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J. Biol. Chem., Vol. 282, Issue 8, 5853-5861, February 23, 2007
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¶12
From the
MacCHESS, Cornell High Energy Synchrotron Source, Cornell University, Ithaca, New York 14853, the Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota 55455, and the ¶Department of Physiology, University of Hong Kong, Hong Kong, China
Received for publication, September 26, 2006 , and in revised form, December 8, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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CD38 cyclizes NAD, a long linear molecule, to a compact cyclic nucleotide, cADPR. The active site of human CD38 has been biochemically and structurally characterized (12, 13). Glu226 is identified as the catalytic residue, because its mutation to other residues essentially eliminates all its catalytic activities (12). Ser193 is also important for catalysis as its mutation to alanine also greatly reduces enzyme activities (14). Conversion of NAD 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 (6). Completely the opposite is observed when NGD, an analog of NAD, is used as substrate. The dominant reaction is now cyclization instead of hydrolysis, producing cyclic GDP-ribose (cGDPR) as the major product (15). Considering the similarity of NGD and NAD, which differ only in the purine rings, it is puzzling why the reactions are so different. Scheme 1 shows two different cyclization reactions catalyzed by human CD38 with NAD and NGD as substrates. It is interesting that, by mutating a conserved active site residue Glu146 to alanine, the mutant acquires greatly increased ADP-ribosyl cyclase activity. With NAD as substrate, the CD38 E146A mutant can produce three times more cADPR than ADPR, indicative of Glu146 role in controlling the cyclization and hydrolysis reactions.
In this study, we employed x-ray crystallography to determine the structures of the enzymatic domain of human CD38 complexed with three relevant ligands, cADPR, cGDPR, and NGD. These complexes, together with previously solved NAD and ADPR complexes (14), allow us to provide a step-by-step description of the catalytic processes involved in the synthesis and hydrolysis of cADPR. The results also provide the first evidence of a conformational change at the active site of CD38 during catalysis.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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A (purchased from Invitrogen) was used for generating all the mutants. The soluble domain of hCD38, starting with arginine 45, was cloned into the vector between the EcoRI and NotI restriction sites in the multiple cloning site of the vector. This subcloning method allows for the ligation of the soluble CD38 in-frame with the -factor secretion signal. Expression is driven by the tightly controlled alcohol oxidase promoter AOX1, which is induced by methanol. The protein is initially expressed as a fusion of the secretion signal peptide, which is cleaved off by endogenous proteolytic enzymes, and the CD38 secreted into the expression medium. Mutations were performed using the QuikChange Site-directed Mutagenesis kit obtained from Stratagene. Complementary primers containing the mutations were used to amplify the entire plasmid using the high fidelity PfuTurbo DNA polymerase. The amplification is linear, and only 20 cycles were used to minimize the chances of generating errors during the process. At the end of the amplification process the reaction mixture contained the newly synthesized vector with nicks at the priming site, and the parent (template) vector, which is hemimethylated due to being amplified in an Escherichia coli strain. Using the endonuclease DpnI, the methylated vector is digested away leaving behind the newly synthesized vector containing the mutation of interest, which was used to transform super-competent XL1-Blue E. coli cells purchased from Stratagene. The positive clones were checked for the presence of the insert, sequenced, and used for the transformation of the X-33 yeast cells.
Primers for the mutations are listed below. Changes in the base pairs are indicated by bold and underlined bases. CD38-E226G: top strand primer, 5'-CAGCACTTTTGGGAGTGTGGGAGTCCATAATTTGCAACCAGAGAA-3'; bottom strand primer, 5'-TTCTCTGGTTGCAAATTATGGACTCCCACACTCCCAAAAGTGCTG-3'; CD38-E226Q: top strand primer, 5'-CAGCACTTTTGGGAGTGTGCAAGTCCATAATTTGCAACCAGAGAA-3'; bottom strand primer, 5'-TTCTCTGGTTGCAAATTATGGACTTGCACACTCCCAAAAGTGCTG-3'; CD38-E226D: top strand primer, 5'-CAGCACTTTTGGGAGTGTGGACGTCCATAATTTGCAACCAGAGAA-3'; bottom strand primer, 5'-TTCTCTGGTTGCAAATTATGGACGTCCACACTCCCAAAAGTGCTG-3'.
The positive clones containing the mutation were linearized by digesting the vector with SacI and using the linearized vector to transform electrocompetent X-33 yeast cells. The transformed cells were selected under 100 µg/ml zeocin. Colonies were formed between 3 and 4 days post-transfection, and six clones from each transfection were selected for expression screening by activity assays and SDS-PAGE. Previously established purification strategies were followed to get pure proteins (12, 16).
Before crystallization, the samples were concentrated to 8 mg/ml and stored at -80 °C. The E226Q mutant was crystallized based on a previously reported recipe (14). E226G and E226D crystals were formed by hanging drop vapor-diffusion technique. For E226G crystals, a 1-µl sample was mixed with a 1-µl reservoir that contains 100 mM MES, 13% PEG 4000, pH 6.0. For E226D crystals, the optimized crystallization condition was determined by increasing the concentration of PEG 4000 from 13 to 15%, while other conditions were the same as that for E226G crystals. At room temperature, crystals usable for data collection appeared within 1 week.
Preparation of cADPR, cGDPR, and NGD Complexes—NGD and cADPR were purchased from Sigma. cGDPR was synthesized by incubating NGD with Aplysia cyclase followed by HPLC column purification as described before (15). The formation of complexes by co-crystallization was not possible, as the residual hydrolysis activities of the mutants were sufficient to break down the ligands during the process. Instead, all complexes were obtained by soaking preformed crystals with solutions containing cADPR, cGDPR, or NGD. The soaking process was performed at 4 °C to prevent damage to the crystals, which could happen in minutes at room temperature. Specifically, the cADPR complexes were obtained by incubating E226Q, E226G, or E226D crystals with 48 mM cADPR solution buffered with the crystallization mother liquor for 3-8 min at 4 °C; the cGDPR complex was obtained by incubating E226Q crystals with 40 mM cGDPR for 5 min; and the NGD complex was obtained by incubating E226Q crystals with 20 mM NGD for 2 min. These soaked crystals were then quickly transferred and preserved in liquid nitrogen until data acquisition.
Data Collection, Reduction, and Structure Refinements—Crystallographic data were collected at the Cornell High-Energy Synchrotron Source A1 station with a fixed wavelength of 0.976 Å. Under the cryo-stream protection at 100 K, each crystal was rotated 360 degrees with 1° oscillation. Data sets were integrated and scaled by using the program package HKL2000 (17). The apo structure of shCD38 (PDB id 1YH3) was used as the initial model for structure solution by the molecular replacement method implemented in MOLREP (18). The initial model of cADPR was derived from its crystal structure (19). The NGD and cGDPR models were manually built in O (20) based on the electron densities and optimized with the program PRODRG (21). Subsequent crystallographic refinements and solvent addition were done with the program REFMAC (18) and ARP/WARP (22). The crystallographic statistics for data reduction and structure refinements are listed in Table 1.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Like wild-type shCD38, the shCD38 mutants were crystallized in space group P1 with two shCD38 molecules in the crystallographic asymmetric unit in a head-to-tail fashion (14). The structures of the complexes were determined by molecular replacement with the wild-type structure as the search model. In all three complex structures, cADPR, cGDPR, and NGD were bound tightly in the catalytic sites defined by the residues Trp125, Trp189, Glu146, Asp155, and Glu226 as proposed previously (12, 23). No secondary binding sites were seen for any of the substrates/products (cADPR, cGDPR, and NGD) either within the protein or between the interfaces of the two shCD38 molecules in the unit cell. This is consistent with our previous findings that NAD and ADPR bind to the same site of the enzyme (14). Therefore, the multiple catalytic reactions of shCD38 occur within the same active site.
The cyclic molecules, cADPR and cGDPR, possess common ribose and phosphate groups, but with different purine rings. The sites of cyclization are also different. In cADPR, the adenine ring is cyclized with the terminal ribose at its N-1 of the adenine, while in cGDPR, it is at the N-7 of the guanine. It is the same N-1/N-7-glycosidic bond that is cleaved, respectively, during the CD38-catalyzed hydrolysis. Consistently, as seen in the complexes (Fig. 1, A and B), the bound cADPR and cGDPR are oriented with the portions containing the N-1/N-7-glycosyl bonds embedded deep toward the bottom of the active site pocket where catalysis occurs.
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cADPR Activation—Human CD38 is the only human enzyme known to hydrolyze cADPR to ADPR. The refined E226Q-cADPR complex structure at 1.98-Å resolution is the first reported structure of cADPR complexed with a protein (Fig. 1A and Fig. 2). The high resolution diffraction data allowed unambiguous identification of the position and orientation of cADPR in the active site from the Fo-Fc omit map (Fig. 2).
The cADPR binding site in the E226Q mutant is formed by residues Arg127, Thr221, Glu146, Asp155, and Trp189. All these residues have direct polar or no-polar interactions with cADPR (Fig. 2). The adenine ring of cADPR adopts an orientation such that it has both hydrophobic interactions with the side chain of Trp189 and hydrophilic interactions with Glu146 and Asp155.
Although the N-1-glycosidic bond of the bound cADPR is facing and is close to the bottom of the active site pocket, it is striking that it is 7.9 Å away from the catalytic residue Gln226 (Glu226 in wild-type), too far to be attacked by it. That Glu226 is the catalytic residue has previously been established by both structural and mutagenesis studies (12, 14). The gap between cADPR and the catalytic residue is filled with five water molecules, which form a stable hydrogen bonds network to prevent the further entry of cADPR (Fig. 2). It is thus clear that this form represents an inactive complex between shCD38 and cADPR and is consistent with E226Q being catalytically impaired. In fact, this inactive form of complex is characteristic of all inactive mutants we have examined. The superposition of the cADPR complex structures obtained from three inactive mutants of shCD38, E226Q, E226G, and E226D reveals essentially the same cADPR structure. It appears, for these Glu226 mutants, that cADPR cannot be correctly positioned in their active sites in close enough distance to Glu226 for catalysis. Repeating the soaking experiments at room temperature gave similar results, indicating this form of cADPR binding is temperature-independent. With wild-type shCD38 crystals, however, even short soaking below 0 °C resulted in its hydrolysis to ADPR. The ability of wild-type shCD38 to hydrolyze cADPR demonstrates that Glu226 is not only catalytic but, surprisingly, is also essential for the binding of cADPR to an active position.
It should be noted that all inactive mutants have some residual catalytic activities (12), indicating that cADPR can transit from the inactive position in the complex to an active one, albeit at a very low rate. Once in the active position, it can then be readily hydrolyzed. This has been verified structurally, as the ADPR product is found in the active site during long term co-crystallization of E226Q with cADPR (14). The complexes of cADPR with the CD38 mutants thus reveal an unexpectedly active role of Glu226 in promoting the binding of cADPR to an active position in the catalytic pocket.
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There are two shCD38 E226Q molecules in the crystallographic asymmetric unit. We observed that cGDPR can only bind to the active site of molecule A. In the active site of molecule B, the electron density can only be modeled as solvent. The inability of molecule B to bind with cGDPR suggests its reduced affinity for cGDPR. It is thus interesting to see the structural differences between the active sites of the two E226Q molecules in the same crystallographic asymmetric unit (molecule A and B). This can be done by structurally aligning the two molecules. The alignment reveals that Glu146 in the apo structure (dark-green) is required to move away from the active site to make sufficient space for the binding of the guanine ring of cGDPR (Fig. 3B). This backward movement of Glu146 (from its dark-green position to gray position) can be as far as 2.66 Å, a distance corresponding to the length of a hydrogen bond (Fig. 3B). As a consequence of the backward shift of Glu146, its neighboring residue Asp147 retreats accordingly (from its dark-green position to gray position). Hence the comparison of the apo and the cGDPR-bound complex indicates the overall effect induced by cGDPR binding is the backward movement of the dipeptide Glu146-Asp147. This movement can be illustrated more dramatically by adding the cADPR complex to the alignment as shown in Fig. 3C. Residues Glu146 and Asp147 in the cADPR complex (magenta) adopt a position between the apo position and cGDPR-bound position. It can be concluded that the entry of either cADPR or cGDPR to the active site requires the consistent movements of Glu146 and Asp147, more extensive with cGDPR than with cADPR.
Comparing the cADPR and the cGDPR complexes, it can be seen that cGDPR bound much tighter then cADPR, with the ribosyl C1' atoms of cGDPR 
3.7 Å deeper into the active site. The tight association is due mainly to the fact that cGDPR is larger than cADPR, such that the guanine ring forms stable interactions with Trp189, Asp155, and Glu146 through both hydrophobic and hydrophilic interactions (Fig. 3A). Particularly is the extensive overlap between the tryptophan ring and the guanine ring with face-to-face 
- interactions between their five-membered rings. This tight binding likewise induces the much larger movement of the dipeptide Glu146-Asp147 than cADPR.
NGD Complex Together with cGDPR/NAD Complexes Defines a Site Responsible for Substrate Specificity—When NGD is used as substrate, human CD38 mainly cyclizes it to cGDPR (15). This is in contrast to when NAD is used as substrate, which is predominately hydrolyzed to ADPR. To find the structural basis for this disparity, we determined E226Q mutant complexed with its substrate NGD at 2.1-Å resolution.
Fig. 4A shows that the nicotinamide group of the bound NGD points toward Glu146 and Asp155 and forms two hydrogen bonds with them (Fig. 4A). The nicotinamide ribose and the diphosphate of NGD interact with protein residues in the same way as their corresponding parts in cGDPR (Figs. 3A and 4A). The Fo-Fc omit electron densities are clear for the nicotinamide terminus and the diphosphate, but not good for its guanine terminus (Fig. 4A). The disordered density reflects the flexibility of the guanine ring of the bound NGD, analogous to the poor density observed for the adenine terminus of bound NAD (14). Structural alignment of the NGD and NAD complexes indicates that NGD and NAD share a common interaction pattern with the enzyme (Fig. 4B). NGD overlaps quite well with NAD except for its guanine terminus. The similarity indicates the structural determinants responsible for the differences in the reactions between NGD and NAD are not in the nicotinamide end, but the purine rings instead. As described above, the guanine ring can form much tighter association with the active site (i.e. Trp189, Asp155, and Glu146) than adenine. Once folded back, the higher affinity should increase the probability of the guanine ring for coupling with the terminal ribose and thus facilitate the cyclization.
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Tightly Controlled cADPR Recognition and Catalysis—All the Glu226 mutants characterized so far are catalytically inactive (12), and the structures of the complex indeed show that cADPR is bound too far from the catalytic Glu226 to be activated. In order for the N-1 glycosidic bond to be cleavable, cADPR must bind closer, at least as close as seen in the cGDPR complex. This can be modeled by aligning the two complexes. All the protein residues of the complexes were fixed and only cADPR was mapped on cGDPR after proper translation and distortion. The nicotinamide ribose and diphosphate of cADPR were aligned with that of cGDPR because they are the structural determinants recognized by the active site as described above. The resultant cADPR model has correct geometry without obstructing contacts with protein residues (Fig. 5A). In this active cADPR model, Gln226 is close enough to form a hydrogen bond to the cADPR 3'-OH group. Ser193 is about3Åto the cADPR C-1' carbon, which is consistent with our previous studies showing that Ser193 is critical for catalysis and in the stabilization of the noncovalent intermediate (14).
From Fig. 5A and Fig. 3C, it can be seen that to be catalyzed, cADPR needs to bind at least 3.7-Å deeper into the active site. In this new position, the N-6 atom of cADPR forms two hydrogen bonds to Glu146 with distances of 2.81 Å and 2.69 Å. This close interaction with Glu146 is consistent with our previous finding that the residue is a critical determinant of the bifurcation between the cyclization and hydrolysis processes (23). Additionally, the 2'-, 3'-OH groups of cADPR are now close enough to form two hydrogen bonds with Glu226. This interaction with the ribose positions cADPR properly such that the carboxyl group of Glu226 is 3.3 Å to the C-1' of the ribose (Fig. 6A), making it suitable as a nucleophile. It can thus be inferred that the strong interactions between Glu226 and cADPR are not only critical for catalysis but also important for positioning cADPR to the catalytic position. Any change of the residue, as in the inactive mutants, renders cADPR incapable of binding deep enough into the active site for catalysis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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NAD enters the active site as a linear molecule with the nicotinamide end positioned and fixed by polar interactions with Asp155 and Glu146 (14). The nicotinamide ring is enforced parallel to the Trp189 ring by the ring-ring stacking interactions between them (Figs. 4A and 6A). Furthermore, the substrate recognition site, composed of residues Arg127, Ser126, Thr221, Phe222, Trp125, and Glu226, binds to the ribose-diphosphate portion of NAD. Meanwhile, the catalytic Glu226 forms two hydrogen bonds with 2'-and 3'-OH groups of the nicotinamide ribose, positioning its C-1' for nucleophilic attack by the OE2 of the carboxyl group of Glu226. The OE2 atom has two lone pairs. One is involved in the formation of H bond with 2'-OH; the other is used for attacking of C-1' carbon from the face of the ring. This results in the nicotinamide group disassociating from the substrate through an SN1 mechanism. The electron pair forming the C-1'-N bond will migrate to the nitrogen side, making C-1' positively charged and forming the oxocarbanium ion intermediate, which is stabilized by joint contributions from Glu226 and Ser193 (both are 3.3 Å away from C-1') (14). The formation of H bond between Glu226 OE2 and 2'-OH prevents a direct covalent linkage between C-1' and the OE2 of Glu226, and promotes the formation of an ionic intermediate instead. This could be the major structural differences between NAD utilizing enzymes and other glycosidases/glycohydrolases.
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Because of the affinity of the docking site formed by Trp125, Ser126, Arg127, and Phe222 (Fig. 4A) for the ribose-diphosphate motif, cADPR has a finite possibility of reentering the active site, resulting in its hydrolysis. The compact size of cADPR, however, makes its binding to the extended docking site not as optimal as for NAD, NGD, or cGDPR. The hydrogen bonding with the Glu226 becomes critical for cADPR to bind deep enough into the active site for catalysis (Fig. 5A). Otherwise, cADPR would be bound peripherally as observed in the complexes with the inactive mutants (Fig. 2). Similar to the process of cADPR formation, the binding of cADPR to its catalytic position also induces the retreat of the same dipeptide (Fig. 6A). Likewise, the cleavage of the N-glycosidic bond of cADPR by Glu226 proceeds also in much the same way as described above. After the bond cleavage, the forward movement of the dipeptide helps the release and unfolding of the adenine ring from the active site (Fig. 6A). Once again, the same ionic intermediate is formed. The unfolding of the adenine ring allows the entry of a water molecule nearby, whose lone pair electron serves as a nucleophile for attacking the intermediate, resulting in the formation of product ADPR (reaction 3). In contrast to cADPR, the linear substrate NAD binds favorably to the docking site and thus is readily hydrolyzed to ADPR (reaction 1, Fig. 6B), which is consistent with hydrolysis being the dominant reaction when NAD is used as a substrate.
As described, both the cyclization and hydrolysis reactions occur via the same intermediate and involve similar catalysis. They are not mutually exclusive, however, nor do they occur sequentially. Which one of the two reactions is dominant would depend on how accessible water is to the intermediate relative to the probability of intramolecular attack by the adenine.
In fact, the dependence on the accessibility of water of the catalysis outcome can explain why CD38 mainly cyclizes NGD instead of hydrolyzing it. As shown in the complexes with NGD and cGDPR, the guanine ring interacts strongly with Trp189, much more so than the adenine ring of cADPR. This higher affinity of the guanine ring is further enhanced by formation of three hydrogen bonds with Glu146, Asp155, and Wat2 (Fig. 3C). Once the nicotinamide ring of the substrate is released, the extensive overlap and stacking of the guanine ring with the tryptophan ring not only increase greatly the probability that the guanine ring is in position for reacting with the intermediate, but also decrease the water access at the same time. Consequently, cyclization is predominant over hydrolysis.
In addition to Glu226, Glu146 is also critically important. Its mutation does not eliminate enzymatic activities but alters the dominance of the reactions CD38 catalyzes. In addition to catalyzing the synthesis and hydrolysis of cADPR, CD38 also catalyzes the synthesis and hydrolysis of another calcium messenger, NAADP (7, 9). The latter occurs only at acidic pH. Changing Glu146 to a neutral residue allows the reactions to occur at neutral or alkaline pH (9). Structural characterizations indicate that the electrostatic repulsion between the Glu146 and the substrates that are negatively charged at neutral pH prohibits their binding and thus inhibits the reactions (9).
Mutation of Glu146 to many other residues, such as alanine, also greatly increases cyclization of NAD and inhibits its hydrolysis (23). This may well be related to the present finding that the dipeptide Glu146-Asp147 undergoes structural rearrangement upon the binding of cADPR or cGDPR (Fig. 3C). The change to alanine is likely to alleviate the strain on the dipeptide during the entry of the adenine ring to the active site, resulting in enhancement of the cyclization process. In any case, the movement of the dipeptide described here represents the first direct evidence for a conformational change at the active site of CD38 during catalysis.
Perhaps the most intriguing possibility raised by this study is that structural changes of the dipeptide may provide a means for regulating CD38 catalysis. It is entirely possible that protein factors or membrane lipids interacting with Asp147 of the dipeptide that is exposed to the surface of CD38 can induce conformational changes of the dipeptide and thus alter the catalysis.
Atomic coordinates and structure factors have been deposited at the Protein Data Bank with the accession codes of 2O3Q (E226Q cADPR complex), 2O3R (E226D-cADPR), 2O3S (E226G-cADPR complex), 2O3T (E226Q-cGDPR complex), and 2O3U (E226Q-NGD complex).
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FOOTNOTES |
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* This work was supported by Grants from the National Institutes of Health (to MacCHESS (RR01646) and H. C. L./Q. H. (GM061568)). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
1 To whom correspondence may be addressed. Tel.: 852-2819-9163; Fax: 852-2819-9230; E-mail: leehc{at}hku.hk.
2 To whom correspondence may be addressed. Tel.: 607-254-8983; Fax: 607-255-9001; E-mail: qh22{at}cornell.edu.
3 The abbreviations used are: cADPR, cyclic ADP-ribose; ADPR, ADP-ribose; NADP, nicotinamide adenine dinucleotide phosphate; NAADP, nicotinic acid adenine dinucleotide phosphate; NGD, nicotinamide guanine dinucleotide; cGDPR, cyclic GDP-ribose; MES, 4-morpholineethanesulfonic acid; PEG, polyethylene glycol; R.m.s., root mean square.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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