DSpace at VNU: Computational Characterization for Catalytic Activities of Human CD38''s Wild Type, E226 and E146 Mutants

DSpace at VNU: Computational Characterization for Catalytic Activities of Human CD38''''s Wild Type, E226 and E146 Mutants...

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

1(Faculty of Chemistry, Hanoi University of Natural Science, VNU, 19 Le Thanh Tong, Hanoi, Vietnam)

2(Hoa Binh University, CC2, My Dinh II, Tu Liem, Hanoi, Vietnam)

Received 15 December / Revised 26 February 2010 / Accepted 4 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.

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

be-tween 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

ar-rangement and electronic nature of its atoms, and the

way these atoms interact with the biological

counter-part (Bohm et al., 1996). Computational chemistry

tools allow one to characterize the structure,

dynam-ics, and energetic of the interactions between the

lig-and lig-and a macromolecule as protein lig-and DNA For

in-stance, molecular mechanics (MM)-based approaches

can efficiently assist the discovery of new drug

candi-dates, and these computationally inexpensive methods

are nowadays routinely used in drug design (Jorgensen,

2004) However, if a description of the electronic

prop-erties 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 ef-fects, charge transfer, etc., and usually estimate molec-ular energies more accurately (Sherwood, 2000;

Jor-gensen 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 form-ing/breaking An excellent overview of target-related applications of first principles quantum chemical

meth-ods 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 pre-dicted in a rather agreement with spectroscopy ex-periments where IC50=10E bind/5.85, Ebind is binding free energy between ligand and protein These soft-wares make an ability to do predictive computations

of complicated biochemical processes In this study,

we employed computational method softwares, namely GLIDE (Schrodinger, 2000), QUANTUM 3.3 (Quan-tum Pharmaceuticals, 2007), WHAT IF (Gert Vriend

et al., 2009) and HYPERCHEM 8.0 (Hypercube, 2007)

to simulate the structure and energy of the enzymatic

domain of wild-type CD38 and its E226 and E146

mu-tants 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

de-posited 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

com-putational 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

CD38 was described first as an antigen that is

in-volved 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

anti-genic functions, CD38 also possesses a multitude of

en-zymatic 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

base-exchange activity that is responsible for synthesizing

NAADP from NADP

The active site of human CD38 has been

biochem-ically and structurally characterized (Munshi et al.,

2000; Liu et al., 2005) Glu226 is identified as the

cat-alytic residue, because its mutation to other residues

essentially eliminates all its catalytic activities

(Mun-shi 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

cat-alyzed 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

ob-served 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

phenylala-nine 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-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 var-ious degrees, whereas the hydrolysis activity was in-hibited greatly E146A showed the highest cyclase ac-tivity, which was more than 3-fold higher than its hy-drolysis 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 hy-drolyzed cADPR effectively, and this activity was corre-spondingly depressed in the mutants When all the mu-tants 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

syn-thesizing 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 en-zymatic 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;

Gra-eff et al., 2001; Liu et al., 2005; Liu et al., 2006; Liu

et al., 2007) The mutants investigated

computation-ally in this study are E146A, E146N, E146G, E146L, E146D, E146F and E146K, E146Q Two latters were obtained by replacing Glu146 by lysine (K) and glu-tamine (Q), respectively The complexes provided a step-by-step description of the catalytic processes in-volved in the synthesis and hydrolysis of cADPR The E226G – a mutant of CD38 received by replacement of Glu226by 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 com-plex E226D-cADPR (code 2o3r), E226G-cADPR (code 2o3s) and other ligand-protein complexes as well de-posited on Protein Data Bank have been used to verify the computational procedure

The calculation procedure includes three core algo-rithms: (i) the replacement of each residue in active sites by other one then makes a geometrical optimiza-tion which simulates the site-directed mutagenesis tech-nique; (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, ex-ecutive file and/or online calculation Depending on the

concrete algorithm the results received by these

soft-wares may be different Though there are many

arti-cles 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

re-liability applied to a narrow branch of proteins

includ-ing 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

eval-uate 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

struc-ture 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

ge-ometry Table 1 presents the comparing results of some

CD38’s mutant models obtained by WHAT IF,

SWISS-PDB 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 1 ˚A In

de-tail, WHAT IF gives rather smaller RMS and LD than

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 struc-ture involving only one mutated residue in minimiza-tion procedure It could be explained that, our calcu-lations 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 do 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 be-ing stable without large torsion unfavorable of the lig-and lig-and keep the other residues’ position unchanged approximately CUPSAT’s predicted stability data is also presented in Table 1 However, E226Q should be taken into account in software development for the long chain and flexible residues The conformation of mu-tation Gln226 received both by WHAT IF and SWISS-PDB 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)) do not show

an obvious difference

Table 1 RMS on all atoms of some mutant predicted models from solved structure deposited in Protein

Data Banka

Mutant

CUPSAT

aAll RMS calculation were done on WHAT IF;bDeposited in Protein Data Bank discarded ligands;∗Predicted stability (kcal/mol);

+ Comparing with CD38 wild-type; ++ Comparing with solved structure in PDB.

Docking – The ligand docking on CD38 and its

mu-tants 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 struc-ture of the ligand-protein complex The mutant – lig-and complexes as: 2hct, 2o3q, 2o3r lig-and 2o3s, 2o3t,

Fig 1 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

cho-sen 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

Sur-prisingly, the docking results depend essentially on the

position of grid center, especially in QUANTUM

calcu-lation 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

en-ergy including Ees(kJ/mol) – the electrostatic and

sol-vation energy, Evdw (kJ/mol) – the short-range

electro-static 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

hydrogen-bonding 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 po-lar 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 cal-culation results are presented in Table 2 As most docking softwares give some predicted docking sites of the ligand Table 2 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

ad-dition, both softwares give RMSD > 2.0 ˚A for the com-plexes of CD38-wild type, especially, with NGD (code 2i66) where the active sites of both molecules were sat-urated with substrate NGD+, and reaction proceeded

in the crystal So that molecule B contains two nu-cleotides, 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 under-line numbers in Table 2) the pose of smaller deviation has lower score (GLIDE) or higher free energy (QUAN-TUM) Fig 2 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 en-ergy 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 ex-tremely flexible ligands Therefore, using QUANTUM and GLIDE as well to predict the protein-ligand com-plex structures, it should be taken care the docking site pairs of small binding free energy difference

(QUAN-TUM) or small score difference (GLIDE).

Table 2 Prediction results of docking ligands on

CD38 and its mutants

Protein Mutant Ligand QUANTUM GLIDE(SP)

RMSD + Ebind RMSD ++ Gscore

2o3r E226D CXR 0.319 8 −37.048 5 0.266 2 −8.75

6.124 7 −34.407 1 2.382 7 −8.60

2o3s E226G CXR 6.188 5 −37.5786 0.248 6 −9.55

0.614 2 −36.730 4 0.577 1 −9.43

2o3t E226Q CGR 7.447 0 −34.216 9 0.158 9 −14.99

0.660 5 −33.903 3 0.387 4 −12.78

2pgj 1yh3 N1C 1.232 5 −34.673 2 0.225 1 −9.59

8.195 3 −30.854 6 8.091 1 −4.13

2pgl E226Q N1C 1.291 0 −35.058 0 0.208 8 −11.76

8.600 5 −30.911 0 5.632 7 −2.94

2o3q E226Q CXR 6.026 1 −36.532 7 1.304 4 −6.22

0.326 4 −34.632 9 0.576 1 −5.49

2hct E226G NMN 0.684 2 −43.252 0 0.736 1 −11.40

5.321 2 −32.191 6 1.053 3 −11.40

2o3u E226Q NGD 1.343 8 −50.992 7 3.093 7 −10.15

4.793 2 −49.669 4 3.759 8 −9.95

1.252 3 −48.643 6

2ef1 1yh3 EPE 1.244 7 −22.601 7 5.203 5 −3.81

3.681 6 −20.216 8 4.865 6 −3.69

2i65 E226Q NAD 0.958 5 −45.958 5

2.424 8 −45.567 5

2i66 1yh3 G1R 1.291 0 −35.058 0 4.971 8 −9.04

8.600 5 −30.911 0 2.599 8 −8.96

2i67 1yh3 APR 3.238 5 −38.199 5 2.358 8 −7.66

6.428 5 −37.983 0 2.181 0 −7.59

+ Comparing with the prepared ligand position; ++ Comparing

with the initial ligand position.

Fig 2 Comparison of cADPR-ligand in two configuration

of largest binding free energy obtained by

QUAN-TUM (sticks) and the poses deposited on Protein

Data Bank (lines) Being of smaller binding free

energy (−34.632 9 KJ/mol) (b) configuration is in

a much better agreement with PDB data than (a)

one of−36.532 7 KJ/mol

In order to characterize the CD38’s catalytic activ-ities 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 pre-dicted by GLIDE and/or QUANTUM The binding free energy of the complexes between protein and lig-and is calculated by QUANTUM The reactions taken into investigation are: cADPR hydrolase, NADase (NAD glycohydrolase), ADP-ribosyl cyclase The lig-ands 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 co-product 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,

tak-ing into account that there are indefiniteness of both GLIDE and QUANTUM in the determination of dock-ing 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 pre-dicted binding free energies is accepted to be 15% as defined by the authors of QUANTUM

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 Thr221and

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 2 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 calcu-lation 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 dis-carded the ligands (E226Q) The binding free energy and its components are presented in Table 3 together with the shortest atomic distances between NAD and key residues

Table 3 (a) Binding free energy and its component

of NAD complexes

E226Q −51.458 6 −26.000 3 −61.921 1 −32.925 7 3.537 12

Wild-type −48.076 3 −17.095 9 −60.077 7 −32.914 5 −3.817 25

Table 3 (b) The shortest atomic distance between

NAD and some key residues

Residue Mutant Ligand atom Residue atom Distance(˚ A)

GLU 146

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 Glu226replacement 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

calcu-lated also the distribution of atomic distances in active

site (Fig 4(a)) It can be seen that the Glu226

replace-ment by Gln226 brings NAD deeper to the active site

and seems to become more constrained and more

sta-bilized 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

crystallo-graphic data is 1.475 4 ˚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 nicoti-namide moiety from the substrate could not be ob-served by the docking software based on rigid bonds model Up to now, we have no computational evi-dence why Glu226’s mutation to other residues essen-tially eliminates all CD38’s catalytic activities as

Mun-shi et al obtained from experiments (MunMun-shi 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 evi-dence that in aqueous solution Gln226of CD38’s E226Q mutant would take not crystallographic pose but com-putational pose However, calculation shown that, in CD38’s E226D and E226G there is also no hydrogen bond between 2’-3’ OH ribosyl with Asp226and 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 reac-tion coordinate of NAD cyclase and hydrolysis reacreac-tion

is not easy to define We start the work by

investigat-ing the activation of the Michaelis complex Followinvestigat-ing

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 crystallo-graphic and computational distances between ribosyl C-1’ carbon and the adenine ring N-1’ is, however,

sur-Fig 3 Stereo representation of NAD and active site (a) Left: the NAD docking pose on E226Q (sticks) and the crystal-lographic data in 2i65 (lines); center: the NAD docking pose on CD38’s wild-type (sticks) and the crystalcrystal-lographic 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 difference between two structures but the hydrogen bond length of ribosyl OH is shorter with Glu146 (2.6 ˚A) than with Glu226 (3.4 ˚A) in the former and is shorter with Gln226 (2.7 ˚A) than with Glu146 (3.0 ˚A) in the latter

Fig 4 (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

exper-imentally the intermediate’s cyclase ability of CD38’s

wild-type It means that, not the structure of

interme-diate 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 con-served 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 ri-bosyl C-1’ or another water molecules attack riri-bosyl

Fig 5 Stereo presentation of docking poses (a) ADPR’s (sticks) crystallographic pose in the active site residues (lines) of CD38’s wild-type The distance between ribosyl C-1’ carbon and the Glu226 hydroxyl group is> 10 ˚A The distance 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 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)

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

ac-tive 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

termi-nus 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

intermedi-ate 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 ex-hibited enhanced cyclase activity to various degrees, whereas the hydrolysis activity was inhibited greatly

In order to continue study on the mechanistic under-standing of human CD38-controlled multiple catalysis the above calculation procedure has been applied to the structure and activation of intermediate Michaelis com-plexes in E146 mutants and the prediction of the mu-tagenesis 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 re-action The experimental data of the reaction activities

can be found in Graeff et al.’s article (2001).

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

or-der 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, Lys129having extensive

polar interactions and Trp125and Trp189which are

non-polar but additionally the parallel displaced π − π

in-teractions between its indol ring (Trp189) and the NAD

pyridine ring

Table 4 presents NAD’s oxocarbenium ion

interme-diate (ADPRI) binding energy and its components on

CD38’s wild-type and its E146 mutants as well

Un-fortunately, there is no obvious relationship between

the catalytic activities and the computational energy data and the distance between ribosyl C-1’ and ade-nine N-1’ However, the relationship between the C-1’-N-1’ distance and cyclase/hydrolyse ratio can be re-garded as linear approximately (Fig 6) There are essentially changes of intermediate’s docking pose be-cause 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, how-ever, 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).

Table 4 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)

Wild-type −36.931 0 −1.298 2 −49.785 2 −27.929 5 −13.777 2 2.95/3.36 2.69/3.87 ++ ++ With Glu 146 ;∗With Trp125 ; $ Ignoring the bonds of> 4.0 A.

14

12

10

8

6

Cyclass/NADase

Fig 6 The computational distance between the

ribo-syl C-1’ and the adenine ring N-1’ versus

cy-clase/hydrolase ratio (Graeffet al.’s data)

The computational docking poses of the

intermedi-ate in active site of CD38’s wild-type and mutants also

show the collaborative role of Trp125 in catalytic

reac-tion Instead of hydrogen bonds with Glu146in CD38’s wild-type, four among six E146 mutants investigated, that is E146D, E146F, E146L and E146N possess hy-drogen bonds with Trp125

cADPR hydrolase – To evaluate the structural

ba-sis of the hydrolyba-sis of cADPR Liu et al (2005) set

up a model cADPR into shCD38, the complex struc-tures 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 hy-drophobic, except for the hydrogen bond interactions

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 Lys129to other neutral or

neg-atively 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

on breaking the bond between ribosyl C-1’ and adenine ring N1 nor the H2O attack on C-1’ and it is not clear the mechanism of cADPR hydrolysis As presented above both QUANTUM and GLIDE give excellent re-sults 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 Unfor-tunately, there is no obvious relation between Vmax – the mutants’ cADPR hydrolase activities measured

ex-perimentally (Graeff et al., 2001) and Ebind, between

Vmax and Ebind’s components as well

Table 5 Binding free energy and its components of cADPR on mutants and cADPR hydrolase activity+

+ 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, Etor – the ligand internal energy change All energies are of kJ/mol; ++ (Graeffet al., 2001).

Analyzing the pocking poses of cADPR, however, we

can identify the structural factors inhibiting the

hy-drolyse activity In E146 mutants, the replacement of

glutamic acid – a polar, acidic and negative

hydropa-thy index residue by either leucine (L), alanine (A)

and glycine (G) – nonpolar, neutral and high

hydropa-thy index residues or polar, neutral/acidic and

nega-tive hydropathy index residue as asparagine (N) and

aspartic acid (D) changes essentially docking pose of

cADPR and consequently the cADPR hydrolase

activ-ity Phenynalanine (F) does not change obviously the

docking pose and consequently the cADPR hydrolysis

activity (Fig 7(a), (b) and (c))

Table 6 presents, in details, the hydrogen bonds

be-tween the active site residues and cADPR in CD38’s

wild-type and mutants There are 3 hydrogen bonds

between cADPR and CD38’s wild-type active site

residues, i.e Asp155 (3.09 ˚A), Glu146 (3.11 ˚A) and

Trp125 (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 be-tween cADPR and active site residues play the role in controlling the cADPR hydrolysis reaction

Table 6 The hydrogen bonds between cADPR and

active site residues (˚ A)

Glu 146 Asp 155 Glu 226 Trp 125 Thr 221 Arg 127 Ser 126

In addition to the E146 mutants, we have also cal-culated binding free energy of cADPR docked on var-ious K129 mutants The obtained energies and their components give also no evidence to confirm the elimi-nation of CD38’s cADPR hydrolase activity However, the complete elimination can be explained by the sig-nificant changing docking pose of cADPR as single re-placing Lys129by nonpolar and neutral residues Stereo