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.

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 Å-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 DNAbinding 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.

Serine hydroxymethyltransferase (SHMT) is a pyridoxal phosphate-dependent enzyme that catalyzes the reversible conversion of serine and tetrahydrofolate to glycine and methylenetetrahydrofolate. This reaction generates single carbon units for purine, thymidine, and methionine biosynthesis. The enzyme is a homotetramer comprising two obligate dimers and four pyridoxal phosphate-bound active sites. The mammalian enzyme is present in cells in both catalytically active and inactive forms. The inactive form is a ternary complex that results from the binding of glycine and 5-formyltetrahydrofolate polyglutamate, a slow tight-binding inhibitor. The crystal structure of a close analogue of the inactive form of murine cytoplasmic SHMT (cSHMT), lacking only the polyglutamate tail of the inhibitor, has been determined to 2.9 A resolution. This first structure of a ligand-bound mammalian SHMT allows identification of amino acid residues involved in substrate binding and catalysis. It also reveals that the two obligate dimers making up a tetramer are not equivalent; one can be described as "tight-binding" and the other as "loose-binding" for folate. Both active sites of the tight-binding dimer are occupied by 5-formyltetrahydrofolate (5-formylTHF), whose N5-formyl carbon is within 4 A of the glycine alpha-carbon of the glycine-pyridoxal phosphate complex; the complex appears to be primarily in its quinonoid form. In the loose-binding dimer, 5-formylTHF is present in only one of the active sites, and its N5-formyl carbon is 5 A from the glycine alpha-carbon. The pyridoxal phosphates appear to be primarily present as geminal diamine complexes, with bonds to both glycine and the active site lysine. This structure suggests that only two of the four catalytic sites on SHMT are catalytically competent and that the cSHMT-glycine-5-formylTHF ternary complex is an intermediate state analogue of the catalytic complex associated with serine and glycine interconversion.

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. 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.

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 ADPribose (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-2F-2de-oxynicotinamide 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.Cyclic ADP-ribose is a novel cyclic nucleotide with Ca 2ϩ -mobilizing activity targeting the endoplasmic reticulum. Its activity was first described in sea urchin eggs (1, 2), and cADPR 3 has since been established as a second messenger molecule responsible for regulating a wide range of physiological functions, from fission in the dinoflagellate (3) to social behavior in mice (Ref. 4 and reviewed in Refs. 5 and 6). The Aplysia ADPribosyl cyclase (cyclase) was the first protein identified that uses NAD, a linear substrate, and ligates its two ends to produce cADPR, with the release of the terminal moiety, nicotinamide (7). The cyclase is a soluble protein of 30 kDa and is present in large amounts in Aplysia ovotestis (7). It is also present in the neurons of the Aplysia buccal ganglion, where it is responsible for the synthesis of endogenous cADPR and the regulation of the evoked synaptic transmission (8). Recently, it is shown that depolarization of Aplysia neurons induces the translocation of the cyclase from the cytosol into the nucleus, providing a mechanism for fine tuning of nuclear Ca 2ϩ signals in neurons (9). CD38 is the major mammalian homolog of the cyclase and is responsible for regulating a wide range of physiological functions. Deletion of the CD38 gene in mice produces multiple defects, including impairment of insulin secretion (10), neutrophil chemota...

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 and has since been shown in a wide variety of cell types, from plants to human (reviewed in Refs. 3-5). NAADP is endogenously present in cells and its level is modulated by various agonists and stimuli, establishing its role as a second messenger (Refs. 6 and 7 and reviewed in Refs. 8 and 9). In contrast to the widespread functional activity of NAADP in cells, only two homologous proteins, mammalian CD38 and Aplysia ADP-ribosyl cyclase, have so far been identified as enzymes responsible for the synthesis of NAADP (10). CD38 is a membrane-bound protein first thought of as a lymphocyte antigen (reviewed in Refs. 11-13) but has since been found to be widely expressed in virtually all tissues examined (14 -17). It is present on the cell surface in some cases but is also localized in various intracellular organelles (15, 18 -20). The Aplysia ADP-ribosyl cyclase, on the other hand, is a soluble protein that shares about 30% sequence identity with CD38 (21) and is found in large quantities in the Aplysia ovotestis (22, 23). Both CD38 and the cyclase are novel multifunctional enzymes capable of synthesizing NAADP from NADP by catalyzing the exchange of nicotinamide in NADP with nicotinic acid (10). Both can also cyclize NAD to produce cyclic ADP-ribose (cADPR) (10, 24), another novel Ca 2ϩ messenger (reviewed in Refs. 3-5). The crystal structures of both proteins have been solved (25,26) and the catalytic glutamate residues at their enzymatic active sites identified by site-directed mutagenesis (27,28). Expectedly, a large degree of structural homology is observed between them (26).As a second messenger, NAADP levels in cells are expected to respond to physiological stimuli. Indeed, agonists and stimuli are shown to activate rapid and transient changes in NAADP levels (7,29,30), indicating the presence in cells of efficient pathways for not only the synthesis but also the degradation of NAADP. As a nucleotide, NAADP is sensitive to degradative enzymes, such as nucleotide phosphodiesterase or alkaline phosphatase (31,32). Whether these general enzymes are responsible for its degradation in cells is not known. NAADP is insensitive to regular NADase (32), and so far, no enzyme has yet been identified that can specifically hydrolyze NAADP. Here we show, unexpectedl...

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.

SUMMARY 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 non-covalent intermediates, depending on the substrate used. The covalent intermediate is resistant to further attack by nucleophiles, resulting in mechanism-based enzyme inactivation. The non-covalent intermediate is stabilized mainly through H-bond interactions, but appears to remain reactive. Our structural results favor the proposal of a non-covalent intermediate during normal enzymatic utilization of NAD by human CD38 and provide structural insights into the design of covalent and non-covalent inhibitors targeting NAD utilization pathways.