ABB Archives of Biochemistry and Biophysics 457 (2007) 187–196 www.elsevier.com/locate/yabbi
Substrate specificity of recombinant dengue 2 virus NS2B-NS3 protease: Influence of natural and unnatural basic amino acids on hydrolysis of synthetic fluorescent substrates I.E. Gouvea a, M.A. Izidoro a, W.A.S. Judice a, M.H.S. Cezari a, G. Caliendo b, V. Santagada b, C.N.D. dos Santos c, M.H. Queiroz c, M.A. Juliano a, P.R. Young d, D.P. Fairlie e, L. Juliano a,* a
Departmento de Biofı´sica, Escola Paulista de Medicina, Universidade Federal de Sa˜o Paulo, Rua Treˆs de Maio, 100 - Sa˜o Paulo 04044-020, Brazil b Dipartimento di Chimica Farmaceutica e Tossicologica, Universita` di Napoli ‘‘Federico II’’, Via D. Montesano, 49-80131 Naples, Italy c Instituto de Biologia Molecular do Parana´, Rua Prof. Algacyr Munhoz Mader 3775, CIC Curitiba 81350-010, Brazil d School of Molecular & Microbial Sciences University of Queensland, St. Lucia, 4072 Brisbane, Australia e Institute for Molecular Bioscience, University of Queensland, St. Lucia, 4072 Brisbane, Australia Received 22 August 2006, and in revised form 30 October 2006 Available online 16 November 2006
Abstract A recombinant dengue 2 virus NS2B-NS3 protease (NS means non-structural virus protein) was compared with human furin for the capacity to process short peptide substrates corresponding to seven native substrate cleavage sites in the dengue viral polyprotein. Using fluorescence resonance energy transfer peptides to measure kinetics, the processing of these substrates was found to be selective for the Dengue protease. Substrates containing two or three basic amino acids (Arg or Lys) in tandem were found to be the best, with Abz– AKRRSQ–EDDnp being the most efficiently cleaved. The hydrolysis of dipeptide substrates Bz–X–Arg–MCA where X is a non-natural basic amino acid were also kinetically examined, the best substrates containing aliphatic basic amino acids. Our results indicated that proteolytic processing by dengue NS3 protease, tethered to its activating NS2B co-factor, was strongly inhibited by Ca2+ and kosmotropic salts of the Hofmeister’s series, and significantly influenced by substrate modifications between S4 and S06 . Incorporation of basic non-natural amino acids in short peptide substrates had significant but differential effects on Km and kcat, suggesting that further dissection of their influences on substrate affinity might enable the development of effective dengue protease inhibitors. 2006 Elsevier Inc. All rights reserved. Keywords: Protease; Peptides; Dengue
The flaviviruses comprise a group of about 70 positivestranded RNA viruses including the causative agents of Dengue, West Nile, Yellow fever, and Japanese Encephalitis infections. Dengue fever and dengue hemorrhagic fever (DHF)1/dengue shock syndrome (DSS) are caused by one of four serotypes of dengue virus (serotypes 1–4), with up to 100 million infections per year. Approximately 2.5 *
Corresponding author. Fax: +55 11 5575 90 40. E-mail address:
[email protected] (L. Juliano). 1 Abbreviations used: DHF, dengue hemorrhagic fever; DSS, dengue shock syndrome; NS, non-structural. 0003-9861/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2006.11.005
billion people are at risk globally, mostly in tropical and sub-tropical regions, reflecting the distribution of the insect vector. Classical dengue fever has been recorded for many centuries; however a marked increase in the more serious complication, DHF/DSS has occurred only over the last few decades. DHF/DSS is associated with significant mortality, particularly in the pediatric population [1–3] and no vaccine or therapeutic treatment is currently available. The dengue virus single positive sense RNA genome is approximately 11 kb and codes for a single polyprotein precursor. This precursor traverses the endoplasmic reticulum (ER) membrane multiple times, and is processed
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co- and post-translationally into three structural proteins, namely, C (core), prM (precursor to membrane), and E (envelope) and seven non-structural (NS) proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 [4]. These processing events are essential for virus replication and require the host proteases called signalase and furin [5,6] as well as a two-component viral protease, NS2B/NS3 [4]. The amino-terminal 180 amino acids of the multifunctional protein NS3 complexed with NS2B is responsible for cleavages on the cytoplasmic side of the ER membrane at the junctions between NS2A-NS2B, NS2B-NS3, NS3-NS4A, and NS4B-NS5 as well as at internal sites within C, NS2A, NS3, and NS4A. The host cell signalase mediates cleavages on the other face of the membrane, in the ER lumen. Later in virion maturation, prM is processed to M protein by a host protease called furin in a post-Golgi compartment to generate M [5,6]. Dengue NS3 protease activity does not depend on the presence of the complete sequence of NS2B co-factor (CF), but does need a central 40 residue relatively hydrophilic domain of NS2B (CF40) to be catalytically active [6–8]. The remaining hydrophobic residues of NS2B are thought to anchor the complex to cellular membranes. Although human hepatitis C virus (HCV) NS3 protease is activated by a shorter synthetic peptide co-factor [9], dengue NS3 protease is only active when co-expressed with the NS2B hydrophilic domain probably due to complementary folding of enzyme with co-factor [8,10]. Dengue 2 virus NS2B/NS3 is a trypsin-like serine protease with a catalytic triad composed of His51, Asp75, and Ser135 folded with two anti-parallel, six-stranded b-barrel symmetry [11]. The dengue NS2B/NS3 protease hydrolyzes its natural polyprotein substrate on the C-terminal side after a pair of basic residues (Lys–Arg, Arg–Arg, or Arg– Lys) or, occasionally, after Gln–Arg, interacting in the subsites S2 and S1 followed by small side chain amino acids (Gly, Ser, or Ala) in the subsite S01 [4]. The best recombinant construct so far described for expression of a soluble, active dengue protease that is resistant to autolysis was based on modeling of the dengue virus NS3 protease complexed with its co-factor [12]. The complex contains the catalytically active NS3 protease (NS3pro) connected to CF40 via a non-cleavable and flexible non-apeptide, Gly4-Ser-Gly4 linker. The fusion protein was designated CF40glyNS3pro, and this chimeric dengue 2 virus protease hydrolyzed synthetic chromogenic peptides containing the non-prime side residues of the cleavage site of dengue virus polyprotein [8]. In the present paper, we report the proteolytic activity of a recombinant dengue 2 virus (strain NGC) chimeric protease (CF40glyNS3pro) on fluorescence resonance energy transfer (FRET) peptides based on native viral polyprotein processing sites. In order to check whether amino acid substitutions between viral strains can affect differences in protease activity, we included the protease CF40glyNS3pro derived from a recent low passage DEN2 isolate from a Brazilian DF case (strain BR/01-EN). The same peptide substrates were also assayed against recombinant human
furin to check for specificity of the dengue polyprotein cleavage sites. As P3 to P1 positions of the cleavage sites in dengue polyproteins have basic amino acids, we explored in more detail the susceptibility to hydrolysis of FRET peptides containing two or three basic amino acids having most of the combinations of Arg and Lys in tandem sequence. Finally, in order to obtain further information for design of NS2-NS3 enzyme inhibitors, we explored the susceptibility to hydrolysis of even shorter fluorescent substrates of general structure Bz–X–Arg–MCA, where X was a nonnatural basic amino acid that combined a positively charged group with an aromatic or aliphatic substituent at the same side chain. Examples of X were 4-aminomethyl-phenylalanine (Amf), 4-guanidine phenylalanine (Gnf), 4-aminomethyl-N-isopropyl-phenylalanine (Iaf), 3-pyridyl-alanine (Pya), 4-piperidinyl-alanine (Ppa), 4-aminomethyl-cyclohexyl-alanine (Ama), and 4-aminocyclohexyl-alanine (Aca) (see structures in Fig. 1). This P2 position seemed suitable for the introduction of non-natural basic amino acids because of the observed restricted specificity of S1 subsite of NS2B-NS3 enzyme to Arg or Lys. Recently, after completing this work, the functional profiling of the proteases of the four dengue serotypes was reported using fluorescent peptide substrate libraries [13], and Niyomrattanakit et al. [14] reported an analysis of the substrate specificity of the NS3 of serotype 2 using internally quenched fluorescent peptides. However, our results using FRET peptide substrates comprising natural dengue virus polyprotein sequences as well as unnatural basic amino acids provide additional information about the specificity of NS2B-NS3 enzyme and further define the best composition of natural and non-natural basic amino acids at the non-prime side substrate residues. Materials and methods Enzymes Recombinant NS3 protease dengue 2 virus, strain NGC (CF40gly NS3pro), was obtained and purified as described previously [8]. Briefly the pQE9.CF40.gly.NS3pro (CF40 fused to NS3pro via a Gly4SerGly4 linker) vector was used for high level, inducible expression of amino-terminal hexahistidine-tagged recombinant enzyme. Cultures of Escherichia coli strain SG13009, transformed with the expression plasmid, were grown in 2 l of LB medium containing 100 lg ml1 ampicillin and 25 lg ml1 kanamycin at 37 C until the A600 nm reached 0.6. The cells were induced for expression by the addition of isopropyl-b-D-thiogalactopyranose to a final concentration of 1 mM and incubated for an additional 3 h at 30 C. The cells were harvested by centrifugation and resuspended in 1 ml of lysis buffer (50 mM Hepes, pH 7.5, 300 mM NaCl, 5% glycerol)/10 ml of original culture and subjected to probe sonication (five 30-s pulses) on ice and then centrifuged at 27,000g for 30 min at 4 C. The supernatant was purified by passage through a 2-ml column of Ni2+ nitrilotriacetic acid– agarose (Qiagen) pre-equilibrated with 50 mM Hepes, pH 7.5, containing 300 mM NaCl. The column was extensively washed with buffer containing 20 mM imidazole, and protein was then eluted from the column in buffer containing 100 mM imidazole. Elution fractions were analyzed by 15% SDS–PAGE. Samples of pre- and post-induced cells as well as soluble and insoluble fractions following lysis were collected and also analyzed by 15% SDS–PAGE. The hydrolytic activities of all samples were also checked on Z-RR-MCA in order to discriminate contamination activity of bacterial
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189
Fig. 1. Structure of the eight basic non-natural amino acids used in this paper. The pK values are indicated for each amino acid. proteases. The same procedure was applied to obtain NS3 protease of dengue 2 virus, strain BR/01-EN. The molar concentrations of these enzymes were determined by active site titration with aprotinin [8]. Recombinant human Furin (hfurin) was obtained, purified, and titrated as earlier described [15,16] and was kindly provided by Dr. Iris Lindberg from the Department of Biochemistry and Molecular Biology of Louisiana State University School of Medicine.
Peptide synthesis All the FRET peptides contained N-(2,4-dinitrophenyl)-ethylenediamine (EDDnp) attached to glutamine, a necessary result of the solid-phase peptide synthesis strategy employed which we detail elsewhere [17]. The peptide series Bz–X–Arg–MCA, and the non-natural basic amino acids X, were obtained as earlier reported [18]. An automated bench-top simultaneous multiple solid-phase peptide synthesizer (PSSM 8 system from Shimadzu) was used for the solid-phase synthesis of all the peptides by the Fmoc-procedure. The final de-protected peptides were purified by semipreparative HPLC using an Econosil C-18 column (10 lm, 22.5 · 250 mm) and a two-solvent system: (A) trifluoroacetic acid (TFA)/H2O (1:1000) and (B) TFA/acetonitrile (ACN)/H2O (1:900:100). The column was eluted at a
flow rate of 5 ml min1 with a 10 (or 30)–50 (or 60)% gradient of solvent B over 30 or 45 min. Analytical HPLC was performed using a binary HPLC system from Shimadzu with a SPD-10AV Shimadzu UV–vis detector and a Shimadzu RF-535 fluorescence detector, coupled to an Ultrasphere C-18 column (5 lm, 4.6 mm · 150 mm) which was eluted with solvent systems A1 (H3PO4/H2O, 1:1000) and B1 (ACN/H2O/H3PO4, 900:100:1) at a flow rate of 1.7 ml min1 and a 10–80% gradient of B1 over 15 min. The HPLC column eluates were monitored by their absorbance at 220 nm and by fluorescence emission at 420 nm, following excitation at 320 nm. The molecular weights and purities of all synthetic peptides were checked by MALDI-TOF mass spectrometry (TofSpec-E, Micromass) and/or peptide sequencing using a protein sequencer PPSQ-23 (Shimadzu, Tokyo, Japan). The concentrations of the solutions of the substrates were determined by colorimetric determination of the 2,4-dinitrophenyl group (extinction coefficient at 365 nm being 17.300 M1 cm1).
Hydrolysis of FRET peptides The hydrolysis of FRET peptides was quantified using a Hitachi F-2500 spectrofluorimeter by measuring the fluorescence at 420 nm following excitation at 320 nm. The inner-filter effect was corrected as
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previously described [19]. When Bz-RR-MCA peptide was used, the condition was changed to 460 and 380 nm, respectively. The recombinant dengue 2 proteases were incubated in 50 mM Tris buffer (pH 9.0, 10 mM NaCl, 20% glycerol) for 2 min at 37 C and the reaction started by addition of the substrate. Furin assays were performed in 100 mM MES buffer, pH 7.0 containing 1 mM CaCl2. The kinetic parameters kcat and Km were calculated by non-linear regression data analysis using the GraFit version 5.0 program (Erithacus Software, Horley, Surrey, UK). The peptide bonds cleaved in each substrate were identified by MALDI-TOF mass spectrometry analysis of the fragments after isolation by HPLC. Ki values were calculated by monitoring the hydrolysis of Z-RRMCA in the presence of inhibitory FRET peptides as described at [20].
Enzyme assays Dengue NS3 pH-profiles were obtained by measuring the kinetic parameters of hydrolysis, kcat, Km and kcat/Km over a pH range of 7.0–11.0 adjusted with 2 M NaOH and HCl. These determinations were carried out in a four-component buffer comprised of 25 mM acetic acid, 25 mM Mes, 75 mM Tris and 25 mM glycine. Enzymatic activity was measured at 37 C, using the fluorimetric assay described above and the data were fitted with the GraFit software to the follow equation: k cat =K m ¼
k cat =K m ðLimitÞ10pHpK a1 102pHpK a1 pK a2
where kcat/Km (limit) is the highest value of this kinetic parameter. The influences of salts (NaCl, Na2SO4, Na2CO3, NaOAc, Na2PO4 and CaCl2), glycerol and detergents were investigated in 50 mM Tris, pH 9.0, using Bz–RR–MCA as substrate. The initial velocity was measured at 37 C with an enzyme NS3NGC concentration of between 22.6 and 116 nM. All the kinetic data are results of at least three determination using different substrate concentrations and the standard errors were less than 5% for all determinations.
HPLC stopped kinetics Peptide concentrations ranging from 10 to 150 lM were treated with dengue NS3 protease at 37 C to achieve 10–15% cleavage. Reactions were stopped by the addition of trifluoroacetic acid to 0.33% and percent cleavage was measured by reverse phase-HPLC.
Docking of the best substrate to active site Docking of the optimized substrate Abz–AKRRSQ–EDDnp was carried out using GOLD v.2.1.2. The crystal structure of the NS3 protease was extracted from the PDB database (code 1DF9). The macromolecular docking of extremely flexible molecules such as peptides into protein active sites is problematic due to the inherent difficulty of sufficiently sampling the conformation space available to the ligands and also the comparatively short energy minimization routines commonly used in virtual screening/ docking packages. In order to overcome the first of these limitations we utilized our knowledge of protease-substrate molecular recognition [21] to loosely constrain the substrates into an approximate extended b-strand. This enabled the side-chains to move and find their optimum location, whilst keeping the backbone in a biologically relevant conformation.
Results Hydrolysis of peptide substrates corresponding to native cleavage sites by dengue 2 virus proteases CF40glyNS3pro chimeric proteases from dengue 2 virus strains, NGC and BR/01-EN, tagged with six consecutive histidines were purified and characterized [8]. There are 10 amino acid differences in the protease encoding a region
of the genome between the two strains, all except one being conservative. They are in the following positions; (BR/01EN versus NGC) NS3proE20D, R28K, R61K, V77I, I114L, T119A, V139I, R141K, D168E, and Y179D. No differences were found in the 40 amino acid CF40 activating segment. Seven FRET peptides containing the amino acid sequences that span the reported cleavage sites of polyprotein processing by dengue 2 virus NS2B/NS3 of NGC strain were synthesized with Abz (ortho-aminobenzoic acid) and EDDnp (N-[2,4-dinitrophenyl)-ethylenediamine) at N- and C-terminal end of each peptide, respectively. The FRET peptides were based on the polyprotein cleavage sites: NS2A/NS2B, NS2B/NS3, NS3/NS4A, NS4B/ NS5, Cint, NS3int, and NS4Aint. Table 1 shows the sequences of these FRET peptides and the kinetic parameters for their hydrolysis by CF40glyNS3pro from NGC and BR/ 01-EN dengue 2 virus strains. Optimal conditions of buffer composition and pH were used for enzyme activity, as described for the hydrolysis of chromogenic peptidyl-pNa substrates [8] that characteristically has low salt concentration (see below the effects of salts in enzyme activity). All assayed peptides were cleaved only at one peptide bond always hydrolyzed after the last C-terminal basic amino acid even for peptides with four consecutive Arg residues. The sites of cleavage in the FRET peptides were the same as those in the polyprotein, indicating that the synthetic peptides containing Abz and EDDnp did not introduce restrictions or different interactions with the viral proteases. The kcat/Km values obtained for the hydrolysis of the seven FRET peptides by CF40glyNS3pro from NGC were approximately two times higher than those obtained from the BR/01-EN dengue 2 virus enzyme and the best and the worst substrates were the same for both enzymes. These results indicate that the structural differences between them do not affect their peptidase specificity but have some impact on catalytic efficiency. Peptides 5 and 6 were hydrolyzed with the highest kcat/ Km values. However, for the hydrolysis of peptide 6 the kcat parameter was the highest among the assayed peptides while for the hydrolysis of peptide 5 the Km value was the lowest. Interestingly, peptide 5 had four consecutive Arg residues, whereas peptide 6 is the only substrate in Table 1 with two Arg residues at prime sites. Although the recognition of basic residues at P2 and P1 positions by the dengue protease is considered the key specificity feature of such flavivirus enzymes [13,14,22–24], it is possible that an interdependence of the protease subsites when occupied by the substrate could justify the hydrolysis of peptide 2 and the resistance of peptide 7. In order to investigate if the Km values obtained were not limited by inner filter effects, HPLC stopped kinetic were performed with peptide 1, the highest Km measurement in fluorescence assays among natural cleaved sites. The value obtained (Km = 39.1 ± 5.7) is in agreement with the fluorescence value (Km = 30.0 ± 1.9)
4500 0.18
697 129 0.33 1.32
2.9 1.6 0.6 0.7 5.1 15.8 Resistant 0.8
kcat/Km (mM s)
5.7 3.4 1.2 1.5 10.2 21.0
1.5
30 8.8 17.7 27 3.9 13.8
26
RTSKKR SWPLNEQ EVKKQRflAGVLWDQ FAAGRKflSLTLNLQ TTSTRRflGTGNIGQ LNRRRRflTAGMIIQ SAAQRRflGRIGRNQ EPEKQRTPQDNQQ HRREKRflSVALQ
2A/2B 2B/3 3/4A 4B/5 Cint NS3int NS4Aint prM/M*
0.17 0.03 0.02 0.04 0.04 0.29 Resistant 0.04
kcat/Km (mM s) Km (lM) kcat (s )
1 2 3 4 5 6 7
fl
Hydrolysis conditions: the proteases were incubated in 50 mM Tris buffer (pH 9.0, 10 mM NaCl, 20% glycerol) for 2 min at 37 C and the reaction started by addition of the substrate. All the kinetic data are results of at least three determination using different substrate concentrations and the standard errors were less than 5% for all determinations.
38 0.44
0.02 Resistant Resistant Resistant 0.23 0.17 Resistant 0.81
kcat/Km (mM s)1 Km (lM) kcat (s1)
hFurin
1
NS3BR/01
1 1
NS3NGC Cleavage in polyprotein
Sequence Abz–peptidyl– EDDnp Peptide No.
Table 1 Kinetic parameters for the hydrolysis by CF40glyNS3pro and hfurin of synthetic FRET peptides substrates based on polyprotein cleavage sites by dengue virus NS2B/NS3 complex and host furin
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We also include in Table 1 the kinetic parameters for the hydrolysis of Abz-HRREKRSVALQ-EDDnp, corresponding to the recognized cleavage site of furin within the dengue prM protein. This FRET peptide was poorly hydrolyzed by CF40glyNS3pro, but it was very susceptible to recombinant human furin (hfurin). All the other peptides in this series (Table 1) were also assayed against hfurin, but only peptide 5 corresponding to the internal site of the core protein (Cint) was significantly hydrolyzed. The other FRET peptides assayed against hfurin were poorly hydrolyzed or resistant to hydrolysis. In order to investigate the contribution of each amino acid of the best substrate in its hydrolytic reaction with CF40glyNS3pro, we synthesized a series of peptides derived from the best substrate in Table 1, namely peptide 6, in which the amino acids were exchanged with those in the corresponding position of peptide 3 (the worst substrate in Table 1). The kinetic parameters for their hydrolysis are shown in Table 2. The biggest decrease in the kcat/Km value was obtained with peptide 10, resulting from a single change in peptide 6 at P1 of an Arg for a Lys. A significant increase in the kcat/Km value was observed with the hydrolysis of peptide 16 which corresponded to a substitution in peptide 6 at P06 of Asn by Leu. The biggest decreases in Km values were observed in peptides 9, 15, and 16, which resulted from the replacements of Gln/Gly at P3, Arg/Asn at P05 and Asn/Leu at P06 , respectively. The variations in the Km values are compensated by the kcat and in most of the cases the kcat/Km values presented in Table 2 do not show large variations, except for peptide 10. Confirming the preference of the S1 subsite of dengue virus protease for Arg instead of Lys, we observed a significant increase in the efficiency of hydrolysis of the peptide 3 homologue in which Lys was substituted by Arg (Abz–FAAGRRSLTLNLQ–EDDnp). The kinetic parameters for hydrolysis of this peptide were: kcat = 0.62 s1, Km = 12.5 lM and kcat/Km = 5.0 s1 mM1. Hydrolysis of FRET hexapeptide substrates containing two or three natural basic amino acids Table 3 shows the kinetic parameters for the hydrolysis of FRET hexapeptides that were designed to contain combinations of two or three basics amino acids in tandem arrangement flanked by small side chain amino acids (peptides 17–28 in Table 3). All the susceptible peptides were hydrolyzed at the carboxyl side of the last basic amino acid of the peptide sequence. The Km values of hydrolysis of this series of FRET peptides are in general significantly higher than those for hydrolysis of the 12 residue FRET peptides derived from the dengue virus polyprotein (Tables 1 and 2). The highest Km values were obtained with substrates containing a pair of Lys at positions P1 and P2 (see the peptides 18, 25, and 26) and the lowest Km values were observed with peptides containing the pair Arg–Lys (see the peptides 19 and 24). In contrast,
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Table 2 Kinetic parameters for the hydrolysis of FRET peptides derived from Abz–SAAQRRGRIGRNQ–EDDnp (peptide 6) by CF40glyNS3pro Peptide No.
3
Positions in the substrates (P6–P01 ) P6
P5
P4
P3
P2
P1
F
A
A
G
R
K
P01 S
Modifications of peptide 6 with the amino acids of peptide 3 6 S A A Q R R G 8 F 9 G 10 K 11 S 12 13 14 15 16
kcat (s1)
Km (lM)
kcat/Km (mM s)1
P02
P03
P04
P05
P06
P07
L
T
L
N
L
Q
0.02
17.7
1.1
R
I
G
R
N
Q
0.29 0.14 0.09 0.04 0.14 0.11 0.34 0.18 0.11 0.16
13.8 7.4 4.0 9.8 5.9 7.2 16.5 6.9 4.6 4.5
21.1 19.4 22.0 4.6 23.5 15.5 20.9 26.5 23.1 35.9
L T L N L
Hydrolysis conditions and the errors of the kinetic parameter determinations were similar as described in Table 1. The standard errors were less than 5% for all determinations.
Table 3 Kinetic parameters for hydrolysis by CF40glyNS3pro of model FRET peptides containing two or three basic amino acids Peptide No.
Sequence Abz–peptidyl–EDDnp
kcat (s1)
17 18 19 20 21 22 23 24 25 26 27 28 29 30
AGRRflSAQ AGKKflSAQ AGRKflSAQ AGKRflSAQ ARRRflSQ AKRRflSQ ARKRflSQ ARRKflSQ ARKKflSQ AKKKflSQ AKRKflSQ AKKRflSQ AGRRPAQ AKRRRPQ Z–RRfl–MCA
0.21 0.02 0.13 0.20 0.38 1.0 0.07 0.09 0.05 0.04 0.04 0.07 Resistant (Ki = 11.8 lM) Resistant (Ki = 15.5 lM) 0.11
Km (lM) 20.0 58.8 21.3 38.0 25.6 31.4 58.0 18.3 67.3 69.2 23.7 53.2
247
kcat/Km (mM s)1 10.5 0.3 6.1 5.3 14.8 31.8 1.2 4.9 0.7 0.6 1.7 1.3
0.5
The arrows indicate the cleavage sites, which were determined by MALDI-TOF mass spectrometry of all reaction mixture after previous desalting and by isolation of the product of hydrolysis and the composition determined by amino acid analysis. Hydrolysis conditions of the kinetic parameter determinations were similar as described in Table 1. The standard errors were less than 5% for all determinations.
the highest kcat values were obtained for the substrates with the pair Arg–Arg (see the peptides 21 and 22). The presence of a third basic amino acid at the P3 position significantly increased only the kcat value of hydrolysis for the peptides containing the Arg–Arg pair at P1 and P2. The best substrate derived from the combinations of the basic amino acids Lys and Arg was Abz-AKRRSQ-EDDnp (peptide 22), which was hydrolyzed with the highest kcat/Km value. In accordance with these results, a similar fluorescent substrate, Bz-nKRR-ACMC (n = norleucine and ACMC = 7-amino-3-carbamoylmethyl-4-methyl coumarin), was described as the best substrate for all four dengue serotype NS2B/NS3 proteases based on the analysis of tetrapeptide positional scanning synthetic combinatorial libraries [13]. It is noteworthy that CF40glyNS3pro does not cleave between two basic residues in all the peptides of Table 3, indicating that the enzyme does not accept basic amino acids
at the P01 position. In order to verify if basic residues in P01 impeded hydrolysis of the substrates, two peptides were synthesized containing Pro after the basic sequence of two and four basic residues (peptides 29 and 30). Both peptides were resistant to hydrolysis but they inhibited the enzyme with Ki values in the lM range (Table 3). Under the same conditions, the commonly used commercially available peptide, Z–RR– MCA, was hydrolyzed with a high Km value (Table 3). Hydrolysis of fluorescent peptide substrates containing basic non-natural amino acids The series of fluorescent peptides Bz–XR–MCA, where X is a basic non-natural amino acid, is shown in Fig. 1. They were assayed as substrates against CF40glyNS3pro and the kinetic parameters for their hydrolysis are shown in Table 4. The peptides containing 4-aminomethyl-phenylalanine (Amf—peptide 32) and 4-aminocyclohexyl-alanine
I.E. Gouvea et al. / Archives of Biochemistry and Biophysics 457 (2007) 187–196 Table 4 Kinetic parameters for hydrolysis of series of peptides derived from Bz–XR–MCA, in which X are Arg or non-natural basic amino acids (see the amino acid structures in Fig. 1) and Bz is benzoyl group X
No. 31 32 33 34 35 36 37 38
Arg Amf Gnf Iaf Pya Ama Ppa Aca
100
Relative Activity (%)
Substrates
CF40glyNS3pro kcat (s1)
Km (lM)
kcatt/Km (mM s)1
0.109 0.013 0.035 0.022 0.002 0.172 0.014 0.013
247 26 490 354 43 298 216 27
0.44 0.50 0.07 0.06 0.05 0.58 0.07 0.48
Hydrolysis conditions of the kinetic parameter determinations were similar as described in Table 1. The standard errors were less than 5% for all determinations.
(Aca—peptide 38) were hydrolyzed with the highest kcat/Km values in this series, primarily as a result of a low Km. The peptide 36 that has trans-4-aminomethylcyclohexyl-alanine (Ama) is hydrolyzed with the highest kcat value in the series. It is noteworthy that the peptide containing 3-pyridylalanine (Pya—peptide 35) was poorly hydrolyzed but the observed Kmvalue was similar to those obtained for peptides 32 and 38. Peptide 33 containing 4-guanidine-phenylalanine (Gnf ) is poorly hydrolyzed with a high Km value in comparison to the other peptides in the series although the side chain of Gnf has the guanidine function. Peptides 34 (Iaf-4-aminomethyl-N-isopropyl–phenylalanine) and 37 (Ppa—4-piperidinyl-alanine) were poorly hydrolyzed also with high Km values.
193
80 60 40 20 0 6
7
8
9
10
11
12
pH Fig. 2. pH dependency profile for Dengue NS3 protease activity. Parameters of hydrolysis, kcat, Km and the relationship kcat/Km were obtained for each pH in conditions described in Materials and methods. kcat/Km were plotted as percentage relative activities assigning the highest catalytic efficiency as 100%. Representative peptide substrates Abz-SAAQRRGRIGRNQ-EDDnp (peptide 6—d) and Abz-FAAGRKSLTLNLQEDDnp (peptide 3—s) are shown.
Table 5 pK values obtained from the pH-profile hydrolysis curves of synthetic FRET peptides substrates based on polyprotein cleavage sites by dengue NS2B/NS3 protease complex Peptide No.
Sequence Abz–peptidyl–EDDnp
pK1
pK2
1 2 3 4 5 6
RTSKKRflSWPLNEQ EVKKQRflAGVLWDQ FAAGRKflSLTLNLQ TTSTRRflGTGNIGQ LNRRRRflTAGMIIQ SAAQRRflGRIGRNQ Z-RR-MCA
8.6 ± 0.2 8.2 ± 0.2 8.3 ± 0.2 8.1 ± 0.1 8.3 ± 0.1 8.5 ± 0.2 8.5 ± 0.1
8.9 ± 0.2 9.4 ± 0.2 9.2 ± 0.2 9.9 ± 0.1 10.2 ± 0.1 10.4 ± 0.2 10.0 ± 0.1
Effects of pH and salts on the protease activity 100
Relative Activity (%)
We determined the pH-profile for the enzyme catalyzed hydrolysis of peptides 1–7 (Table 1) and of Z-RR-MCA. All presented a similar shape profile to those obtained for the hydrolysis of the substrates Abz-SAAQRRGRIGRNQ-EDDnp (peptide 6—NS3int) or Abz-FAAG RKSLTLNLQ-EDDnp (peptide 3—NS3/NS4A), as shown in Fig. 2. The pK1and pK2 for all of the obtained profiles are presented in Table 5. No difference was observed for the pK1 values, whereas the pK2 values of peptides 1–3 were approximately one pK unit lower that those of peptides 4–6. These two sets of peptides differ in their basic amino acid composition; peptides 1–3 have Lys while peptides 4–6 have only Arg. This difference in the pK2 values could be related to deprotonation of the e-NH2-group of the Lys side chain. The pKa of the free amino acid is 10, but in peptides 1–3 Lys is flanked by other basic residues with electrostatic repulsion potentially reducing its pK [24]. A pK2 value of 10 for the short peptide Z–RR–MCA pH-profile hydrolysis gives support to this interpretation. The anion-dependent activity of CF40glyNS3pro in the presence of citrate, sulfate, phosphate, acetate, and chloride in their sodium form are shown in Fig. 3. All the
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Salt (mM) Fig. 3. Dengue NS3 salt inhibition profile. Relative activities determined by measuring the initial velocity of hydrolysis of the peptide Z–RR–MCA by NS3 in the presence of NaOAc (h), NaCl (d), Na2SO4 (s), sodium citrate (,) and CaCl2 (j). The assay conditions were as described in Materials and methods.
anions inhibited the enzyme and, interestingly, the decrease in activity by salts followed the Hofmeister series: cit rate > SO2 4 > acetate Cl . CF40glyNS3pro was also strongly inhibited by CaCl2 to an extent similar to citrate.
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Fig. 4. Colour representations of the active site region of Dengue NS3 protease with the catalytic triad shown in pink. Enzyme subsites are labelled S5 –S02 .
Modeling of Abz–AKRRSQ–EDDnp interaction with NS3 subsites The substrate Abz–AKRRSQ–EDDnp (peptide 22) which presented the highest kcat/Km value was modeled into the Dengue 2 NS3 binding site occupying the S5–S02 subsites (Fig. 4).The substrate occupies a shallow solvent exposed active site, with the exception of the S1 pocket which is sufficiently deep to almost completely conceal the P1 arginine side-chain from solvent. The P2 arginine side-chain docks in an orientation directed towards Asn152 in the shallow S2 pocket. The P3 lysine side chain makes hydrophobic contacts through the b- and c-methylenes to Val155 before terminating in solvent. The S4–S5 region is hydrophobic. The P01 serine side-chain docks well into the small S1 0 pocket. The subsite pockets in the S3 0 –S6 0 region are not well defined, and because of this uncertainty, no constraints could be used in this region. Consequently, the substrate docked in multiple orientations with one example shown in Fig. 4 where the dinitrophenyl group is occupying a small pocket bounded by Ala99, Glu103, Phe130, Pro132, and Thr134. Discussion The hydrolytic activities of the proteases from dengue strains NGC and BR/01-EN on FRET peptides corresponding to endogenous cleavage sites of the viral polyprotein have similar specificity, which is expected due to their high degree of similarity, although NGC enzyme is a more efficient peptidase. The seven synthetic peptides (Table 1) had different distributions of basic residues, yet were selective for the dengue protease over hfurin, except for the sequence derived from the internal core segment (Cint). Although cleavages of the dengue polyprotein by NS2B/ NS3 and furin occur in different host cell compartments, the amino acid sequences of the cleavage sites are also selective for each of these enzymes. This result contrasts to the high hydrolytic activity of West Nile virus NS3 peptidase on furin substrates [10]. The kcat/Kmvalues associated with peptides 1–4 (Table 1) are 2–10 times higher than the corresponding reported
chromogenic peptidyl-pNa substrates containing the same peptide sequences [8]. These differences can be attributed essentially to lower Km values for the hydrolysis of FRET peptide substrates, and this is clearly related to the occupancy of both prime and non-prime enzyme subsites [8] by these substrates. The earlier reported dengue 2 protease construct NS2B–NS3pro [25] hydrolyzed dansyl-labeled fluorescent peptides corresponding to polyprotein cleavage sites [26] but with significantly lower kcat/Kmvalues than we obtained with the CF40glyNS3pro enzyme construct. The main differences are in Km values that are likely the result of differences in the structure of the dengue 2 protease recombinants, since the peptide sequences of the assayed substrates are very similar. The S1 subsite of CF40glyNS3pro has a restricted preference for basic residues as shown by the cleavage sites in the FRET peptides in Tables 1 and 2, which is in accord with the reported hydrolysis of peptides derived from dengue 2 polyprotein sequences [4,8,25] and also with the analysis of positional scanning synthetic combinatorial libraries [13]. The S2 subsite of the enzyme is less selective, but also has a marked preference for basic amino acids, although dengue NS2B/NS3 protease hydrolyses peptides with Gln in this position (peptide 2 of this study) or peptides containing Thr, as shown by synthetic combinatorial libraries [12,13]. However, the efficiency of hydrolysis of peptides containing basic or non-basic residues at the P2 position seems to depend on the nature of the amino acids in other positions, as demonstrated by the resistance to hydrolysis of Abz–EPEKQRTPQDNQQ–EDDnp (peptide 7 in Table 1) and the susceptibility to hydrolysis of Abz–EVKKQRAGVLWDQ–EDDnp (peptide 2 in Table 1). Peptide 5 was hydrolyzed with the lowest Km value and this can be related to the presence of four Arg in positions P1 to P4. This is in accordance with the low Ki values reported for inhibition of dengue virus NS2B/NS3 protease by synthetic peptides containing three consecutive basic residues, that were designed based on the products of hydrolysis of the dengue polyprotein substrate [27]. It is noteworthy that peptide 1 containing three basic residues at positions P1–P3 was hydrolyzed with the highest Km value in the series. This may simply be a result of the basic amino acid composition of substrates in their P1–P3positions, or could be due to the differences in prime site residues that influence the enzyme-substrate interaction. The role of amino acids in substrate prime site positions is observed by comparing peptides 2 and 7. These substrates have the same amino acids at positions P3–P2–P1, but peptide 2 is significantly hydrolyzed, whereas peptide 7 is resistant. We also explored the extended binding site of CF40glyNS3pro from P6 to P06 by substituting amino acids of the worst substrate (peptide 3) into corresponding positions in the best substrate found in Table 1 (peptide 6). Significant variations in the Km and kcat values were observed for the hydrolysis of the resulting peptides, but for the majority they compensated each other resulting in only
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small variations in kcat/Km. The only exception was the substitution of Arg by Lys at P1 which resulted in a 7-fold decrease in the kcat value, confirming the high preference of the enzyme for Arg in this position. Molecular models of substrate binding with the NS3 dengue protease, in the absence of the NS2B activating co-factor, suggested that the critical enzyme–substrate interactions were restricted to the four residues spanning P2 to P02 [11]. However, the data presented in Table 2 show that the substrate binding site in CF40glyNS3pro might also involve residues P6 and P06 . In accordance with this view, CF40glyNS3pro was reported to be more effective on the hydrolysis of the substrate Boc–GRR–MCA than on the hydrolysis of Na-benzoyl-L-Arg-p-nitroanilide (BAPNA) [7] that contains only a P1 side chain. The crystal structure of dengue virus NS3pro without the NS2B activator in complex with a Bowman–Birk inhibitor was reported [11] and a unique bifurcated S1 pocket detected. Several non-substrate inhibitors were synthesized containing biguanidine structures that potentially mimic this interaction [28], but they are poor inhibitors with Ki values higher than 40 lM. The substrate Abz-SAAQRRGRIGRNQ-EDDnp (peptide 6) and some of its analogues (peptides 9, 11, and 13–16) were hydrolyzed with kcat/Km 2–3 times higher that the best internally quenched peptides recently described [14]. This significant difference between the susceptibility of our substrates could be related to the nature of the quencher group used. Shiryaev et al. [14] used nitro-tyrosine, with is deprotonated at pH 9 and the reported specificity of dengue NS3 protease could have significant interference of the negatively charged side chain of nitro-tyrosine that was not observed with our EDDnp that has no charge at any pH. We explored in more detail the susceptibility to hydrolysis of FRET peptides containing two or three basic amino acids having most of the combinations of Arg and Lys in tandem sequence (Table 3). All the combinations of Arg and Lys in the substrates presented in Table 3 resulted in substrates hydrolyzed with Km values systematically higher than those observed with the hydrolysis of the peptides derived from the polyprotein. This further indicates that, in addition to interactions between basic residues of the substrates with enzyme subsites S1–S3, the affinities of substrates and possibly inhibitors require other interactions than in these positions, as already suggested above. All the peptides containing Lys in P1 resulted in substrates that hydrolyzed with lower kcat, however if the peptides with Lys in P1 had Arg at P2 they were hydrolyzed with the lowest Km (peptides 19, 24, and 27 in Table 3). The best combination of basic amino acid was obtained with Abz–AKRRSQ–EDDnp (peptide 22) that was hydrolyzed with the highest kcat value in this series. It is noteworthy that all of the substrates containing three basic residues were always hydrolyzed after the last C-terminal basic amino acid, indicating that Arg or Lys is not accepted at the S01 subsite. This view is supported by the resistance to hydrolysis of CF40glyNS3pro of the peptides Abz–AGRRPAQ–EDDnp and Abz–AKRRRPQ–
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EDDnp, and this occurs even with the latter peptide that contains the sequence KRR also present in the best substrate (peptide 22). The peptides Abz–AGRRPAQ–EDDnp and Abz–AKRRRPQ–EDDnp significantly inhibited the enzyme (Kivalues are shown in Table 3), therefore it is possible that the last Arg of these peptides fits into the S1 subsite of the enzyme but the Arg–Pro bond is resistant to hydrolysis. The reported cleavages of peptides containing Arg at the P01 site in the positional scanning synthetic combinatorial libraries [13] contrast with this observation. As the verification of the cleavage sites in this kind of library is not done, the cleavages in these two libraries probably occurred after the Arg–Arg and Arg–Lys pair of basic amino acids and not between them. The libraries designed to put Arg or Lys in the P01 position in fact put them at P1 position, and this occurs due to a limitation in the analysis of the hydrolysis of these libraries since the cleavage sites of their peptides cannot be checked. The peptide series Bz–X–Arg–MCA, in which X is a nonnatural basic amino acid with an amine or guanidine substituents bound to an aliphatic or aromatic group, were hydrolyzed by CF40glyNS3pro with kcat/Km values lower than for Bz–Arg–Arg–MCA (peptide 32). The exceptions were peptides containing a primary amine substituent in position 4 of the phenyl group (Amf in peptide 32) or aliphatic cyclohexyl group (Ama, peptide 36 and Aca, peptide 38). It is noteworthy from the results presented in Tables 1– 3 that the guanidine substituent of Arg fits better into the S2 subsite of CF40glyNS3pro than the primary amine of Lys. However, the inverse seems to occur when the basic substituent is attached to a aromatic or aliphatic ring. Another interesting result is the relatively low Km value for the hydrolysis of the peptide containing 3-pyridyl-alanine (Pya, peptide 35), in which the amino function is deprotonated at pH 9. All these observations are likely to be important for future design of dengue protease inhibitors. The pH-profiles of hydrolysis obtained with all FRET peptides of Table 1 present their optimum value around pH 9 which did not change even after modifying the salt composition of the buffers and by addition of different detergents (data not shown). Abz and EDDnp groups seem also not be involved in the determination of the optimum pH since Z–RR–MCA hydrolysis pH-profile was similar to those of the FRET peptides. The peptide acetyl–SAAQRRGRIGRN–amide was synthesized without the fluorescent donor–acceptor groups and its pHprofile hydrolysis was similar to all the assayed substrates (data not shown). The high activity of CF40glyNS3pro at alkaline pH can not be the physiological environment of the enzyme inside the host cell and the observed behavior of CF40glyNS3pro could not represent the real function of the native protease. It is possible that NS3 requires further interactions with an unknown activator. A similar situation was described for human kallikrein 6 that presented optimum activity at pH 9.0 but in the presence of glycosaminoglycan or kosmotropic salts, the pH shifted to 7.5 [29].
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The large inhibitory effect of Ca2+ on protease activity compared with the other salts is noteworthy. This result suggests that Ca2+ may interact with CF40glyNS3pro modifying its structure and impairing its peptidase activity. Ca2+ may, therefore, play a significant role in the control of virus replication in the host cell. The susceptibility of CF40glyNS3pro activity to kosmotropic salts such as citrate and sulfate is a further indication that the active conformation of this enzyme is likely to be sensitive to its physiological environment. The model of Abz-AKRRSQEDDnp docked with the NS3 protease is consistent with the kinetic parameters of hydrolysis of this peptide, which is the best substrate among all the examined peptides, although the enzyme we used contained the activating central 40 residue, relatively hydrophilic domain of NS2B (CF40) that is necessary for a high catalytic activity. In conclusion, CF40glyNS3pro may have an extended binding site and that the affinity of substrates and possibly inhibitors depends on a number of interactions that extend at least from positions P6 to P06 . One interpretation of this information is that the substrate extends outside of the catalytic site and may fold back onto, and interact with, residues on the exterior surface of the enzyme-co-factor construct. Finally, our results have also indicated that the incorporation of basic non-natural amino acids in short peptide sequences affected the affinity and catalytic efficiency. Further dissection of such effects may enable use of these and other unnatural amino acids to develop efficient inhibitors for this viral enzyme. Acknowledgment This work was supported by the Brazillian research agencies Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) and the National Health & Medical Research Council (NHMRC) and Australian Research Council (ARC) of Australia. We thank Martin Stoermer for performing the peptide docking study. References [1] W.J. McBride, H. Bielefeldt-Ohmann, Microbes Infect. 2 (2000) 1041–1050. [2] T. Srichaikul, S. Nimmannitya, Baillieres Best Pract. Res. Clin. Haematol. 13 (2000) 261–276. [3] J.R. Stephenson, Bull. World Health Organ. 83 (2005) 308–314. [4] T.J. Chambers, R.C. Weir, A. Grakoui, D.W. McCourt, J.F. Bazan, R.J. Fletterick, C.M. Rice, Proc. Natl. Acad. Sci. USA 87 (1990) 8898–8902.
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