Identification of ADP-ribosylation sites of CD38 mutants by precursor ion scanning mass spectrometry.

Protein ADP-ribosylation, including mono- and poly-ADP-ribosylation, is increasingly recognized to play important roles in various biological pathways. Molecular understanding of the functions of ADP-ribosylation requires the identification of the sites of modification. Although tandem mass spectrometry (MS/MS) is widely recognized as an effective means for determining protein modifications, identification of ADP-ribosylation sites has been challenging due to the labile and hydrophilic nature of the modification. Here we applied precursor ion scanning-triggered MS/MS analysis on a hybrid quadrupole linear ion trap mass spectrometer for selectively detecting ADP-ribosylated peptides and determining the auto-ADP-ribosylation sites of CD38 (cluster of differentiation 38) E226D and E226Q mutants. CD38 is an enzyme that catalyzes the hydrolysis of nicotinamide adenine dinucleotide (NAD) to ADP-ribose. Here we show that NAD can covalently label CD38 E226D and E226Q mutants but not wild-type CD38. In this study, we have successfully identified the D226/Q226 and K129 residues of the two CD38 mutants being the ADP-ribosylation sites using precursor ion scanning hybrid quadrupole linear ion trap mass spectrometry. The results offer insights about the CD38 enzymatic reaction mechanism. The precursor ion scanning method should be useful for identifying the modification sites of other ADP-ribosyltransferases such as poly(ADP-ribose) polymerases.

Design, synthesis and biological characterization of novel inhibitors of CD38.

Human CD38 is a novel multi-functional protein that acts not only as an antigen for B-lymphocyte activation, but also as an enzyme catalyzing the synthesis of a Ca(2+) messenger molecule, cyclic ADP-ribose, from NAD(+). It is well established that this novel Ca(2+) signaling enzyme is responsible for regulating a wide range of physiological functions. Based on the crystal structure of the CD38/NAD(+) complex, we synthesized a series of simplified N-substituted nicotinamide derivatives (Compound 1-14). A number of these compounds exhibited moderate inhibition of the NAD(+) utilizing activity of CD38, with Compound 4 showing the highest potency. The crystal structure of CD38/Compound 4 complex and computer simulation of Compound 7 docking to CD38 show a significant role of the nicotinamide moiety and the distal aromatic group of the compounds for substrate recognition by the active site of CD38. Biologically, we showed that both Compounds 4 and 7 effectively relaxed the agonist-induced contraction of muscle preparations from rats and guinea pigs. This study is a rational design of inhibitors for CD38 that exhibit important physiological effects, and can serve as a model for future drug development.

Microcrystallography, high-pressure cryocooling and BioSAXS at MacCHESS.

The Macromolecular Diffraction Facility at the Cornell High Energy Synchrotron Source (MacCHESS) is a national research resource supported by the National Center for Research Resources of the US National Institutes of Health. MacCHESS is pursuing several research initiatives designed to benefit both CHESS users and the wider structural biology community. Three initiatives are presented in further detail: microcrystallography, which aims to improve the collection of diffraction data from crystals a few micrometers across, or small well diffracting regions of inhomogeneous crystals, so as to obtain high-resolution structures; pressure cryocooling, which can stabilize transient structures and reduce lattice damage during the cooling process; and BioSAXS (small-angle X-ray scattering on biological solutions), which can extract molecular shape and other structural information from macromolecules in solution.

Mechanism of cyclizing NAD to cyclic ADP-ribose by ADP-ribosyl cyclase and CD38.

Mammalian CD38 and its Aplysia homolog, ADP-ribosyl cyclase (cyclase), are two prominent enzymes that catalyze the synthesis and hydrolysis of cyclic ADP-ribose (cADPR), a Ca(2+) messenger molecule responsible for regulating a wide range of cellular functions. Although both use NAD as a substrate, the cyclase produces cADPR, whereas CD38 produces mainly ADP-ribose (ADPR). To elucidate the catalytic differences and the mechanism of cyclizing NAD, the crystal structure of a stable complex of the cyclase with an NAD analog, ribosyl-2'F-2'deoxynicotinamide adenine dinucleotide (ribo-2'-F-NAD), was determined. The results show that the analog was a substrate of the cyclase and that during the reaction, the nicotinamide group was released and a stable intermediate was formed. The terminal ribosyl unit at one end of the intermediate formed a close linkage with the catalytic residue (Glu-179), whereas the adenine ring at the other end stacked closely with Phe-174, suggesting that the latter residue is likely to be responsible for folding the linear substrate so that the two ends can be cyclized. Mutating Phe-174 indeed reduced cADPR production but enhanced ADPR production, converting the cyclase to be more CD38-like. Changing the equivalent residue in CD38, Thr-221 to Phe, correspondingly enhanced cADPR production, and the double mutation, Thr-221 to Phe and Glu-146 to Ala, effectively converted CD38 to a cyclase. This study provides the first detailed evidence of the cyclization process and demonstrates the feasibility of engineering the reactivity of the enzymes by mutation, setting the stage for the development of tools to manipulate cADPR metabolism in vivo.

Structural basis for enzymatic evolution from a dedicated ADP-ribosyl cyclase to a multifunctional NAD hydrolase.

Cyclic ADP-ribose (cADPR) is a universal calcium messenger molecule that regulates many physiological processes. The production and degradation of cADPR are catalyzed by a family of related enzymes, including the ADP-ribosyl cyclase from Aplysia california (ADPRAC) and CD38 from human. Although ADPRC and CD38 share a common evolutionary ancestor, their enzymatic functions toward NAD and cADPR homeostasis have evolved divergently. Thus, ADPRC can only generate cADPR from NAD (cyclase), whereas CD38, in contrast, has multiple activities, i.e. in cADPR production and degradation, as well as NAD hydrolysis (NADase). In this study, we determined a number of ADPRC and CD38 structures bound with various nucleotides. From these complexes, we elucidated the structural features required for the cyclization (cyclase) reaction of ADPRC and the NADase reaction of CD38. Using the structural approach in combination with site-directed mutagenesis, we identified Phe-174 in ADPRC as a critical residue in directing the folding of the substrate during the cyclization reaction. Thus, a point mutation of Phe-174 to glycine can turn ADPRC from a cyclase toward an NADase. The equivalent residue in CD38, Thr-221, is shown to disfavor the cyclizing folding of the substrate, resulting in NADase being the dominant activity. The comprehensive structural comparison of CD38 and APDRC presented in this study thus provides insights into the structural determinants for the functional evolution from a cyclase to a hydrolase.

Covalent and noncovalent intermediates of an NAD utilizing enzyme, human CD38.

Enzymatic utilization of nicotinamide adenine dinucleotide (NAD) has increasingly been shown to have fundamental roles in gene regulation, signal transduction, and protein modification. Many of the processes require the cleavage of the nicotinamide moiety from the substrate and the formation of a reactive intermediate. Using X-ray crystallography, we show that human CD38, an NAD-utilizing enzyme, is capable of catalyzing the cleavage reactions through both covalent and noncovalent intermediates, depending on the substrate used. The covalent intermediate is resistant to further attack by nucleophiles, resulting in mechanism-based enzyme inactivation. The noncovalent intermediate is stabilized mainly through H-bond interactions, but appears to remain reactive. Our structural results favor the proposal of a noncovalent intermediate during normal enzymatic utilization of NAD by human CD38 and provide structural insights into the design of covalent and noncovalent inhibitors targeting NAD-utilization pathways.

Hierarchical and helical self-assembly of ADP-ribosyl cyclase into large-scale protein microtubes.

Proteins are macromolecules with characteristic structures and biological functions. It is extremely challenging to obtain protein microtube structures through self-assembly as proteins are very complex and flexible. Here we present a strategy showing how a specific protein, ADP-ribosyl cyclase, helically self-assembles from monomers into hexagonal nanochains and further to highly ordered crystalline microtubes. The structures of protein nanochains and consequently self-assembled superlattice were determined by X-ray crystallography at 4.5 A resolution and imaged by scanning electron microscopy. The protein initially forms into dimers that have a fixed size of 5.6 nm, and then, helically self-assembles into 35.6 nm long hexagonal nanochains. One such nanochain consists of six dimers (12 monomers) that stack in order by a pseudo P6(1) screw axis. Seven nanochains produce a series of large-scale assemblies, nanorods, forming the building blocks for microrods. A proposed aging process of microrods results in the formation of hollow microstructures. Synthesis and characterization of large scale self-assembled protein microtubes may pave a new pathway, capable of not only understanding the self-assembly dynamics of biological materials, but also directing design and fabrication of multifunctional nanobuilding blocks with particular applications in biomedical engineering.

Conformational Closure of the Catalytic Site of Human CD38 Induced by Calcium (dagger) (double dagger).

First identified on the surface of lymphoids as a type II transmembrane protein, CD38 has now been established to have dual functions not only as a receptor but also as a multifunctional enzyme, catalyzing the synthesis of and hydrolysis of a general calcium messenger molecule, cyclic ADP-ribose (cADPR). The receptorial functions of CD38 include the induction of cell adhesion, differentiation, apoptosis, and cytokine production upon antibody ligation. Here we determined the crystal structure of calcium-loaded human CD38 at 1.45 A resolution which reveals that CD38 undergoes dramatic structural changes to an inhibited conformation in the presence of calcium. The structural changes are highly localized and occur in only two regions. The first region is part of the active site and consists of residues 121-141. In the presence of calcium, W125 moves 5 A into the active site and forms hydrophobic interactions with W189. The movement closes the active site pocket and reduces entry of substrates, resulting in inhibition of the enzymatic activity. The structural role of calcium in inducing these conformational changes is readily visualized in the crystal structure. The other region that undergoes calcium-induced changes is at the receptor region, where a highly ordered helix is unraveled to a random coil. The results suggest a novel conformational coupling mechanism, whereby protein interaction targeted at the receptor region can effectively regulate the enzymatic activity of CD38.

Conformational Closure of the Catalytic Site of Human CD38 Induced by Calcium.

First identified on the surface of lymphoids as a type II transmembrane protein, CD38 has now been established to have dual functions not only as a receptor but also as a multifunctional enzyme,catalyzing the synthesis of and hydrolysis of a general calcium messenger molecule, cyclic ADP-ribose(cADPR). The receptorial functions of CD38 include the induction of cell adhesion, differentiation,apoptosis, and cytokine production upon antibody ligation. Here we determined the crystal structure of calcium-loaded human CD38 at 1.45 A resolution which reveals that CD38 undergoes dramatic structural changes to an inhibited conformation in the presence of calcium. The structural changes are highly localized and occur in only two regions. The first region is part of the active site and consists of residues 121-141.In the presence of calcium, W125 moves 5 A into the active site and forms hydrophobic interactions with W189. The movement closes the active site pocket and reduces entry of substrates, resulting in inhibition of the enzymatic activity. The structural role of calcium in inducing these conformational changes is readily visualized in the crystal structure. The other region that undergoes calcium-induced changes is at the receptor region, where a highly ordered helix is unraveled to a random coil. The results suggest a novel conformational coupling mechanism, whereby protein interaction targeted at the receptor region can effectively regulate the enzymatic activity of CD38.

Catalysis-associated conformational changes revealed by human CD38 complexed with a non-hydrolyzable substrate analog.

Cyclic ADP-ribose (cADPR) is a calcium mobilization messenger important for mediating a wide range of physiological functions. The endogenous levels of cADPR in mammalian tissues are primarily controlled by CD38, a multifunctional enzyme capable of both synthesizing and hydrolyzing cADPR. In this study, a novel non-hydrolyzable analog of cADPR, N1-cIDPR (N1-cyclic inosine diphosphate ribose), was utilized to elucidate the structural determinants involved in the hydrolysis of cADPR. N1-cIDPR inhibits CD38-catalyzed cADPR hydrolysis with an IC(50) of 0.26 mM. N1-cIDPR forms a complex with CD38 or its inactive mutant in which the catalytic residue Glu-226 is mutated. Both complexes have been determined by x-ray crystallography at 1.7 and 1.76 A resolution, respectively. The results show that N1-cIDPR forms two hydrogen bonds (2.61 and 2.64 A) with Glu-226, confirming our previously proposed model for cADPR catalysis. Structural analyses reveal that both the enzyme and substrate cADPR undergo catalysis-associated conformational changes. From the enzyme side, residues Glu-146, Asp-147, and Trp-125 work collaboratively to facilitate the formation of the Michaelis complex. From the substrate side, cADPR is found to change its conformation to fit into the active site until it reaches the catalytic residue. The binary CD38-cADPR model described here represents the most detailed description of the CD38-catalyzed hydrolysis of cADPR at atomic resolution. Our structural model should provide insights into the design of effective cADPR analogs.

Structural basis for formation and hydrolysis of the calcium messenger cyclic ADP-ribose by human CD38.

Human CD38 is a multifunctional ectoenzyme responsible for catalyzing the conversions from nicotinamide adenine dinucleotide (NAD) to cyclic ADP-ribose (cADPR) and from cADPR to ADP-ribose (ADPR). Both cADPR and ADPR are calcium messengers that can mobilize intracellular stores and activate influx as well. In this study, we determined three crystal structures of the human CD38 enzymatic domain complexed with cADPR at 1.5-A resolution, with its analog, cyclic GDP-ribose (cGDPR) (1.68 A) and with NGD (2.1 A) a substrate analog of NAD. The results indicate that the binding of cADPR or cGDPR to the active site induces structural rearrangements in the dipeptide Glu(146)-Asp(147) by as much as 2.7 A) providing the first direct evidence of a conformational change at the active site during catalysis. In addition, Glu(226) is shown to be critical not only in catalysis but also in positioning of cADPR at the catalytic site through strong hydrogen bonding interactions. Structural details obtained from these complexes provide a step-by-step description of the catalytic processes in the synthesis and hydrolysis of cADPR.

Structural basis for the mechanistic understanding of human CD38-controlled multiple catalysis.

The enzymatic cleavage of the nicotinamide-glycosidic bond on nicotinamide adenine dinucleotide (NAD(+)) has been proposed to go through an oxocarbenium ion-like transition state. Because of the instability of the ionic intermediate, there has been no structural report on such a transient reactive species. Human CD38 is an ectoenzyme that can use NAD(+) to synthesize two calcium-mobilizing molecules. By using NAD(+) and a surrogate substrate, NGD(+), we captured and determined crystal structures of the enzyme complexed with an intermediate, a substrate, and a product along the reaction pathway. Our results showed that the intermediate is stabilized by polar interactions with the catalytic residue Glu(226) rather than by a covalent linkage. The polar interactions between Glu(226) and the substrate 2',3'-OH groups are essential for initiating catalysis. Ser(193) was demonstrated to have a regulative role during catalysis and is likely to be involved in intermediate stabilization. In addition, a product inhibition effect by ADP-ribose (through the reorientation of the product) or GDP-ribose (through the formation of a covalently linked GDP-ribose dimer) was observed. These structural data provide insights into the understanding of multiple catalysis and clues for drug design.

Acidic residues at the active sites of CD38 and ADP-ribosyl cyclase determine nicotinic acid adenine dinucleotide phosphate (NAADP) synthesis and hydrolysis activities.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a novel metabolite of NADP that has now been established as a Ca(2+) messenger in many cellular systems. Its synthesis is catalyzed by multifunctional enzymes, CD38 and ADP-ribosyl cyclase (cyclase). The degradation pathway for NAADP is unknown and no enzyme that can specifically hydrolyze it has yet been identified. Here we show that CD38 can, in fact, hydrolyze NAADP to ADP-ribose 2'-phosphate. This activity was low at neutrality but greatly increased at acidic pH. This novel pH dependence suggests that the hydrolysis is determined by acidic residues at the active site. X-ray crystallography of the complex of CD38 with one of its substrates, NMN, showed that the nicotinamide moiety was in close contact with Glu(146) at 3.27 A and Asp(155) at 2.52 A. Changing Glu(146) to uncharged Gly and Ala, and Asp(155) to Gln and Asn, by site-directed mutagenesis indeed eliminated the strong pH dependence. Changing Asp(155) to Glu, in contrast, preserved the dependence. The specificity of the two acidic residues was further demonstrated by changing the adjacent Asp(147) to Val, which had minimal effect on the pH dependence. Crystallography confirmed that Asp(147) was situated and directed away from the bound substrate. Synthesis of NAADP catalyzed by CD38 is known to have strong preference for acidic pH, suggesting that Glu(146) and Asp(155) are also critical determinants. This was shown to be case by mutagensis. Likewise, using similar approaches, Glu(98) of the cyclase, which is equivalent to Glu(146) in CD38, was found to be responsible for controlling the pH dependence of NAADP synthesis by the cyclase. Based on these findings, a catalytic model is proposed.

Structural and function analyses of the global regulatory protein SarA from Staphylococcus aureus.

The sarA locus in Staphylococcus aureus controls the expression of many virulence genes. The sarA regulatory molecule, SarA, is a 14.7-kDa protein (124 residues) that binds to the promoter region of target genes. Here we report the 2.6 A-resolution x-ray crystal structure of the dimeric winged helix SarA protein, which differs from the published SarA structure dramatically. In the crystal packing, multiple dimers of SarA form a scaffold, possibly via divalent cations. Mutations of individual residues within the DNA-binding helix-turn-helix and the winged region as well as within the metal-binding pocket implicate basic residues R84 and R90 within the winged region to be critical in DNA binding, whereas acidic residues D88 and E89 (wing), D8 and E11 (metal-binding pocket), and cysteine 9 are essential for SarA function. These data suggest that the winged region of the winged helix protein participates in DNA binding and activation, whereas the putative divalent cation binding pocket is only involved in gene function.

Crystal structure of human CD38 extracellular domain.

Human CD38 is a multifunctional protein involved in diverse functions. As an enzyme, it is responsible for the synthesis of two Ca2+ messengers, cADPR and NAADP; as an antigen, it is involved in regulating cell adhesion, differentiation, and proliferation. Besides, CD38 is a marker of progression of HIV-1 infection and a negative prognostic marker of B-CLL. We have determined the crystal structure of the soluble extracellular domain of human CD38 to 1.9 A resolution. The enzyme's overall topology is similar to the related proteins CD157 and the Aplysia ADP-ribosyl cyclase, except with large structural changes at the two termini. The extended positively charged N terminus has lateral associations with the other CD38 molecule in the crystallographic asymmetric unit. The analysis of the CD38 substrate binding models revealed two key residues that may be critical in controlling CD38's multifunctionality of NAD hydrolysis, ADP-ribosyl cyclase, and cADPR hydrolysis activities.

Crystal structure of a new class of glutathione transferase from the model human hookworm nematode Heligmosomoides polygyrus.

The crystal structure of GST Nu2-2 (HpolGSTN2-2) from the model hookworm nematode Heligmosomoides polygyrus has been solved by the molecular replacement method and refined to a resolution of 1.71 A, providing the first structural data from a class of nematode-specific GSTs. By structural alignment with two Sigma class GSTs, glutathione could be rationally docked into the G-site of the enzyme. By comparing with all mammalian GST classes, a novel, long, and deep cleft was identified at the H-site, providing a potential site for ligand binding. This new GST class may support the establishment of infection parasitic nematodes by passively neutralizing chemical toxins derived from host environment. The structure serves as a starting point for structure-based drug/inhibitor design that would aim to selectively disrupt nematode chemical defenses.

Crystal structure of a heat-resilient phytase from Aspergillus fumigatus, carrying a phosphorylated histidine.

In order to understand the structural basis for the high thermostability of phytase from Aspergillus fumigatus, its crystal structure was determined at 1.5 A resolution. The overall fold resembles the structure of other phytase enzymes. Aspergillus niger phytase shares 66% sequence identity, however, it is much less heat-resistant. A superimposition of these two structures reveals some significant differences. In particular, substitutions with polar residues appear to remove repulsive ion pair interactions and instead form hydrogen bond interactions, which stabilize the enzyme; the formation of a C-terminal helical capping, induced by arginine residue substitutions also appears to be critical for the enzyme's ability to refold to its active form after denaturation at high temperature. The heat-resilient property of A.fumigatus phytase could be due to the improved stability of regions that are critical for the refolding of the protein; and a heat-resistant A.niger phytase may be achieved by mutating certain critical residues with the equivalent residues in A.fumigatus phytase. Six predicted N-glycosylation sites were observed to be glycosylated from the experimental electron density. Furthermore, the enzyme's catalytic residue His59 was found to be partly phosphorylated and thus showed a reaction intermediate, providing structural insight, which may help understand the catalytic mechanism of the acid phosphatase family. The trap of this catalytic intermediate confirms the two-step catalytic mechanism of the acid histidine phosphatase family.

ADP-ribosyl cyclase; crystal structures reveal a covalent intermediate.

ADP-ribosyl cyclase catalyzes the elimination of nicotinamide from NAD and cyclization to cADPR, a known second messenger in cellular calcium signaling pathways. We have determined to 2.0 A resolution the structure of Aplysia cyclase with ribose-5-phosphate bound covalently at C3' and with the base exchange substrate (BES), pyridylcarbinol, bound to the active site. In addition, further refinement at 2.4 A resolution of the structure of nicotinamide-bound cyclase, which was previously reported, reveals that ribose-5-phosphate is also covalently bound in this structure, and a second nicotinamide site was identified. The structures of native and mutant Glu179Ala cyclase were also solved to 1.7 and 2.0 A respectively. It is proposed that the second nicotinamide site serves to promote cyclization by clearing the active site of the nicotinamide byproduct. Moreover, a ribosylation mechanism can be proposed in which the cyclization reaction proceeds through a covalently bound intermediate.