds involving charged side chains The A-chain is intramolecularly cross-linked by five side-chain electrostatic interactions grouped into three separate clusters (D1a–K9, K14a– D14–R4–E8 and E13–R14d) Besides the covalent disulfide connection between Cys1 and Cys122, seven salt bridges, grouped into five clusters (D1a–R206, E8–K202–E14c, D14–R137, K135–E14e–K186d and K14a–E23), interconnect A- with B-chain; almost 90% FEBS Journal 273 (2006) 159–169 ª 2005 FEBS No claim to original US government works R De Cristofaro et al Fig Kinetics of disulfide scrambling of WT and DK9 thrombin Kinetics of reduced A-chain release from WT (s) and DK9 thrombin (h) Continuous lines were drawn according to a single exponential equation with the best-fit first order rate constant equal to 1.69 ± 0.03 · 10)2Ỉmin)1 for the WT form, and 2.96 ± 0.05 · 10)2Ỉmin)1 for DK9 thrombin The kinetics of disappearance of the intact adduct of A with B chain for WT (d) and DK9 thrombin (n) is also shown The single exponential decay rate constant was equal to 1.70 ± 0.06 and 3.01 ± 0.02 · 10)2Ỉmin)1 for WT and DK9 thrombin, respectively The vertical bars are the standard errors of the experimental measurements of the total electrostatic energy of the A–B-chain interaction has been calculated to be due to these salt clusters [17] We calculated the total number of stabilizing A–B interchain electrostatic ⁄ H-bonding interactions along the whole MD simulations, by using gromacs routines (g_saltbr and g_hbond), and found that in the WT form they are % 10% more than in the DK9 mutant (25969 and 23471 in WT and DK9, respectively) These computational results suggest that in this case the higher the flexibility of the A-chain the higher the number of electrostatic ⁄ H-bonding contacts between the A- and B-chain in the WT thrombin, in agreement with slower release of the light chain under mild reducing conditions, as shown by the disulfide scrambling experiments Conclusions The results obtained in this study provide knowledge about the perturbations triggered by deleting the Lys9 residue in the A-chain of thrombin [1,2] Measurements of the pH dependence of both steady-state amidase activity and binding of the high-affinity inhibitor a-NAPAP showed pKa values of the catalytic His57 higher in DK9 mutant than in WT thrombin Application of the Brønsted theory on acid ⁄ base-catalyzed Functions of the thrombin A-chain reactions indicated that in the thrombin amidase cycle the His57 imidazolium form acts as a general acid to facilitate amine expulsion from the tetrahedral intermediate, and this process is the rate-limiting step for the overall acylation reaction The increased basicity of the His57 N (nitrogen in the DK9 mutant would oppose this function, resulting in a decrease in its catalytic competence, as shown experimentally by both in vitro and in vivo data) Based on disulfide scrambling denaturation experiments, we inferred that in the DK9 mutant a refolded A-chain should reduce the structural stability of the whole a-thrombin molecule, weakening A–B interchain contacts Previously reported MD simulations showed a transition of the A-chain from a boomerang-like shape (WT) to a handle-like shape (DK9) [2] Computational studies highlighted lower conformational flexibility in the A-chain resulting in fewer A–B interchain electrostatic ⁄ H-bonding contacts in the DK9 mutant These A-chain folding effects should be allosterically transmitted to the active site cleft: (a) altering the geometry and protonation state of the residues involved in catalysis and inhibitor binding, and (b) limiting the allosteric effects triggered by sodium binding X-ray structures of human thrombin show that the A-chain closely follows the contour of the catalytic B-chain, hinging the two interacting six-stranded barrel-like domains of the B-chain [17] A well-structured network of ionic and H-bond interactions stabilize the correct orientation of the two barrels in the catalytic B-chain Deletion of Lys9 may cause a re-registration of this ionic network In particular, in the WT forms Asp14 makes a very strong salt bridge with Arg137 In the DK9 mutant this salt bridge should be severely perturbed, because Asp14, as a consequence of Lys9 deletion, preferentially interacts with Lys202, which is electrostatically linked to Glu14C in WT thrombin [17] Destruction of the salt bridge Asp14–Arg137 could alter the environment in which two Trp residues, namely Trp207 (at vdW distance from Arg137) and Trp29, are located, modifying its polarity and inducing conformational changes in the two Trp side chains which would be in DK9 more exposed to the solvent, as our calculations on MD conformations proved It is known that the higher the conformational flexibility of a fluorescent molecule the lower its fluorescence quantum yield [13], and Trp207 predominantly contributes to the global fluorescence of thrombin [12] Taking these reports and our data into account, it is reasonable to hypothesize that even a subtle perturbation in the polarity and ⁄ or flexibility of the environment of the Trp residues could significantly affect the fluorescence of the DK9 thrombin Moreover, the hydropho- FEBS Journal 273 (2006) 159–169 ª 2005 FEBS No claim to original US government works 165 Functions of the thrombin A-chain R De Cristofaro et al bic cluster below Arg137, comprising the side chains of Phe181, Phe199, Phe227, and Tyr228, close to the active site, may be destabilized, with repercussions for the catalytic pocket, as shown in crystal structure studies [10] In particular, a perturbed conformational change of Trp215, linked to Na+ binding and likely responsible for the reduced fractional change in fluorescence in the Na+-bound DK9 conformer, may be another evidence that the conformational transition caused by Lys9 deletion in the A-chain are sensed by catalytic subsites of the mutant enzyme These findings lead us to propose that a significant uncoupling between Na+ binding and conformational changes sensed by the fluorescence change of Trp215 takes place in DK9 thrombin Globally taken, the experimental and computational studies reported herein provide mechanistic support to the phenomenological evidence that the A-chain would affect both the conformation and the catalytic activity of the thrombin B-chain, thus corroborating the belief of an extraordinary conformational plasticity of this enzyme Experimental procedures Site-directed mutagenesis and construction of expression vectors Site-directed mutagenesis, expression, activation and purification of WT and DK9 thrombin form were obtained as recently detailed [1,2] The active-site titration of thrombin forms, obtained by using p-nitro-phenyl guanidinobenzoate gave a concentration of 95 ± 5% with respect to that measured spectrophotometrically at 280 nm using E ẳ 1.83 mgặmL)1 SDS ⁄ PAGE showed a single band of ~ 36 kDa for all the thrombin forms Effect of pH on thrombin amidase activity and binding of the inhibitor a-NAPAP to the thrombin active site The effects of pH (5.5–10) on the hydrolysis of d-Phe-PipArg-pNA substrate by both WT and DK9 mutant thrombin and binding of the inhibitor a-NAPAP to the enzyme active site were analyzed The experiments were carried out in an appropriate triple buffer (25 mm Bis ⁄ Tris, 25 mm Tris, 50 mm CHES, 0.15 m NaCl, 0.1% PEG 6000) This buffer system allowed us to keep the ionic strength of the solution nearly constant over the entire pH range [18] Calculations performed with a program written in basic showed that using the triple-buffer system in presence of 0.15 m monovalent salts, the ionic strength falls to a value % 2.5% lower than that at extreme pH values The Michaelis–Menten 166 Scheme constants of d-Phe-Pip-Arg-pNA (Instrumentation Laboratory, Milan, Italy) hydrolysis, as well as the equilibrium dissociation constant of a-NAPAP (Sigma-Aldrich, St Louis, MO) binding to thrombin, were calculated as detailed previously [8] The kinetic scheme for the catalytic cycle of thrombin in the steady-state analysis is given in Scheme E, ES and EP are the free, Michaelis–Menten and acylated enzyme forms, whereas k1, k-1, k2 and k3 are the kinetic constants for substrate binding, dissociation, acylation and deacylation, respectively Within the overall acylation step of the canonical Scheme (k2), tetrahedral intermediate (TI) formation (kB) or breakdown (kA) may be rate limiting [7,19–21] (Scheme 2) Recently, a kinetic study showed that DK9 thrombin catalyses the hydrolysis of a synthetic amide substrate with < a k2 < k3 [2] Under these conditions, k2 % kcat Furthermore, for both WT and DK9 thrombin Km % Kd, that is the equilibrium dissociation constant of substrate’s binding to thrombin The effect of protons on this kinetic scheme was analyzed by an appropriate extension of Scheme 1, whereby binding and dissociation of protons were considered much more rapid than all binding and catalytic steps of the substrate [3,4] Thus the kinetic Scheme was expanded, assuming the existence of two ionizable thrombin groups involved in catalysis, as emerged by a best-fit minimization procedure of the experimental data taken over a 5.5–10 pH range Accordingly, Scheme was expanded as shown in Scheme In Scheme 1K and 2K are the equilibrium association constant for proton binding to the unprotonated and mono-protonated thrombin form, respectively, whereas the ‘s’ and ‘p’ subscript refer to ES and EP thrombin species, and ‘1’ and ‘2’ superscript refer to the mono- and diprotonated thrombin species, respectively Using the linkage scheme analysis detailed previously [3,4], the pH effects on the Michaelis–Menten parameters kcat, Km, and kcat ⁄ Km of WT and DK9 mutant thrombin hydrolysis of d-Phe-PipArg-pNA were analyzed as follows: obs Km ẳ 0Km ỵ KHị1 ỵ KHị ỵ K s Hị1 ỵ K s Hị 2ị where Km values were approximated to the equilibrium dissociation constant of the substrate binding to thrombin [2], Km is the asymptotic Km value in absence of protons, and K and 2K are the equilibrium association constant of proton (H) binding to the first and second ionizable group for free, and ES thrombin species (the latter denoted by the FEBS Journal 273 (2006) 159–169 ª 2005 FEBS No claim to original US government works R De Cristofaro et al Functions of the thrombin A-chain Scheme was studied in the presence of seven fixed NAPAP concentrations (1–64 nm) and were simultaneously analyzed using a simple scheme, whereby both binding of NAPAP and d-Phe-Pip-Arg-pNA to the thrombin active site were mutually exclusive [8] In the analysis of pH effects on NAPAP binding, the Km values were replaced in Eqn (2) by the Ki values, calculated by the above competitive scheme [8] Fitting of catalytic parameters was constrained to an internally consistent picture, such that, at the three protonation levels, values of kcat, Km, and kcat ⁄ Km must be closely related according to Eqns (2–4) Global and simultaneous analysis of the experimental data by grafit software allowed computation of the pKa values of the two groups both in the free and substrate-bound species of thrombin forms, along with the kinetic parameters’ values pertaining to the three protonated thrombin species The values of standard errors (± SD) were also obtained in the fitting procedure Denaturation by urea Scheme ‘s’ subscript), respectively Likewise, the observed kcat values were analyzed as a function of pH as follows [3,4]: obs kcat ẳ kcat ỵ kcat K s ỵ 2Ks ịH ỵ kcat K s K s ịH2 ỵ K s Hị1 ỵ K s Hị 3ị where 0kcat, 1kcat, and 2kcat refer to the kcat value pertaining to unprotonated, mono- and diprotonated thrombin form, respectively Hence, the pH dependence of the observed kcat ⁄ Km value (referred to as r) is: obs r ẳ ẵr0 ỵ r1 K Hị ỵ r2 K K à H2 ފ=Z ð4Þ where the superscript 0, 1, and refer to the unprotonated, mono- and diprotonated thrombin form, respectively, and Z ¼ (1 +1K H) (1 +2K H) In the data sets of NAPAP inhibition of d-Phe-Pip-ArgpNA hydrolysis at each pH value, the steady-state velocity of cleavage of seven substrate concentrations (0.5–32 lm) Urea-induced denaturation curves of both WT and DK9 thrombin were obtained in 10 mm Bis ⁄ Tris, 0.15 m NaCl, pH 6.80, by monitoring the fluorescence emission at 342 nm (excitation at 280 nm) in a Varian Eclipse spectrofluorometer (Leini, Italy) Spectra were taken with an excitation ⁄ emission slit of nm The results were expressed as the percentage of the measure fluorescence at any urea concentration compared with that obtained in the absence of the denaturating agent Denaturation by disulfide scrambling Denaturation of both WT and DK9 thrombin was performed by disulfide scrambling, that is the unfolding of thrombin by urea in the presence of low concentrations of the reducing agent b-mercaptoethanol, as reported previously [16] Briefly, both WT and DK9 thrombin (50 lgỈmL)1 in 0.1 m Bis ⁄ Tris buffer, pH 6.8) were treated with m urea in the presence of 0.2 mm b-mercaptoetha- FEBS Journal 273 (2006) 159–169 ª 2005 FEBS No claim to original US government works 167 Functions of the thrombin A-chain R De Cristofaro et al nol Denaturation was performed at 25 °C for h to allow the reaction to reach equilibrium To monitor the kinetics of denaturation, 50 lL of the samples were removed at different time intervals, mixed with an equal volume of 4% TFA, and analyzed by RP-HPLC, using a Bio-Rad C18 Hi-Pore RP-318, 250 · 4.6 mm column The eluant was composed of solvent A (0.1%) trifluoroacetic acid, and solvent B was acetonitrile ⁄ water (9 : v ⁄ v) containing 0.08% trifluoroacetic acid The gradient was 20–40% solvent B for 10 min, linearly increased from 40 to 55% for 40 min, kept at 55% for 10 min, and reduced to 20% solvent B in 20 min, while the flow rate was 0.5 mLỈmin)1 The HPLC instrument was a PU-2080 instrument connected to a UV-2075 spectrophotometer (Jasco Europe s.r.l., Cremella, Italy) The chromatographic peaks were analyzed and quantified using borwin-1 software (Jasco Europe) The results were expressed as a percentage with respect to the peak area measured at the start The kinetic data referring to the disappearance of the native enzyme, [A + B%], were fitted to the single exponential decay equation: ẵA ỵ B%t ẳ 100 expktị 5ị where [A + B%]t is the percentage of the native enzyme present at time t, and k is the first-order rate constant of this process The appearance of the free A-chain at time t, referred to as [A%]t, was fitted to single exponential relation: ẵA%t ẳ 100 expktịị Computational methods gromacs 3.2.1 software [22], running on a Linux PC cluster, was used for the MD simulations and analysis of the trajectories Using the gromacs utilities, the ED analysis was carried out in to separate the motions of the examined protein models into an essential subspace, describing most of the functional motions, and into a physically constrained subspace, describing irrelevant local fluctuations Eigenvectors defining the direction of higher displacement are extracted in decreasing order of the corresponding eigenvalue, and the first few eigenvectors capture most of the essential motions of the proteins The cross-correlation function between the projection of protein segments pairs onto the first eigenvector obtained from the ED analysis of both segments was also calculated to estimate correlation of the essential motions ð6Þ where k is the first-order rate constant of the A chain release At the end of the denaturation process three stable B chain isomers were produced, in agreement with previous studies, although obtained at different pH [16] The kinetic Eqns (5–6) were fitted to the experimental data using the grafit program (Erithacus Software Ltd, Staines, UK) Acknowledgements Financial support from Italian Ministry of Education, Universities and Research (MIUR) is gratefully acknowledged by CA and RDC (PRIN 2003, Grant no 2003064812) We thank Dr Vincenzo De Filippis (University of Padova, Italy) for helpful comments and critical reading of the manuscript References Binding of sodium ion Steady-state fluorescence titration measurements were carried out using 500 nm WT and mutant thrombin, as described previously [9] Fluorescence emission spectra (kex ¼ 280 nm) were recorded at 25 °C in a cm quartz cell, using a Varian Eclipse spectrofluorometer (Leini, Italy), in mmolỈL)1 Tris, pH 8.0 and increasing amount of NaCl Emission spectra between 300 and 400 nm did not show any significant peak shift Titrations were performed by acquiring the changes in fluorescence intensity at the peak of the emission spectrum at 342 nm The concentration of Na+ was increased by aspirating a defined volume of thrombin solution dissolved in the cuvette in the above buffer containing 0.2 m tetramethylammonium chloride and by adding the same volume of a thrombin solution (at the same concentration) dissolved in the same buffer but con- 168 taining 0.2 m NaCl This procedure allowed us to keep constant both the concentration of thrombin and the ionic strength of the solution The decrease in fluorescence signal, usually defined as ‘bleaching effect’, due to the iterative exposure of the sample to high intensity light beam, was restricted to < 3% of the initial intensity and was always taken into consideration in the analysis of the titration data Akhavan S, Mannucci PM, Lak M, Mancuso G, Mazzucconi MG, Rocino A, Jenkins PV & Perkins SJ (2000) Identification and three-dimensional structural analysis of nine novel mutations in patients with prothrombin deficiency Thromb Haemost 84, 989–997 De Cristofaro R, Akhavan S, Altomare C, Carotti A, Peyvandi F & Mannucci PM (2004) A natural prothrombin mutant reveals an unexpected influence of A-chain structure on the activity of human alphathrombin J Biol Chem 279, 13035–13043 Di Cera E, De Cristofaro R, Albright DJ & Fenton JW II (1991) Linkage between proton binding and amidase activity in human a-thrombin: effect of ions and temperature Biochemistry 30, 7913–7924 FEBS Journal 273 (2006) 159–169 ª 2005 FEBS No claim 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Transition States of Biochemical Processes (Gandour RD & Schowen RL, eds), pp 355–423 Plenum Press, New York 22 Lindahl E, Hess B & van der Spoel D (2001) Gromacs 3.0: a package for molecular simulation and trajectory analysis J Mol Mod 7, 306–317 FEBS Journal 273 (2006) 159–169 ª 2005 FEBS No claim to original US government works 169 ... a single band of ~ 36 kDa for all the thrombin forms Effect of pH on thrombin amidase activity and binding of the inhibitor a-NAPAP to the thrombin active site The effects of pH (5.5–10) on the. .. conformational transitions linked to its binding are of more limited extension Fig Titration by steady-state fluorescence of Na+ binding to 75 nM WT (d) and DK9 thrombin (s) Na+ binding was investigated... sodium binding [10] In particular, the distance between the sulfur atom of Cys182 and the Cb of ˚ Tyr225 was reduced by % A in WT thrombin by + Na binding and mediated the conformational transitions