NM23͞NDP kinases play an important role in development and cancer but their biological function is unknown, despite an intriguing collection of biochemical properties including nucleosidediphosphate kinase (NDP kinase), DNA binding and transcription, a mutator function, and cleavage of unusually structured DNA by means of a covalent enzyme-DNA complex. To assess the role of the nuclease in human NM23-H2, we sought to identify the amino acid responsible for covalent catalysis. By sequencing a DNA-linked peptide and by site-directed mutagenesis, we identified lysine-12, a phylogenetically conserved residue, as the amino acid forming the covalent complex with DNA. In particular, the -amino group acts as the critical nucleophile, because substitution with glutamine but not arginine completely abrogated covalent adduct formation and DNA cleavage, whereas the DNA-binding properties remained intact. These findings and chemical modification data suggest that phosphodiester-bond cleavage occurs by a DNA glycosylase͞lyase-like mechanism known as the signature of base excision DNA repair nucleases. Involvement of NM23͞NDP kinase in a DNA repair pathway would be consistent with its role in normal and tumor cell development. Additionally, lysine-12, which is known in the x-ray crystallographic structure to lie in the catalytic pocket involved in the NDP kinase phosphorylation reaction, was found essential also for the NDP kinase activity of NM23-H2, suggesting that the two catalytic activities of NM23-H2 are fundamentally connected. N M23 is a large family of structurally and functionally conserved proteins consisting of four to six identical subunits of 16 to 20 kDa each, known also as NDP kinases (nucleoside diphosphate kinase; EC 2.7.4.6; refs. 1-4). In Escherichia coli, the gene for NDP kinase functions as a mutator gene (5), whereas the homologue in Drosophila, known as altered wing discs (AWD), is required for development (6). Multiple NM23͞ NDP kinase genes exist in vertebrates, including six in humans (NM23-H1 to NM23-H8; ref. 7), where they also play a role in development (8). And, NM23-H1 and NM23-H2 have both been implicated in the metastasis (9, 10) and pathogenesis (11, 12) of tumors.NDP kinases catalyze phosphoryl-group transfer between nucleoside di-and triphosphates through a conserved histidine as the phosphorylated intermediate (1-3), and they play a role in maintaining intracellular nucleotide concentrations. NDP kinases have broad substrate specificities and well understood catalytic mechanisms (13), and several crystal structures, both with and without bound nucleotide substrates, have been solved, including two of human NM23-H2͞NDP kinase B (14, 15).It has generally been assumed, however, that NM23͞NDP kinases are far more than housekeeping enzymes, because experiments in vivo have indicated the existence of additional mechanisms (16,17). In 1993, our laboratory reported that NM23-H2͞NDP kinase B is a DNA-binding protein identical to the c-MYC transcription factor PuF, suggesting that NM23-H2͞ PuF may r...
Adnectins are targeted biologics derived from the tenth type III domain of human fibronectin (¹⁰Fn3), a member of the immunoglobulin superfamily. Target-specific binders are selected from libraries generated by diversifying the three ¹⁰Fn3 loops that are analogous to the complementarity determining regions of antibodies. The crystal structures of two Adnectins were determined, each in complex with its therapeutic target, EGFR or IL-23. Both Adnectins bind different epitopes than those bound by known monoclonal antibodies. Molecular modeling suggests that some of these epitopes might not be accessible to antibodies because of the size and concave shape of the antibody combining site. In addition to interactions from the Adnectin diversified loops, residues from the N terminus and/or the β strands interact with the target proteins in both complexes. Alanine-scanning mutagenesis confirmed the calculated binding energies of these β strand interactions, indicating that these nonloop residues can expand the available binding footprint.
CD40–CD40L interactions play a critical role in regulating immune responses. Blockade of CD40L by Abs, such as the anti-CD40L Ab 5c8, demonstrated positive clinical effects in patients with autoimmune diseases; however, incidents of thromboembolism (TE) precluded further development of these molecules. In this study, we examined the role of the Fc domain interaction with FcγRs in modulating platelet activation and potential for TE. Our results show that the interaction of the 5c8 wild-type IgG1 Fc domain with FcγRs is responsible for platelet activation, as measured by induction of PAC-1 and CD62P. A version of 5c8 with a mutated IgG1 tail was identified that showed minimal FcγR binding and platelet activation while maintaining full binding to CD40L. To address whether Fc effector function is required for immunosuppression, a potent Ab fragment, termed a “domain Ab” (dAb), against murine CD40L was identified and fused to a murine IgG1 Fc domain containing a D265A mutation that lacks Fc effector function. In vitro, this dAb–Fc demonstrated comparable potency to the benchmark mAb MR-1 in inhibiting B cell and dendritic cell activation. Furthermore, the anti-CD40L dAb–Fc exhibited a notable efficacy comparable to MR-1 in various preclinical models, such as keyhole limpet hemocyanin–induced Ab responses, alloantigen-induced T cell proliferation, “heart-to-ear” transplantation, and NZB × NZW F1 spontaneous lupus. Thus, our data show that immunosuppression and TE can be uncoupled and that a CD40L dAb with an inert Fc tail is expected to be efficacious for treating autoimmune diseases, with reduced risk for TE.
Nucleoside-diphosphate (NDP) kinase (NTP:nucleoside-diphosphate phosphotransferase) catalyzes the reversible transfer of ␥-phosphates from nucleoside triphosphates to nucleoside diphosphates through an invariant histidine residue. It has been reported that the high-energy phosphorylated enzyme intermediate exhibits a protein phosphotransferase activity toward the protein histidine kinases CheA and EnvZ, members of the two-component signal transduction systems in bacteria. Here we demonstrate that the apparent protein phosphotransferase activity of NDP kinase occurs only in the presence of ADP, which can mediate the phosphotransfer from the phospho-NDP kinase to the target enzymes in catalytic amounts (ϳ1 nM). These findings suggest that the protein kinase activity of NDP kinase is probably an artifact attributable to trace amounts of contaminating ADP. Additionally, we show that Escherichia coli NDP kinase, like its human homologue NM23-H2/PuF/NDP kinase B, can bind and cleave DNA. Previous in vivo functions of E. coli NDP kinase in the regulation of gene expression that have been attributed to a protein phosphotransferase activity can be explained in the context of NDP kinase-DNA interactions. The conservation of the DNA binding and DNA cleavage activities between human and bacterial NDP kinases argues strongly for the hypothesis that these activities play an essential role in NDP kinase function in vivo. Nucleoside-diphosphate (NDP)1 kinase (EC 2.7.4.6) catalyzes the exchange of a ␥-phosphate between nucleoside triand diphosphates via a ping-pong mechanism with a highenergy phosphohistidine intermediate:where Mg⅐NDP and Mg⅐NЈTP are the Mg 2ϩ complexes with nucleoside (or 2Ј-deoxynucleoside) di-and triphosphates. Histidine phosphorylation by Mg⅐NTP is fully reversible with an equilibrium constant of about 0.25, and it is very efficient and has a turnover number Ͼ 1000 s Ϫ1 . Thus, the phosphorylation and dephosphorylation steps take less than 1 ms, whereas the phosphorylated form is stable for a few hours in the absence of a nucleoside diphosphate acceptor (catalytic mechanism is reviewed in Ref. 1). NDP kinases are a large family of proteins found in many organisms with a high level of sequence and structure homology throughout the whole family. The enzyme is composed of four or six identical subunits (16 -20 kDa each) with an ␣/ sandwich or ferredoxin fold (reviewed in Ref. 2). In both crystals and solution, NDP kinases exist in two different quaternary structures: eukaryotic enzymes are hexamers (a trimer of dimers with dihedral D3 symmetry), and some bacterial enzymes are tetramers (a pair of dimers with pyramidal D2 symmetry) (3). Each subunit in the hexamer or tetramer has an independent active center that comprises the nucleophilic histidine and the nucleotide-binding site able to bind both di-and triphosphates. The binding site forms a cleft on the protein surface about 20 Å long, 6 Å wide, and 10 Å deep, with the phosphoaccepting histidine located at the bottom of this cleft. Amino acid compositions o...
Escherichia coli nucleoside diphosphate kinase (eNDK) is an XTP:XDP phosphotransferase that plays an important role in the regulation of cellular nucleoside triphosphate concentrations. It is also one of several recently discovered DNases belonging to the NM23͞NDK family. E. coli cells disrupted in the ndk gene display a spontaneous mutator phenotype, which has been attributed to the mutagenic effects of imbalanced nucleotide pools and errors made by replicative DNA polymerases. Another explanation for the increased mutation rates is that endk ؊ cells lack the nuclease activity of the NDK protein that is essential for a DNA repair pathway. Here, we show that purified, cloned endk is a DNA repair nuclease whose substrate is uracil misincorporated into DNA. We have identified three new catalytic activities in eNDK that act sequentially to repair the uracil lesion: (i) uracil-DNA glycosylase that excises uracil from single-stranded and from U͞A and U͞G mispairs in double-stranded DNA; (ii) apyrimidinic endonuclease that cleaves double-stranded DNA as a lyase by forming a covalent enzyme-DNA intermediate complex with the apyrimidinic site created by the glycosylase; and (iii) DNA repair phosphodiesterase that removes 3-blocking residues from the ends of duplex DNA. All three of these activities, as well as the nucleoside-diphosphate kinase, reside in the same protein. Based on these findings, we propose an editing function for eNDK as a mechanism by which the enzyme prevents mutations in DNA. N ucleoside diphosphate kinases (NDKs) (EC 2.7.4.6) have been recognized for decades as a large and conserved family of enzymes that synthesize nucleoside triphosphates from nucleoside diphosphates and ATP. Given their broad substrate specificities and the nucleotide pool perturbations caused by the absence of NDK in Escherichia coli, it has been suggested that their function in vivo is to maintain the necessary nucleoside triphosphate concentrations (1, 2). The x-ray crystal structures of many NDKs, with and without nucleotide substrates, are well known (3). During the last decade, additional DNA metabolic activities have been demonstrated for NDKs, including sequence-dependent DNA binding and transcription (4-8) and site-specific cleavage of . Moreover, the DNase activity of human NM23-H2͞NDK (h2NDK) has been implicated in base excision repair (BER) on the basis of its DNA cleavage mechanism, which uses the -amino group of a conserved lysine as the active site nucleophile forming a covalent Schiff base intermediate with DNA, the hallmark mechanism of DNA glycosylase͞apyrimidinic (AP) lyase BER enzymes (10,14,15). Thus far, however, h2NDK has been associated only with the cleavage of unusually structured but otherwise undamaged DNA (9-11, 14), and not with the repair of specific DNA lesions.Mammalian NDKs are also known as NM23 (nonmetastatic) on the basis of their involvement in tumorigenesis and tumor progression (16,17). The Drosophila NDK is AWD, a developmental protein encoded by the altered wing disk gene (18). In E. col...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.