Botulinum neurotoxin D uses synaptic vesicle protein SV2 and gangliosides as receptors

Botulinum neurotoxins (BoNTs) include seven bacterial toxins (BoNT/A-G) that target presynaptic terminals and act as proteases cleaving proteins required for synaptic vesicle exocytosis. Here we identified synaptic vesicle protein SV2 as the protein receptor for BoNT/D. BoNT/D enters cultured hippocampal neurons via synaptic vesicle recycling and can bind SV2 in brain detergent extracts. BoNT/D failed to bind and enter neurons lacking SV2, which can be rescued by expressing one of the three SV2 isoforms (SV2A/B/C). Localization of SV2 on plasma membranes mediated BoNT/D binding in both neurons and HEK293 cells. Furthermore, chimeric receptors containing the binding sites for BoNT/A and E, two other BoNTs that use SV2 as receptors, failed to mediate the entry of BoNT/D suggesting that BoNT/D binds SV2 via a mechanism distinct from BoNT/A and E. Finally, we demonstrated that gangliosides are essential for the binding and entry of BoNT/D into neurons and for its toxicity in vivo, supporting a double-receptor model for this toxin.     

Nucleotide interactions with membrane-bound transporter associated with antigen processing proteins

The transporter associated with antigen processing (TAP) contains two nucleotide-binding domains (NBD) in the TAP1 and TAP2 subunits. When expressed as individual subunits or domains, TAP1 and TAP2 NBD differ markedly in their nucleotide binding properties. We investigated whether the two nucleotide-binding sites of TAP1/TAP2 complexes also differed in their nucleotide binding properties. To facilitate electrophoretic separation of the subunits when in complex, we used TAP complexes in which one of the subunits was expressed as a fluorescent protein fusion construct. In binding experiments at 4 degrees C using the photo-cross-linkable nucleotide analogs 8-azido-[gamma-(32)P]ATP and 8-azido-[alpha-(32)P]ADP, TAP2 was found to have reduced affinity for nucleotides compared with TAP1, when the two proteins were separately expressed. Complex formation with TAP1 enhanced the binding affinity of the TAP2 nucleotide-binding site for both nucleotides. Binding analyses with mutant TAP complexes that are deficient in nucleotide binding at one or both sites provided evidence for the existence of two ATP-binding sites with relatively similar affinities in TAP1/TAP2 complexes. TAP1/TAP2 NBD interactions appear to contribute at least in part to enhanced nucleotide binding at the TAP2 site upon TAP1/TAP2 complex formation. Binding analyses with mutant TAP complexes also demonstrate that the extent of TAP1 labeling is dependent upon the presence of a functional TAP2 nucleotide-binding site.     

Signals involved in targeting membrane proteins to synaptic vesicles

1. Synaptic vesicles (SVs) mediate fast regulated secretion of classical neurotransmitters. In order to perform their task SVs rely on a restrict set of membrane proteins. The mechanisms responsible for targeting these proteins to the SV membrane are still poorly understood.2. Likewise, little is known about the intracellular routes taken by these proteins in their way to SV membrane. Recently, several domains and motifs necessary for correct localization of SV proteins have been identified.3. In this review we summarize the sequence motifs that have been identified in the cytoplasmic domains of SV proteins that are involved in endocytosis and targeting of SVs. We suggest that the vesicular acetylcholine transporter, a protein found predominantly in synaptic vesicles, is perhaps a model protein to understand the pathways and interactions that are used for synaptic vesicle targeting.     

UNC-31/CAPS docks and primes dense core vesicles in C. elegans neurons

UNC-31 or its mammalian homologue, Ca²⁺-dependent activator protein for secretion (CAPS), is indispensable for exocytosis of dense core vesicle (DCV) and synaptic vesicle (SV). From N- to the C-terminus, UNC-31 contains putative functional domains, including dynactin 1 binding domain (DBD), C2, PH, (M)UNC-13 homology domain (MHD) and DCV binding domain (DCVBD), the last four we examined in this study. We employed UNC-31 null mutant C. elegans worms to examine whether UNC-31 functions could be rescued by ectopic expression of full length UNC-31 vs each of these four domain-deleted mutants. Full length UNC-31 cDNA rescued the phenotypes of C. elegans null mutants in response to Ca²⁺-elevation in ALA neurons. Surprisingly, MHD deletion also rescued UNC-31 exocytotic function in part because the relatively high Ca²⁺ level (pre-flash Ca²⁺ was 450 nM) used in the capacitance study could bypass the MHD defect. Nonetheless, the three other domain-truncation cDNAs had almost no rescue on Ca²⁺ evoked secretion. Importantly, this genetic null mutant rescue strategy enabled physiological studies at levels of whole organism to single cells, such as locomotion assay, pharmacological study of neurotransmission at neuromuscular junction, in vivo neuropeptide release measurement and analysis of vesicular docking. Our results suggest that each of these UNC-31 domains support distinct sequential molecular actions of UNC-31 in vesicular exocytosis, including steps in vesicle tethering and docking that bridge vesicle with plasma membrane, and subsequently priming vesicle by initiating the formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) core complex.     

The synaptic vesicle protein 2C mediates the uptake of botulinum neurotoxin A into phrenic nerves

Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release by selectively cleaving core components of the vesicular fusion machinery. The synaptic vesicle proteins Synaptotagmin-I and -II act as receptors for BoNT/B and BoNT/G. Here we show that BoNT/A also interacts with a synaptic vesicle protein, the synaptic vesicle glycoprotein 2C (SV2C), but not with the homologous proteins SV2A and SV2B. Binding of BoNT/A occurs at the membrane juxtaposed region preceding transmembrane domain 8. A peptide comprising the intravesicular domain between transmembrane domains 7 and 8 specifically reduces the neurotoxicity of BoNT/A at phrenic nerve preparations demonstrating the physiological relevance of this interaction.     

CAPS Activity in Priming Vesicle Exocytosis Requires CK2 Phosphorylation

CAPS (Ca²⁺-dependent activator protein for secretion) functions in priming Ca²⁺-dependent vesicle exocytosis, but the regulation of CAPS activity has not been characterized. Here we show that phosphorylation by protein kinase CK2 is required for CAPS activity. Dephosphorylation eliminated CAPS activity in reconstituting Ca²⁺-dependent vesicle exocytosis in permeable and intact PC12 cells. Ser-5, -6, and -7 and Ser-1281 were identified by mass spectrometry as the major phosphorylation sites in the 1289 residue protein. Ser-5, -6, and -7 but not Ser-1281 to Ala substitutions abolished CAPS activity. Protein kinase CK2 phosphorylated CAPS in vitro at these sites and restored the activity of dephosphorylated CAPS. CK2 is the likely in vivo CAPS protein kinase based on inhibition of phosphorylation by tetrabromo-2-benzotriazole in PC12 cells and by the identity of in vivo and in vitro phosphorylation sites. CAPS phosphorylation by CK2 was constitutive, but the elevation of Ca²⁺ in synaptosomes increased CAPS Ser-5 and -6 dephosphorylation, which terminates CAPS activity. These results identify a functionally important N-terminal phosphorylation site that regulates CAPS activity in priming vesicle exocytosis.Regulated neurotransmitter secretion is central to intercellular communication in the nervous system. Two types of secretory vesicles mediate neurotransmitter release; that is, synaptic vesicles that release transmitters such as glutamate at synapses and dense-core vesicles that release modulatory transmitters and neuropeptides at non-synaptic sites. Both types of secretory vesicles are recruited to docking sites on the plasma membrane where they are primed to a ready release state to undergo fusion in response to Ca²⁺ elevations. Many of the proteins that mediate the targeting, docking, priming, and Ca²⁺-dependent fusion of vesicles with the plasma membrane function in both synaptic vesicle and dense-core vesicle pathways (1). CAPS-1² (also known as Cadps1) is a 1289-residue protein that reconstitutes Ca²⁺-triggered dense-core vesicle exocytosis in permeable neuroendocrine cells at a priming step (2–4). CAPS is required for secretion of a subset of transmitters in Caenorhabditis elegans (5) and Drosophila melanogaster (6) and for priming dense-core vesicle exocytosis in neuroendocrine cells (7) and synaptic vesicle exocytosis in neurons (8). Vesicle priming reactions are extensively modulated during physiological demand (9), but mechanisms that regulate CAPS function remain to be identified.Reversible protein phosphorylation is a major mechanism for the regulation of cellular processes including vesicle exocytosis. Many proteins that function in evoked vesicle exocytosis are phosphoproteins (10, 11). The neuronal SNARE proteins syntaxin 1A, VAMP-2, and SNAP-25 are phosphorylated by several protein kinases in vitro (12–14). Protein kinase C and protein kinase A sites on SNAP-25 affect refilling rates and size, respectively, of the primed pool of vesicles in chromaffin cells (15, 16). Several SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-binding proteins such as munc18, RIM1, and rabphilin undergo regulated phosphorylation, but it is not known whether phosphorylation affects function (10, 11, 17).Because the function of CAPS at a priming step in vesicle exocytosis may be regulated, we determined whether CAPS is phosphorylated. We show that CAPS is a phosphoprotein with functionally essential N-terminal phosphorylated Ser residues. Ser-5, -6, and -7 in CAPS were substrates for protein kinase CK2 in vitro and in vivo as well as for a Ca²⁺-dependent dephosphorylation mechanism. The results indicate that phosphorylation by protein kinase CK2 is necessary for CAPS activity in priming vesicle exocytosis and that regulated dephosphorylation may constitute a mechanism for terminating CAPS activity.     

SV2 mediates entry of tetanus neurotoxin into central neurons

Tetanus neurotoxin causes the disease tetanus, which is characterized by rigid paralysis. The toxin acts by inhibiting the release of neurotransmitters from inhibitory neurons in the spinal cord that innervate motor neurons and is unique among the clostridial neurotoxins due to its ability to shuttle from the periphery to the central nervous system. Tetanus neurotoxin is thought to interact with a high affinity receptor complex that is composed of lipid and protein components; however, the identity of the protein receptor remains elusive. In the current study, we demonstrate that toxin binding, to dissociated hippocampal and spinal cord neurons, is greatly enhanced by driving synaptic vesicle exocytosis. Moreover, tetanus neurotoxin entry and subsequent cleavage of synaptobrevin II, the substrate for this toxin, was also dependent on synaptic vesicle recycling. Next, we identified the potential synaptic vesicle binding protein for the toxin and found that it corresponded to SV2; tetanus neurotoxin was unable to cleave synaptobrevin II in SV2 knockout neurons. Toxin entry into knockout neurons was rescued by infecting with viruses that express SV2A or SV2B. Tetanus toxin elicited the hyper excitability in dissociated spinal cord neurons - due to preferential loss of inhibitory transmission - that is characteristic of the disease. Surprisingly, in dissociated cortical cultures, low concentrations of the toxin preferentially acted on excitatory neurons. Further examination of the distribution of SV2A and SV2B in both spinal cord and cortical neurons revealed that SV2B is to a large extent localized to excitatory terminals, while SV2A is localized to inhibitory terminals. Therefore, the distinct effects of tetanus toxin on cortical and spinal cord neurons are not due to differential expression of SV2 isoforms. In summary, the findings reported here indicate that SV2A and SV2B mediate binding and entry of tetanus neurotoxin into central neurons.     

A role for synaptotagmin (p65) in regulated exocytosis

Proteins that are specifically localized to synaptic vesicles in the nervous system have been proposed to mediate aspects of synaptic transmission. Antibodies raised against the cytoplasmic domains of five of these proteins, vamp, rab3A, synaptophysin, synaptotagmin, and SV2, were used to investigate their function. Microinjection of monoclonal and polyclonal antibodies raised against synaptotagmin (p65), but not the other vesicle proteins, decreases K⁺/Ca²⁺-mediated dopamine β-hydroxylase surface staining, a measure of regulated secretion in PC12 cells. Microinjection of a soluble fragment of synaptotagmin encompassing one of the domains homologous to the C2 regulatory region of protein kinase C, but lacking the membrane anchor, also inhibits evoked dopamine β-hydroxylase surface staining. These results provide support for the hypothesis that synaptotagmin, a Ca²⁺- and phospholipid-binding protein, is important for regulated exocytosis in neurons.     

Levetiracetam reverses synaptic deficits produced by overexpression of SV2A

Levetiracetam is an FDA-approved drug used to treat epilepsy and other disorders of the nervous system. Although it is known that levetiracetam binds the synaptic vesicle protein SV2A, how drug binding affects synaptic functioning remains unknown. Here we report that levetiracetam reverses the effects of excess SV2A in autaptic hippocampal neurons. Expression of an SV2A-EGFP fusion protein produced a ～1.5-fold increase in synaptic levels of SV2, and resulted in reduced synaptic release probability. The overexpression phenotype parallels that seen in neurons from SV2 knockout mice, which experience severe seizures. Overexpression of SV2A also increased synaptic levels of the calcium-sensor protein synaptotagmin, an SV2-binding protein whose stability and trafficking are regulated by SV2. Treatment with levetiracetam rescued normal neurotransmission and restored normal levels of SV2 and synaptotagmin at the synapse. These results indicate that changes in SV2 expression in either direction impact neurotransmission, and suggest that levetiracetam may modulate SV2 protein interactions.     

Association of botulinum neurotoxin serotypes a and B with synaptic vesicle protein complexes

Botulinum neurotoxins (BoNTs) elicit flaccid paralysis through cleavage of SNARE proteins within peripheral neurons. There are seven serotypes of the BoNTs, termed A-G, which differ in the SNARE protein and/or site that is cleaved. BoNTs are single-chain toxins that comprise an N-terminal zinc metalloprotease domain that is disulfide linked to the C-terminal translocation/receptor binding domain. SV2 and synaptotagmin have been identified as receptors for BoNT serotypes A and B, respectively. Using affinity chromatography, BoNTs A and B were observed to bind synaptic vesicle protein complexes in synaptosome lysates. Tandem LC-MS/MS identified SV2, synaptotagmin I, synaptophysin, vesicle-associated membrane protein 2 (VAMP2), and the vacuolar proton pump as components of the BoNT-receptor complex. Density gradient analysis showed that BoNT serotypes A and B exhibited unique interactions with the synaptic vesicle protein complexes. The association of BoNT serotypes A and B with synaptic vesicle protein complexes implicates a physiological role for protein complexes in synaptic vesicle biology and provides insight into the interactions of BoNT and neuronal receptors.     

Molecular machines in the synapse: overlapping protein sets control distinct steps in neurosecretion

Activity regulated neurotransmission shapes the computational properties of a neuron and involves the concerted action of many proteins. Classical, intuitive working models often assign specific proteins to specific steps in such complex cellular processes, whereas modern systems theories emphasize more integrated functions of proteins. To test how often synaptic proteins participate in multiple steps in neurotransmission we present a novel probabilistic method to analyze complex functional data from genetic perturbation studies on neuronal secretion. Our method uses a mixture of probabilistic principal component analyzers to cluster genetic perturbations on two distinct steps in synaptic secretion, vesicle priming and fusion, and accounts for the poor standardization between different studies. Clustering data from 121 perturbations revealed that different perturbations of a given protein are often assigned to different steps in the release process. Furthermore, vesicle priming and fusion are inversely correlated for most of those perturbations where a specific protein domain was mutated to create a gain-of-function variant. Finally, two different modes of vesicle release, spontaneous and action potential evoked release, were affected similarly by most perturbations. This data suggests that the presynaptic protein network has evolved as a highly integrated supramolecular machine, which is responsible for both spontaneous and activity induced release, with a group of core proteins using different domains to act on multiple steps in the release process.     

Two distinct ATP-binding domains are needed to promote protein export by Escherichia coli SecA ATPase

Six putative ATP-binding motifs of SecA protein were altered by oligonucleotide-directed mutagenesis to try to define the ATP-binding regions of this multifunctional protein. The effects of the mutations were analysed by genetic and biochemical assays. The results show that SecA contains two essential ATP-binding domains. One domain is responsible for high-affinity ATP binding and contains motifs AO and BO, located at amino acid residues 102-109 and 198-210, respectively. A second domain is responsible for low-affinity ATP binding and contains motifs A3 and a predicted B motif located at amino acid residues 503-511 and 631-653, respectively. The ATP-binding properties of both domains were essential for SecA-dependent translocation ATPase and in vitro protein translocation activities. The significance of these findings for the mechanism of SecA-dependent protein translocation is discussed.     

Allosteric control of simian virus 40 T-antigen binding to viral origin DNA.

Simian virus 40 (SV40) large tumor antigen (T antigen) possesses several biochemical activities localized in different domains of the protein. These activities include sequence-specific binding to two major sites, I and II, in the SV40 control region, ATPase, and nucleotide-binding activity. In the present communication, we present evidence that specific binding of immunopurified T antigen to SV40 DNA is markedly inhibited by low concentrations of ATP, dATP, GTP, and dGTP. The inhibition is reversible after removal of the nucleotide, suggesting that simple nucleotide binding rather than a covalent modification of T antigen in the presence of ATP is responsible for the inhibition. The results suggest that T antigen may assume two conformations, one active and one inactive in binding to the SV40 origin of replication. In the presence of purine nucleoside triphosphates, the inactive conformation is favored.     

Combining modelling and mutagenesis studies of synaptic vesicle protein 2A to identify a series of residues involved in racetam binding

LEV (levetiracetam), an antiepileptic drug which possesses a unique profile in animal models of seizure and epilepsy, has as its unique binding site in brain, SV2A (synaptic vesicle protein 2A). Previous studies have used a chimaeric and site-specific mutagenesis approach to identify three residues in the putative tenth transmembrane helix of SV2A that, when mutated, alter binding of LEV and related racetam derivatives to SV2A. In the present paper, we report a combined modelling and mutagenesis study that successfully identifies another 11 residues in SV2A that appear to be involved in ligand binding. Sequence analysis and modelling of SV2A suggested residues equivalent to critical functional residues of other MFS (major facilitator superfamily) transporters. Alanine scanning of these and other SV2A residues resulted in the identification of residues affecting racetam binding, including Ile273 which differentiated between racetam analogues, when mutated to alanine. Integrating mutagenesis results with docking analysis led to the construction of a mutant in which six SV2A residues were replaced with corresponding SV2B residues. This mutant showed racetam ligand-binding affinity intermediate to the affinities observed for SV2A and SV2B.     

UNC-13 interaction with syntaxin is required for synaptic transmission

Neurotransmitter secretion at synapses is controlled by several processes-morphological docking of vesicles at release sites, priming of docked vesicles to make them fusion competent, and calcium-dependent fusion of vesicles with the plasma membrane . In worms, flies, and mice, mutants lacking UNC-13 have defects in vesicle priming . Current models propose that UNC-13 primes vesicles by stabilizing Syntaxin's "open" conformation by directly interacting with its amino-terminal regulatory domain . However, the functional significance of the UNC-13/Syntaxin interaction has not been tested directly. A truncated protein containing the Munc homology domains (MHD1 and MHD2) and the carboxy-terminal C2 domain partially rescued both the behavioral and secretion defects of unc-13 mutants in C. elegans. A double mutation in MHD2 (F1000A/K1002A) disrupts the UNC-13/Syntaxin interaction. The rate of endogenous synaptic events and the amplitude of nerve-evoked excitatory post-synaptic currents (EPSCs) were both significantly reduced in UNC-13S(F1000A/K1002A). However, the pool of primed (i.e., fusion-competent) vesicles was normal. These results suggest that the UNC-13/Syntaxin interaction is conserved in C. elegans and that, contrary to current models, the UNC-13/Syntaxin interaction is required for nerve-evoked vesicle fusion rather than synaptic-vesicle priming. Thus, UNC-13 may regulate multiple steps of the synaptic-vesicle cycle.     

SV2B regulates synaptotagmin 1 by direct interaction

SV2 proteins are abundant synaptic vesicle proteins expressed in two major (SV2A and SV2B) and one minor (SV2C) isoform. SV2A and SV2B have been shown to be involved in the regulation of synaptic vesicle exocytosis. Previous studies found that SV2A, but not SV2B, can interact with the cytoplasmic domain of synaptotagmin 1, a Ca2+ sensor for synaptic vesicle exocytosis. To determine whether SV2B can interact with full-length synaptotagmin 1, we performed immunoprecipitations from brain protein extracts and found that SV2B interacts strongly with synaptotagmin 1 in a detergent-resistant, Ca2+ -independent manner. In contrast, an interaction between native SV2A and synaptotagmin 1 was not detectable under these conditions. The SV2B-synaptotagmin 1 complex also contained the synaptic t-SNARE proteins, syntaxin 1 and SNAP-25, suggesting that SV2B may participate in exocytosis by modulating the interaction of synaptotagmin 1 with t-SNARE proteins. Analysis of retinae in SV2B knock-out mice revealed a strong reduction in the level of synaptotagmin 1 in rod photoreceptor synapses, which are unique in that they express only the SV2B isoform. In contrast, other synaptic vesicle proteins were not affected by SV2B knock out, indicating a specific role for SV2B in the regulation of synaptotagmin 1 levels at certain synapses. These experiments suggest that the SV2B-synaptotagmin 1 complex is involved in the regulation of synaptotagmin 1 stability and/or trafficking. This study has demonstrated a new role of SV2B as a regulator of synaptotagmin 1 that is likely mediated by direct interaction of these two synaptic proteins.     

Evidence for a second nucleotide binding site in rat elongation factor eEF-2 specific for adenylic nucleotides

The rat elongation factor eEF-2 catalyzes the translocation step of protein synthesis. Besides its well-characterized GTP/GDP binding properties, we have previously shown that ATP and ADP bind to eEF-2 [Sontag, B., Reboud, A. M., Divita, G., Di Pietro, A., Guillot, D., and Reboud, J. P. (1993) Biochemistry 32, 1976-1980]. However, whether the adenylic and guanylic nucleotide binding sites were different or not remained unclear. To further characterize these sites, eEF-2 was incubated in the presence of N-methylanthraniloyl (Mant) fluorescent derivatives of GTP, GDP, ATP, and ADP. This led to an increase in the probe fluorescence and to a partial quenching of eEF-2 tryptophans in each case. The Mant-derivatives and the unmodified corresponding nucleotides were shown to bind to eEF-2 with a similar affinity. Competition experiments between Mant-labeled and unmodified nucleotides suggested the presence of two different sites binding either guanylic or adenylic nucleotides. A F?rster's transfer between tryptophan residues and the Mant-probe is obtained with both the adenylic and the guanylic Mant-nucleotides, and comparison of the transfer efficiencies confirmed the presence of a second binding site specific for adenylic nucleotides. A sequence alignment of EF-Gs with eEF-2s from different species suggests the presence of potential Walker A and B motifs in an insert of the G-domain of eEF-2s from higher eukaryotes. Our results raise the possibility that a site specific for adenylic nucleotides and located in this insert has appeared in the course of evolution although its physiological function is still unknown.     

Glycosylated SV2A and SV2B mediate the entry of botulinum neurotoxin E into neurons

Botulinum neurotoxin E (BoNT/E) can cause paralysis in humans and animals by blocking neurotransmitter release from presynaptic nerve terminals. How this toxin targets and enters neurons is not known. Here we identified two isoforms of the synaptic vesicle protein SV2, SV2A and SV2B, as the protein receptors for BoNT/E. BoNT/E failed to enter neurons cultured from SV2A/B knockout mice; entry was restored by expressing SV2A or SV2B, but not SV2C. Mice lacking SV2B displayed reduced sensitivity to BoNT/E. The fourth luminal domain of SV2A or SV2B alone, expressed in chimeric receptors by replacing the extracellular domain of the low-density lipoprotein receptor, can restore the binding and entry of BoNT/E into neurons lacking SV2A/B. Furthermore, we found disruption of a N-glycosylation site (N573Q) within the fourth luminal domain of SV2A rendered the mutant unable to mediate the entry of BoNT/E and also reduced the entry of BoNT/A. Finally, we demonstrate that BoNT/E failed to bind and enter ganglioside-deficient neurons; entry was rescued by loading exogenous gangliosides into neuronal membranes. Together, the data reported here demonstrate that glycosylated SV2A and SV2B act in conjunction with gangliosides to mediate the entry of BoNT/E into neurons.     

ATP and AMP Mutually Influence Their Interaction with the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) at Separate Binding Sites

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter protein family. In the presence of ATP and physiologically relevant concentrations of AMP, CFTR exhibits adenylate kinase activity (ATP + AMP ⇆ 2 ADP). Previous studies suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for this activity. Two other ABC proteins, Rad50 and a structural maintenance of chromosome protein, also have adenylate kinase activity. All three ABC adenylate kinases bind and hydrolyze ATP in the absence of other nucleotides. However, little is known about how an ABC adenylate kinase interacts with ATP and AMP when both are present. Based on data from non-ABC adenylate kinases, we hypothesized that ATP and AMP mutually influence their interaction with CFTR at separate binding sites. We further hypothesized that only one of the two CFTR ATP-binding sites is involved in the adenylate kinase reaction. We found that 8-azidoadenosine 5′-triphosphate (8-N₃-ATP) and 8-azidoadenosine 5′-monophosphate (8-N₃-AMP) photolabeled separate sites in CFTR. Labeling of the AMP-binding site with 8-N₃-AMP required the presence of ATP. Conversely, AMP enhanced photolabeling with 8-N₃-ATP at ATP-binding site 2. The adenylate kinase active center probe P¹,P⁵-di(adenosine-5′) pentaphosphate interacted simultaneously with an AMP-binding site and ATP-binding site 2. These results show that ATP and AMP interact with separate binding sites but mutually influence their interaction with the ABC adenylate kinase CFTR. They further indicate that the active center of the adenylate kinase comprises ATP-binding site 2.