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DSpace at VNU: Computational Characterization for Catalytic Activities of Human CD38''''s Wild Type, E226 and E146 Mutants...
Interdiscip Sci Comput Life Sci (2010) 2: 193–204 DOI: 10.1007/s12539-010-0091-0 Computational Characterization for Catalytic Activities of Human CD38’s Wild Type, E226 and E146 Mutants My H NGUYEN1 , Van U DANG1,2∗ , Boi V LUU1 (Faculty of Chemistry, Hanoi University of Natural Science, VNU, 19 Le Thanh Tong, Hanoi, Vietnam) (Hoa Binh University, CC2, My Dinh II, Tu Liem, Hanoi, Vietnam) Received 15 December / Revised 26 February 2010 / Accepted March 2010 Abstract: A series of the complexes of human CD38’s wild type, E226 and E146 mutants as well have been simulated The biosoftwares well simulate the penetration of nicotinamide-adenine-dinucleotide (NAD) into the active site The nicotinamide end of NAD penetrates deep into the active site consistent with cleavage of the nicotinamide-glycosidic bond which is the first step of catalysis creating a Michaelis complex regarded as the intermediate product of NAD cyclase and hydrolysis reaction The breaking down hydrogen bond between 2’-3’ OH ribosyl and the residues replaced Glu226 makes NAD to be less constrained in active site and nicotinamide (NA) becomes more difficult to be cleaved and eliminates the mutant catalytic activities The large majority of the substrate NAD is hydrolyzed to ADPR while the conversion of NAD to cADPR is not the dominant reaction catalyzed by wild-type human CD38 The more strongly kept ribosyl group by hydrogen bonds the more NADase and the less cyclase activity Breaking hydrogen bonds of ribosyl 2’- and 3’-OH by mutation will loosen it to promote the cyclase The cyclic adenosine diphosphate-ribose (cADPR) could also penetrate deeply into active site to make some hydrogen bonds with Glu146 and Glu226 ; however, its docking poses are affected by a residue located at the entrance of the catalytic pocket (Lys129 ) These results are in good agreement with the previous crystallographic analysis and the experiments quantified the catalytic activities of human CD38 and its mutants Key words: human CD38, mutant, nicotinamide-adenine-dinucleotide, cyclase, hydrolysis reaction Introduction A fundamental postulate in the classical drug design paradigm is that the effect of a drug in the human body is a consequence of the molecular recognition between a ligand (the drug) and a macromolecule (the target) The pharmacological activity of the ligand at its site of action is ultimately due to the spatial arrangement and electronic nature of its atoms, and the way these atoms interact with the biological counterpart (Bohm et al., 1996) Computational chemistry tools allow one to characterize the structure, dynamics, and energetic of the interactions between the ligand and a macromolecule as protein and DNA For instance, molecular mechanics (MM)-based approaches can efficiently assist the discovery of new drug candidates, and these computationally inexpensive methods are nowadays routinely used in drug design (Jorgensen, 2004) However, if a description of the electronic properties is deemed necessary, there is no substitute for quantum mechanics (QM) Indeed, since QM based ap∗ Corresponding author E-mail: hbuniv@gmail.com proaches also account for quantum electronic effects, they describe bonds forming/breaking, polarization effects, charge transfer, etc., and usually estimate molecular energies more accurately (Sherwood, 2000; Jorgensen et al., 1988; Brooks et al., 1983) QM methods are also fundamental to studying biological reactions, as quantum electronic effects must be taken into account to properly describe the phenomena of bonds forming/breaking An excellent overview of target-related applications of first principles quantum chemical methods in drug design is presented by Cavalli et al (2006) By various commercial and/or academic software’s combining QM and MM based tools not only the position and pose of ligands binding on protein but also the inhibition constant as IC50 could be predicted in a rather agreement with spectroscopy experiments where IC50=10E bind /5.85, Ebind is binding free energy between ligand and protein These softwares make an ability to predictive computations of complicated biochemical processes In this study, we employed computational method softwares, namely GLIDE (Schrodinger, 2000), QUANTUM 3.3 (Quantum Pharmaceuticals, 2007), WHAT IF (Gert Vriend et al., 2009) and HYPERCHEM 8.0 (Hypercube, 2007) 194 to simulate the structure and energy of the enzymatic domain of wild-type CD38 and its E226 and E146 mutants complexed with relevant ligands related to their multitude of catalytic activities The object ligand – CD38 complexes are introduced briefly Fortunately, there are series of experimental study on structure and activity of CD38 and its mutants are publicized and atomic coordinates and structure factors have been deposited at the Protein Data Bank (PDB) These data and a large amount of other ligand-protein complexes deposited in PDB have been used to assess the computational procedure The calculated results include the cADPR hydrolase, NADase (NAD glycohydrolase) – the intermediate Michaelis complex, the activation of the intermediate Michaelis complex and E146 mutants’ cyclase and hydrolysis activity Ligand-protein complexes CD38 was described first as an antigen that is involved in a host of lymphocyte functions including differentiation, proliferation, and apoptosis (Malavasi et al., 1994) Its expression has since been found to be widespread among nonhematopoietic tissues as well (Khoo and Chang, 1999) In addition to the antigenic functions, CD38 also possesses a multitude of enzymatic activities (Lee, 2006) It catalyzes not only the hydrolysis of NAD and cADPR to ADPR, but also the cyclization of NAD a long linear molecule, and its analog, NGD, to produce a compact cyclic nucleotide, cADPR and cGDPR, respectively CD38 has also baseexchange activity that is responsible for synthesizing NAADP from NADP The active site of human CD38 has been biochemically and structurally characterized (Munshi et al., 2000; Liu et al., 2005) Glu226 is identified as the catalytic residue, because its mutation to other residues essentially eliminates all its catalytic activities (Munshi et al., 2000) Ser193 is also important for catalysis as its mutation to alanine also greatly reduces enzyme activities (Liu et al., 2006) NAD will be conversed to cADPR; however is not the dominant reaction catalyzed by wild-type human CD38 In fact, the large majority of the substrate NAD is hydrolyzed to ADPR (Howard et al., 1993) Completely the opposite is observed when NGD, an analog of NAD, is used as a substrate The dominant reaction is now cyclization instead of hydrolysis, producing cGDPR as the major product (Graeff et al., 1994) Considering the similarity of NGD and NAD, which differ only in the purine rings, it is puzzling why the reactions are so different (Liu et al., 2007) Glu146 is a conserved residue present in the active site of CD38 Its replacement with phenylalanine greatly enhanced the cyclization activity to a level similar to that of the NAD hydrolysis activity A series of additional replacements was made at the Glu146 po- Interdiscip Sci Comput Life Sci (2010) 2: 193–204 sition including alanine (E146A), asparagine (E146N), glycine (E146G), aspartic acid (E146D), phenylalanine (E146F) and leucine (E146L) (Graeff et al., 2001) All the mutants exhibited enhanced cyclase activity to various degrees, whereas the hydrolysis activity was inhibited greatly E146A showed the highest cyclase activity, which was more than 3-fold higher than its hydrolysis activity All mutants also cyclized NGD to produce cGDPR This activity was enhanced likewise, with E146A showing more than 9-fold higher activity than the wild type In addition to NAD, CD38 also hydrolyzed cADPR effectively, and this activity was correspondingly depressed in the mutants When all the mutants were considered, the two cyclase activities and the two hydrolase activities were correlated linearly The Glu146 replacements, however, only minimally affected the base-exchange activity that is responsible for synthesizing NAADP (Graeff et al., 2001) Unfortunately, E146-mutant’s structure has not yet been deposited in Protein Data Bank Homology modeling was used to assess possible structural changes at the active site of E146A (Graeff et al., 2001) In this study, we employed the structures of the enzymatic domain of human CD38’s wild-type and its mutants complexed with the relevant ligands, that is NAD, ADPR, cADPR, NGD, GDPR, cGDPR, EPE, NMN and N1C by x-ray crystallography (Howard et al., 1993; Graeff et al., 1994; Munshi et al., 2000; Graeff et al., 2001; Liu et al., 2005; Liu et al., 2006; Liu et al., 2007) The mutants investigated computationally in this study are E146A, E146N, E146G, E146L, E146D, E146F and E146K, E146Q Two latters were obtained by replacing Glu146 by lysine (K) and glutamine (Q), respectively The complexes provided a step-by-step description of the catalytic processes involved in the synthesis and hydrolysis of cADPR The E226G – a mutant of CD38 received by replacement of Glu226 by glycine – complexed with NMN – a substrate of CD38 (code in PDB is 2hct), the complex of E226Q mutant of CD38 and cADPR (code 2o3q), the complex E226D-cADPR (code 2o3r), E226G-cADPR (code 2o3s) and other ligand-protein complexes as well deposited on Protein Data Bank have been used to verify the computational procedure Computational procedures The calculation procedure includes three core algorithms: (i) the replacement of each residue in active sites by other one then makes a geometrical optimization which simulates the site-directed mutagenesis technique; (ii) docking ligand on mutants to determine the docking poses; (iii) calculating the binding energy of the obtained complexes Fortunately, all three algorithms could be received on web in the form of source code, executive file and/or online calculation Depending on the Interdiscip Sci Comput Life Sci (2010) 2: 193–204 195 concrete algorithm the results received by these softwares may be different Though there are many articles presented the studies on the reliability of various bio –chemistry softwares applied to a large amount of proteins of different kinds and shown the ability of each software, this article pays attention to the software’s reliability applied to a narrow branch of proteins including the complexes between various ligands and CD38 and its mutants as well Relating to the computational characterization for the CD38’s multitude of catalytic activities, we should choose the softwares could give well prediction of the mutant structure based on native protein structure – the site-directed mutagenesis - and of the docking pose of ligand on active sites Mutant prediction – SWISS-PDB Viewer4.0 (Guex and Peitsch, 2008) can be very useful to quickly evaluate the putative effect of a mutation before actually doing the simulation work CUPSAT (Parthiban et al., 2006) gives also ability to predict the stability of the mutant WHAT IF gives on-line prediction of mutant structure and comparison of a model to a resolved structure as well (Gert Vriend et al., 2009) We have also used HYPERCHEM 8.0 (Hypercube, 2007) to make a single residue replacement and optimize the mutant geometry Table presents the comparing results of some CD38’s mutant models obtained by WHAT IF, SWISSPDB Viewer and HYPERCHEM to the solved structure deposited in PDB of the relative mutant All mutant atom structure data are of in the form of mutant-ligand complex with various ligands Therefore, the ligands have been discarded in comparing calculation The RMS on all atoms in all cases is about ˚ A In detail, WHAT IF gives rather smaller RMS and LD than Table RMS on all atoms of some mutant predicted models from solved structure deposited in Protein Data Banka CUPSAT PDBb Mutant ΔΔG∗ Torsion favor E226D 1.58 – 2o3r E226G 1.64 – 2o3s E226Q a All SWISS-PDB and HYPERCHEM Taking into account that with the exception of the mutated residue, energy minimization procedure locked all rest residues and that all residues of the predicted mutant are involved in RMS calculation, the mutant predicted structures are in a very good agreement with the experiment data Using HYPERCHEM and SWISS-PDBViewer softwares we have also involved all residues in energy minimization procedures However, after very long CPU time the obtained structure is not in a better agreement with deposited structure than the previous predicted structure involving only one mutated residue in minimization procedure It could be explained that, our calculations take into account the mutants created by single site directed mutagenesis technique at active sites only Comparing the coordinate deposited in Protein Data Bank of CD38’s wild type and its mutants at single active sites gives RMSD of ∼1 ˚ A It means that the single residue replacement at active sites not affect obviously to the structure of protein with the exception of mutated residue The space made by active sites is large enough in order to hold substitute residues being stable without large torsion unfavorable of the ligand and keep the other residues’ position unchanged approximately CUPSAT’s predicted stability data is also presented in Table However, E226Q should be taken into account in software development for the long chain and flexible residues The conformation of mutation Gln226 received both by WHAT IF and SWISSPDB differentiates essentially with the crystallographic one (Fig 1(a)), while the mutation residues of a shorter chain as Asp (Fig 1(b)) and Gly (Fig 1(c)) not show an obvious difference 0.83 + RMS2++ RMS1+ WHAT IF HYPER SWISS-PDB 0.955 0.953 0.968 0.971 0.955 0.955 1.176 1.058 2hct 1.017 1.011 1.016 1.016 2o3q 0.934 0.934 1.028 0.966 2o3t 0.998 0.991 1.134 1.079 2o3u 1.023 1.023 1.258 1.186 2pgl 1.056 1.049 1.219 1.214 RMS calculation were done on WHAT IF; b Deposited in Protein Data Bank discarded ligands; ∗ Predicted stability (kcal/mol); with CD38 wild-type; ++ Comparing with solved structure in PDB + Comparing Docking – The ligand docking on CD38 and its mutants is predicted on QUANTUM 3.3 and GLIDE as well There are little differences between two softwares in the preparation of the proteins and ligands In both cases we applied the rigid protein model and follow the docking calculation procedure to receive model structure of the ligand-protein complex The mutant – ligand complexes as: 2hct, 2o3q, 2o3r and 2o3s, 2o3t, 196 Fig Interdiscip Sci Comput Life Sci (2010) 2: 193–204 Illustration of the mutant prediction in active site (a) Mutate prediction of E226Q (sticks) and the crystallographic data of 2i65, 2pgl, 2o3q, 2o3u and 2o3t (lines); (b) Mutation prediction of E226D (sticks) and the crystallographic data of 2o3r (lines); (c) Mutation prediction of E226G (sticks) and the crystallographic data of 2o3s and 1hct (lines) All mutations received by WHAT IF on-line All crystallographic data deposited on Protein Data Bank 2o3u, 2i65, 2pgl and wild-type CD38 – ligand complexes as: 2pgj, 2ef1, 2i66 and 2i67 deposited in PDB were chosen to be testing samples of software reliability As the workspace structure consists of a receptor only, there is no default center for the enclosing box The box will not be displayed until you have specified a grid center by selecting residues or proposed ligand position Surprisingly, the docking results depend essentially on the position of grid center, especially in QUANTUM calculation of long chain and flexible ligands In the cases we are not sure of the proposed ligand position we may select the center of the grid box by selecting any atom that lies approximately in the middle of the active site and all active site atoms that are on the surface of the protein should be covered by a grid box, or at least all important chemical groups of the active site should lie inside the grid box Perhaps a micro genetic algorithm loop should be used to find the grid center giving the pose of maximum binding free energy Among the output data of QUANTUM we can find IC50 ((Mol/L), Ebind (kJ/mol) – the binding free energy including Ees (kJ/mol) – the electrostatic and solvation energy, Evdw (kJ/mol) – the short-range electrostatic and exchange and Van der Waals energies, TdS (kJ/mol) – the entropy contribution, Etor (kJ/mol) – the ligand internal energy change We received also the total charge Q, mass M, number of flexible bonds of the ligand and RMSD (A) – the root mean square distance between the initial and final position In another way, GLIDE gives GLIDE score includes standard precision (SP) and extra precision (XP) GLIDE score is given by: Score = a∗ vdW + b∗ Coul + Lipo + Hbond + Metal +Rewards+RotB+Site, where vdW is van der Waals interaction energy, Coul is Coulomb interaction energy, Lipo is lipophilic-contact plus phobic-attractive term, HBond is hydrogenbonding term, Metal is metal-binding term (usually a reward), Rewards is various reward or penalty terms, RotB is penalty for freezing rotatable bonds, Site is polar interactions in the active site and a=0.063, b=0.120 for Standard Precision (SP) Glide 4.5 In order to compare the reliability of the softwares we used RMSD (˚ A) – the root mean square distance between the solved position deposited in PDB and the model position given by docking software The calculation results are presented in Table As most docking softwares give some predicted docking sites of the ligand Table presented the RMSD and score of two or occasionally three best ones It is clearly that GLIDE gives excellent results for cycle ligands and in most cases gives RMSD lower than QUANTUM In addition, both softwares give RMSD > 2.0 ˚ A for the complexes of CD38-wild type, especially, with NGD (code 2i66) where the active sites of both molecules were saturated with substrate NGD+ , and reaction proceeded in the crystal So that molecule B contains two nucleotides, a GDP-ribose intermediate and a hydrolyzed product, GDPR, whereas molecule A contains GDPR dimer (Liu et al., 2006) The docking calculation of only one GDPR molecule would never give good agreement with crystallographic data It should be noted that in some cases (the underline numbers in Table 2) the pose of smaller deviation has lower score (GLIDE) or higher free energy (QUANTUM) Fig displays, for example, two highest score docking poses of cADPR on E226Q mutant obtained by QUANTUM The complex has the accession code of 2o3q at the Protein Data Bank In most of these cases the docking pose pairs of small binding free energy difference (∼2 KJ/mol for QUANTUM) or small score difference (∼0.5 for GLIDE) It can be regarded approximately as the indefiniteness of the docking data given by the software in the cases of very large and extremely flexible ligands Therefore, using QUANTUM and GLIDE as well to predict the protein-ligand complex structures, it should be taken care the docking site pairs of small binding free energy difference (QUAN- Interdiscip Sci Comput Life Sci (2010) 2: 193–204 197 Results TUM) or small score difference (GLIDE) Table Prediction results of docking ligands on CD38 and its mutants Protein Mutant Ligand QUANTUM RMSD+ 2o3r 2o3s 2o3t 2pgj E226D E226G CXR CXR E226Q CGR 1yh3 N1C GLIDE(SP) RMSD++ Gscore 0.319 −37.048 0.266 −8.75 6.124 −34.407 2.382 −8.60 6.188 −37.5786 0.248 −9.55 0.614 −36.730 0.577 −9.43 7.447 −34.216 0.158 −14.99 0.660 −33.903 0.387 −12.78 1.232 −34.673 0.225 −9.59 8.195 −30.854 8.091 −4.13 Ebind 2pgl E226Q N1C 1.291 −35.058 0.208 −11.76 8.600 −30.911 5.632 −2.94 2o3q E226Q CXR 6.026 −36.532 1.304 −6.22 0.326 −34.632 0.576 −5.49 −11.40 2hct E226G NMN 0.684 −43.252 0.736 5.321 −32.191 1.053 −11.40 2o3u E226Q NGD 1.343 −50.992 3.093 −10.15 4.793 −49.669 3.759 −9.95 1.252 −48.643 2ef1 2i65 EPE 1.244 −22.601 5.203 −3.81 3.681 −20.216 4.865 −3.69 E226Q NAD 0.958 −45.958 1yh3 In order to characterize the CD38’s catalytic activities we have predicted mutants by WHAT IF from the atom coordinate crystallographic data of CD38’s wild-type deposited in Protein Data Bank (code 1yh3) Then, the docking poses of the various ligands are predicted by GLIDE and/or QUANTUM The binding free energy of the complexes between protein and ligand is calculated by QUANTUM The reactions taken into investigation are: cADPR hydrolase, NADase (NAD glycohydrolase), ADP-ribosyl cyclase The ligands involved in calculation are NAD, NGD, ADPR, GDPR, cADPR, cGDPR, NA and NMN as well NAD and NGD are reactants; ADPR, GDPR, cADPR and cGDPR are products in catalytic reaction NA is coproduct of cyclising reaction and hydrolysis as well NMN is a substrate of CD38 (Liu et al., 2005) and is also a mimic substrate of NAD and can be hydrolyzed by CD38 (Sauve et al., 2000) As presented below, taking into account that there are indefiniteness of both GLIDE and QUANTUM in the determination of docking pose of long chain and flexible ligands, not only one but some highest score poses in each case should be taken into account and the common tolerance of predicted binding free energies is accepted to be 15% as defined by the authors of QUANTUM Discussion 2.424 −45.567 4.971 −9.04 8.600 −30.911 2.599 −8.96 3.238 −38.199 2.358 −7.66 2.181 −7.59 2i66 1yh3 G1R 1.291 −35.058 2i67 1yh3 APR 6.428 −37.983 + Comparing with the prepared ligand position; with the initial ligand position Fig ++ Comparing Comparison of cADPR-ligand in two configuration of largest binding free energy obtained by QUANTUM (sticks) and the poses deposited on Protein Data Bank (lines) Being of smaller binding free energy (−34.632 KJ/mol) (b) configuration is in a much better agreement with PDB data than (a) one of −36.532 KJ/mol N ADase (N AD glycohydrolase) – the intermediate Michaelis complex – By incubating preformed CD38 E226Q crystals with NAD+ Liu et al (2006) found that NAD+ can easily diffuse into the active site and form the Michaelis complex The linear NAD+ is constrained by the enzyme and is stabilized in the active site by extensive polar interaction involving residues Asp155 , Glu146 , Gln226 , Trp125 , Ser126 , Arg127 , and Thr221 and a structural water molecule The docking simulation of the complex of E226Q and NAD gives a good agreement with the 2i65 deposited in Protein Data Bank (Table RMSD=0.958 5) Taking into account that there is no structural data of NAD complex with CD38’s wild-type deposited on Protein Data Bank and the above calculation shown that the computational conformation of mutate Gln226 residue is not in good agreement with crystallographic data (see Fig 1), in order to interpret satisfactorily the single residue replacement effect on the intermediate Michaelis complex we compare the structure of NAD docked computationally on CD38’s wild-type (code 1yh3) and on the complex 2i65 discarded the ligands (E226Q) The binding free energy and its components are presented in Table together with the shortest atomic distances between NAD and key residues 198 Table Mutants E226Q Interdiscip Sci Comput Life Sci (2010) 2: 193–204 (a) Binding free energy and its component of NAD complexes Ebind Ees Evdw TdS Etor −51.458 −26.000 −61.921 −32.925 3.537 12 Wild-type −48.076 −17.095 −60.077 −32.914 −3.817 25 Table Residue GLU146 GLU226 GLY226 ASP226 (b) The shortest atomic distance between NAD and some key residues Residue atom Distance(˚ A) Mutant Ligand atom Wild O OE2 2.614 C OE2 3.222 O CD 3.616 E226G O OE2 2.886 E226D O OE2 2.587 O CD 3.452 O OE1 3.536 C OE2 3.541 Wild E226G E226D O OE2 3.024 O OE1 3.398 O CD 3.605 O CA 7.189 O N 7.442 O OD2 4.693 C OD2 5.438 C OD2 5.719 O CG 5.722 The interpretation of catalytic activities based on the degree of ligand penetration into the active sites seems to be satisfactory The Glu226 replacement by glutamine (Q) seems not to affect significantly to the docking pose of NAD (Fig 3(a)) The nicotinamide end of NAD also penetrates deep into the active site consistent with cleavage of the nicotinamide-glycosidic bond, which is the first step of catalysis In order to assess the effect of Glu226 replacement by glutamine we compared the ligand-protein atomic shortest distances obtained by docking software between CD38’s wild-type and E226Q complex of NAD It can be seen that NAD could rather approaches to the Gln226 of E226Q than the Glu226 of CD38’s wild-type (Fig 3(b)) We calculated also the distribution of atomic distances in active site (Fig 4(a)) It can be seen that the Glu226 replacement by Gln226 brings NAD deeper to the active site and seems to become more constrained and more stabilized by the residue replacement It should be also noted that there is no obvious computational evidence for stretching the labile nicotinamide-ribosyl bonds in NAD The bond length of 1.483 ˚ A can be found in both cases of CD38’wildtype and E226Q while the crystallographic data is 1.475 ˚ A in 2i65 The computation well simulates the penetration of NAD into the active site creating a Michaelis complex which can be regarded as the intermediate product of NAD cyclase and hydrolysis reaction The activation of the complex to promote the dissociation of the nicotinamide moiety from the substrate could not be observed by the docking software based on rigid bonds model Up to now, we have no computational evidence why Glu226 ’s mutation to other residues essentially eliminates all CD38’s catalytic activities as Munshi et al obtained from experiments (Munshi et al., 2000) However, the conformation of Gln226 in active site obtained by WHAT IF and SWISS-PDB (Fig 1(a)) could give us the answer Docking NAD on the CD38’s computational mutant E226Q gives another picture on the active site There is no hydrogen bond between 2’-3’ OH ribosyl with Gln226 (7.79 ˚ A) but with Glu146 only So that, though NAD could also penetrate into active site in the case of the mutant E226Q but it is less constrained and stabilized than in wild-type In fact, there is a certain difference between complex structure in crystal and in liquid Unfortunately, we have no evidence that in aqueous solution Gln226 of CD38’s E226Q mutant would take not crystallographic pose but computational pose However, calculation shown that, in CD38’s E226D and E226G there is also no hydrogen bond between 2’-3’ OH ribosyl with Asp226 and Gly226 , respectively (Table 3(b)) So that we can affirm that the breaking down hydrogen bonds between 2’-3’ OH ribosyl and the residues replaced Glu226 makes NAD to be less constrained in active site and NA becomes more difficult to be cleaved and eliminates the mutant catalytic activities The activated intermediate Michaelis complex – a transition state – As intermediate Michaelis complex reveals products with divergent conformation, the reaction coordinate of NAD cyclase and hydrolysis reaction is not easy to define We start the work by investigating the activation of the Michaelis complex Following the NAD docking poses received by calculation it can be seen that the adenine terminus of NAD is out of the active site and is not expected to be stabilized by the enzyme (Liu et al., 2006) and contributes significantly to the RMSD (see Fig 3(b)) The nicotinamide end of NAD penetrates deep into and becomes stabilized by the active site So that, the adenine end of NAD is more flexible and is easy to leave In order to approach computationally the transition state we used the so-called ‘fragment docking technique’ by cutting off the nicotinamide end and docking the rest oxocarbenium ion intermediate of NAD and NGD on the protein The calculation result is presented in Fig 5(a) and Fig 5(b) comparing the docking pose of intermediate and ADPR in CD38’s wild-type There is no essential difference between the crystallographic and computational docking poses of ADPR Both crystallographic and computational distances between ribosyl C-1’ carbon and the adenine ring N-1’ is, however, sur- Interdiscip Sci Comput Life Sci (2010) 2: 193–204 199 Fig Stereo representation of NAD and active site (a) Left: the NAD docking pose on E226Q (sticks) and the crystallographic data in 2i65 (lines); center: the NAD docking pose on CD38’s wild-type (sticks) and the crystallographic data in 2i65 (lines); right: the NAD docking pose on CD38’s wild-type (sticks) and on E226Q (lines) (b) The active sites (lines) and NAD docked on CD38’s wild-type (sticks-left) and on E226Q (sticks-right) There is no obvious A) than difference between two structures but the hydrogen bond length of ribosyl OH is shorter with Glu146 (2.6 ˚ A) in the former and is shorter with Gln226 (2.7 ˚ A) than with Glu146 (3.0 ˚ A) in the latter with Glu226 (3.4 ˚ Fig (a) The intermolecular atomic distance population of NAD – active site residue (b) Stereo representation of NAD and active site in CD38’s computational E226Q mutant prisingly much shorter (4.86 ˚ A) than the corresponding distance in the intermediate complexes (9.30 ˚ A) and the distance between ribosyl C-1’ carbon and guanine ring N7 (8.65 ˚ A) as well (Fig 5(d)) which shows experimentally the intermediate’s cyclase ability of CD38’s wild-type It means that, not the structure of intermediate but the molecular interaction between ligand and active site is dominant in catalytic activities and it is not clear that where will take place the intermediate cyclization, inside or outside of the active site? For the ADPR intermediate, the structurally conserved water molecules (crosses in Fig 5(b)) not only contributes to the stabilization of the products but also attend to NAD hydrolyse either by the migration of water molecule in hydrogen bonding with 2’-OH to ribosyl C-1’ or another water molecules attack ribosyl 200 Fig Interdiscip Sci Comput Life Sci (2010) 2: 193–204 Stereo presentation of docking poses (a) ADPR’s (sticks) crystallographic pose in the active site residues (lines) of A The distance CD38’s wild-type The distance between ribosyl C-1’ carbon and the Glu226 hydroxyl group is > 10 ˚ between ribosyl C-1’ carbon and the adenine ring N-1’ is 4.86 ˚ A (b) The computational pose of NAD oxocarbenium ion intermediate (sticks) in the active site residues (lines) of CD38’s wild-type The shortest distance between ribosyl 2’- or 3’-OH group and the residues Glu226 ’s carboxyl group is 2.95 ˚ A The distance between structurally conserved water molecule (cross) and another ribosyl –OH group is 2.42 ˚ A, between ribosyl C-1’ carbon and the adenine ring N-1’ is 9.30 ˚ A (c) and (d) Docking pose of NGD oxocarbenium ion intermediate in the complex crystal of CD38’s wild-type The distance between ribosyl C-1’ carbon and guanine ring N7 is 8.65 ˚ A There are three hydrogen bonds A) and Glu146 (3.62 between ribosyl 2’- or 3’-OH group and the carboxyl groups of residues Glu226 (3.22 and 3.19 ˚ ˚ A) C-1’ In the GDPR intermediate complex (Fig 5(d)), there are three hydrogen bonds would locate between ribosyl 2’- or 3’-OH group and the carboxyl groups of residues Glu226 (3.22 and 3.19 ˚ A) and Glu146 (3.62 ˚ A) as well So that, the hydrolyse can be completed in active site and the large majority of the substrate NAD is hydrolyzed to ADPR while the conversion of NAD to cADPR is not the dominant reaction catalyzed by wild-type human CD38 as the outside adenine terminus of NAD may be more difficult to connect with the ribosyl C-1’ located deeply and stabilized in actives site though it is flexible Docked in the same active site GDPR intermediate gives a more favorable interaction energy condition than ADPR (Fig 5(d)) though the distance between ribosyl C-1’ carbon and guanine ring N7 is large (8.65 ˚ A) Michaelis complexes intermediate in CD38’s E146 mutants and cyclase/hydrolyse activity – Graeff et al (2001) shown that the replacement of Glu146 by Phe, Ala, Asn, Gly, Asp and Leu created the mutants exhibited enhanced cyclase activity to various degrees, whereas the hydrolysis activity was inhibited greatly In order to continue study on the mechanistic understanding of human CD38-controlled multiple catalysis the above calculation procedure has been applied to the structure and activation of intermediate Michaelis complexes in E146 mutants and the prediction of the mutagenesis effect on the NADase and hydrolysis reaction hoping that the analysis of the energy and structural data of the intermediate could provide further insights into the understanding mechanism of the catalytic reaction The experimental data of the reaction activities can be found in Graeff et al.’s article (2001) Interdiscip Sci Comput Life Sci (2010) 2: 193–204 As the crystallographic structure of related mutants and the complexes have not yet deposited on Protein Data Bank we need predict, firstly, the mutants by SWISS-PDBViewer and/or WHAT IF then dock NAD and oxocarbennium ion intermediate as well on CD38’ E146 mutants Taking into account that we have no previous information on the ligand docking poses, in order to reduce the effect of grid center on docking results we select the middle of the active site to be the center of the grid box and all active site atoms should be covered by a grid box The active site of CD38’s wild-type and mutants includes 10 residues Arg127 , Asp155 , Ser126 , Ser193 , Glu146 , Glu226 , Thr221 , Lys129 having extensive polar interactions and Trp125 and Trp189 which are nonpolar but additionally the parallel displaced π − π interactions between its indol ring (Trp189 ) and the NAD pyridine ring Table presents NAD’s oxocarbenium ion intermediate (ADPRI) binding energy and its components on CD38’s wild-type and its E146 mutants as well Unfortunately, there is no obvious relationship between Table 201 the catalytic activities and the computational energy data and the distance between ribosyl C-1’ and adenine N-1’ However, the relationship between the C-1’N-1’ distance and cyclase/hydrolyse ratio can be regarded as linear approximately (Fig 6) There are essentially changes of intermediate’s docking pose because of Glu146 replacement The hydrogen bonds are also rearranged (Table 4) which is dominant for CD38’s catalytic activities Two extreme cases are CD38’s wild-type and E146A The former is stabilized strongly by four hydrogen bonds between ribosyl 2’ and 3’-OH group and Glu146 and Glu226 as well The latter, however, has no hydrogen bond It means that the more strongly kept ribosyl group by hydrogen bonds the more NADase and less cyclase activity The hydrogen bonds will prevent the ribosyl group penetrated into active site to approach the N-1’ of adenine ring which located flexibly outside Breaking hydrogen bonds of ribosyl 2’ and 3’-OH will loosen it to promote the cyclase It is in a good agreement with Graeff et al.’s experimental data (Graeff et al., 2001) Oxocarbenium ion intermediate’s binding energy and its components on CD38’s wild-type and its mutants (KJ/mol) and the distance of hydrogen bonds involving ribosyl 2’-3’OH (˚ A) Mutants Ebind Ees Evdw TdS Etor E146A −38.569 −6.435 −51.287 −25.233 E146D −39.051 1.084 −54.211 −27.646 Hydrogen bond (˚ A) Glu226 Other −6.080 NA NA$ −13.571 3.06/3.03 2.87∗ 2.62∗ E146F −39.058 −3.323 −51.911 −29.252 −13.076 2.69/3.11 E146G −33.148 6.600 −49.164 −21.394 −11.979 NA NA E146L −35.008 −1.756 −48.320 −22.788 −7.720 2.69/2.89 3.38∗ E146N −37.790 0.631 −51.730 −28.486 −15.178 2.86/3.10 3.04∗ Wild-type −36.931 −1.298 −49.785 −27.929 −13.777 2.95/3.36 2.69/3.87++ ++ With Glu146 ; ∗ With Trp125 ; $ Ignoring the bonds of > 4.0 A 14 tion Instead of hydrogen bonds with Glu146 in CD38’s wild-type, four among six E146 mutants investigated, that is E146D, E146F, E146L and E146N possess hydrogen bonds with Trp125 Distance (A) 12 10 Fig Cyclass/NADase The computational distance between the ribosyl C-1’ and the adenine ring N-1’ versus cyclase/hydrolase ratio (Graeff et al.’s data) The computational docking poses of the intermediate in active site of CD38’s wild-type and mutants also show the collaborative role of Trp125 in catalytic reac- cADPR hydrolase – To evaluate the structural basis of the hydrolysis of cADPR Liu et al (2005) set up a model cADPR into shCD38, the complex structures of CD157 with its substrate analog Etheno-NADP (PDB ID: 1ish) were aligned to CD38 based on a least square optimization for all atoms in three conserved residues (Glu226 , Trp125 and Trp189 ) within the active site and the conjugate gradient energy minimization method was used to fit the position of cADPR Liu et al (2005) proposed that polar interaction is essential for the hydrolysis of cADPR In the CD38-cADPR model, most interactions between cADPR and CD38 are hydrophobic, except for the hydrogen bond interactions 202 Interdiscip Sci Comput Life Sci (2010) 2: 193–204 involving Lys129 , Glu226 , Glu146 , and cADPR Glu226 is shown to be critical not only in catalysis but also in positioning of cADPR at the catalytic site through strong hydrogen bonding interaction (Liu et al., 2007) As the single mutation of Lys129 to other neutral or negatively charged residues completely eliminates CD38’s cADPR hydrolase activity (Tohgo et al., 1997), Lys129 located at the entrance of the catalytic pocket is also essential for cADPR hydrolysis It is possible that the breaking of the hydrogen bond will disable the binding and the entry of cADPR into the catalytic pocket and prevents the hydrolysis reaction from occurring (Liu et al., 2005) So that, there is neither obvious evidence Table on breaking the bond between ribosyl C-1’ and adenine ring N1 nor the H2 O attack on C-1’ and it is not clear the mechanism of cADPR hydrolysis As presented above both QUANTUM and GLIDE give excellent results in docking cycle ligands QUANTUM software is also used to simulate the docking poses and binding free energy of cADPR docked on the active site of CD38’s various E146 mutants (Table 5) Evdw and TdS are dominant components of binding free energies Unfortunately, there is no obvious relation between Vmax – the mutants’ cADPR hydrolase activities measured experimentally (Graeff et al., 2001) and Ebind , between Vmax and Ebind’s components as well Binding free energy and its components of cADPR on mutants and cADPR hydrolase activity+ Mutants Ebind Ees Evdw TdS Etor V++ max 700±720 CD38 −31.500 3.389 −47.209 −14.201 −1.882 E146F −32.316 1.943 −47.787 −14.626 −1.099 300±260 E146N −34.673 −2.393 −48.369 −18.805 −2.715 300±180 200±490 E146L −32.271 −1.725 −45.791 −15.966 −0.720 E146A −33.401 −2.249 −46.745 −17.810 −2.218 800±60 E146G −34.031 −5.148 −46.448 −18.234 −0.668 360±90 E146D −30.290 0.841 −43.808 −15.522 −2.845 ND + The abbreviations used are: Ebind – the binding free energy, Ees – the electrostatic and solvation energy, Evdw – the short range electrostatic and exchange and Van der Waals energies, TdS – entropy contribution, E tor – the ligand internal energy change All energies are of kJ/mol; ++ (Graeff et al., 2001) Analyzing the pocking poses of cADPR, however, we can identify the structural factors inhibiting the hydrolyse activity In E146 mutants, the replacement of glutamic acid – a polar, acidic and negative hydropathy index residue by either leucine (L), alanine (A) and glycine (G) – nonpolar, neutral and high hydropathy index residues or polar, neutral/acidic and negative hydropathy index residue as asparagine (N) and aspartic acid (D) changes essentially docking pose of cADPR and consequently the cADPR hydrolase activity Phenynalanine (F) does not change obviously the docking pose and consequently the cADPR hydrolysis activity (Fig 7(a), (b) and (c)) Table presents, in details, the hydrogen bonds between the active site residues and cADPR in CD38’s wild-type and mutants There are hydrogen bonds between cADPR and CD38’s wild-type active site residues, i.e Asp155 (3.09 ˚ A), Glu146 (3.11 ˚ A) and 125 ˚ Trp (3.76 A) The ligand located far from Glu226 (6.17 ˚ A) It seems to be different with Liu et al’s model The single residue replacement in active site pocket changed essentially hydrogen bond map E146D is an extreme case which remains only one hydrogen bond and as Graeff et al.’s experiment data (Graeff et al., 2001), entirely eliminates the cADPR hydrolase So quantitatively we can say that the hydrogen bonds between cADPR and active site residues play the role in controlling the cADPR hydrolysis reaction Table Mutants The hydrogen bonds between cADPR and active site residues (˚ A) Residue Glu146 Asp155 Glu226 Trp125 Thr221 Arg127 Ser126 E146A – 3.90 – 2.88 2.95 – E146D – – 2.98 – – – – E146F – – 3.55 – 3.09 3.14 3.93 E146N – – – 2.95 2.97 – 3.23 E146L – – – 3.00 3.06 – 3.19 E146G – – – 2.90 – – 3.26 Wild-type 3.11 3.09 – 3.76 – – – 3.20 In addition to the E146 mutants, we have also calculated binding free energy of cADPR docked on various K129 mutants The obtained energies and their components give also no evidence to confirm the elimination of CD38’s cADPR hydrolase activity However, the complete elimination can be explained by the significant changing docking pose of cADPR as single replacing Lys129 by nonpolar and neutral residues Stereo Interdiscip Sci Comput Life Sci (2010) 2: 193–204 Fig 203 Stereo representation of cADPR complexes in active site (a) and (b): CD38’s wild-type and E146F, respectively, have the same degree of cADPR hydrolase activity (c): E146L, the replacement of a nonpolar, neutral and high hydropathy index residue decreases cADPR hydrolase activity (d), (e) and (f): K129L, K129F, and K129G the LYS129 replacement essentially eliminates cADPR hydrolase activity representation of cADPR complexes (Fig 7(d), (e) and (f)) shown that, the single replacement of Lys129 by a nonpolar, neutral and high hydropathy index residue such as leucine (L) and phenylalanine (F) moves adenine group far from Glu146 while by a nonpolar, neutral and negative hydropathy index residues as glycine (G) turn also adenine group far from Glu146 but in an other side In details, there is only one hydrogen bond between cADPR and Asp155 (2.81 ˚ A) Conclusion Results described in this study characterized computationally human CD38’s multitude of catalytic activities The interpretation of catalytic activities based on the degree of ligand penetration into the active sites seems to be satisfactory The computation software well simulates the penetration of NAD into the active site The adenine terminus of NAD is out of the active site and is not stabilized by the enzyme and contributes significantly to the RMSD The nicotinamide end of NAD also penetrates deep into the active site consistent with cleavage of the nicotinamide-glycosidic bond which is the first step of catalysis creating a Michaelis complex which can be regarded as the intermediate product of NAD cyclase and hydrolysis reaction (ADPRI) The breaking down hydrogen bond between 2’-3’ OH ribosyl and the residues replaced Glu226 makes NAD to be less constrained in active site and NA becomes more diffi- cult to be cleaved and eliminates the mutant catalytic activities As analyzing the docking poses of ADPRI and GDPRI in the active site of CD38’s wild-type and mutants we can state that, not the conformation of the intermediate but the molecular interaction between it and active site is dominant in catalytic activities The hydrolyse can be completed in active site and the large majority of the substrate NAD is hydrolyzed to ADPR while the conversion of NAD to cADPR is not the dominant reaction catalyzed by wild-type human CD38 as the outside adenine terminus of NAD may be more difficult to connect with the ribosyl C-1’ located deeply and stabilized in actives site though it is flexible The more strongly kept ribosyl group by hydrogen bonds the more NADase and less cyclase activity Breaking hydrogen bonds of ribosyl 2’ and 3’-OH by mutation will loosen it to promote the cyclase The cADPR a product of NAD cyclase reaction could also penetrate deeply into active site to make some hydrogen bonds with Glu146 and Glu226 However, as its conformation is not as favorable as NAD, the docking poses are affected by a residue located at the entrance of the catalytic pocket (Lys129 ) (Liu et al., 2005) Overall, by analyzing the computational structure data we can also explain the mechanistic understanding of the Michaelis complex revealing products with divergent conformations Based on the experimental data publicized by Univ Minnesota Group and the 204 crystallographic structural data deposited on Protein Data Bank we can state that though the exactness of bioinformatic softwares we used is only moderate (15% of binding free energy), the calculation is useful for predicting at least qualitatively the mutagenesis effect on catalytic activities of human CD38 In order to continue the prediction study at quantitative degrees, we should deal with the potential energy surface of ligands in active site by using another software (Mousseau et al., 2001), and the most difficult work would be finding the transition state Acknowledgments We thank National Program of Fundamental Sciences and the Project of Pharmaceutical Chemistry (Vietnam National University, Hanoi) for research support, Qun Liu (Cornell University) et al for giving CD38’s mutants crystallographic data and their related articles, WHAT IF’s authorities for on-line calculation, Hypercube, Inc for evaluation license of HyperChem 8.0 References [1] Bă ohm, H.J., Klebe, G 1996 What 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