Allosteric substrate activation of SAMHD1 shapes deoxynucleotide triphosphate imbalances by interconnecting the depletion and biosynthesis of different dNTPs.

SAMHD1 is a dNTPase that impedes replication of HIV-1 in myeloid cells and resting T lymphocytes. Here we elucidate the substrate activation mechanism of SAMHD1 that depends on dNTP binding at allosteric sites and the concomitant tetramerization of the enzyme. The study reveals that SAMHD1 activation involves an inactive tetrameric intermediate with partial occupancy of the allosteric sites. The equilibrium between the inactive and active tetrameric states, which is coupled to cooperative binding/dissociation of at least two allosteric dNTP ligands, controls the dNTPase activity of the enzyme, which, in addition, depends on the identity of the dNTPs occupying the four allosteric sites of the active tetramer. We show how such allosteric regulation determines deoxynucleotide triphosphate levels established in the dynamic equilibria between dNTP production and SAMHD1-catalyzed depletion. Notably, the mechanism enables a distinctive functionality of SAMHD1, which we call facilitated dNTP depletion, whereby elevated biosynthesis of some dNTPs results in more efficient depletion of others. The regulatory relationship between the biosynthesis and depletion of different dNTPs sheds light on the emerging role of SAMHD1 in the biology of dNTP homeostasis with implications for HIV/AIDS, innate antiviral immunity, T cell disorders, telomere maintenance and therapeutic efficacy of nucleoside analogs.

Crucial roles of the BRCA1-BARD1 E3 ubiquitin ligase activity in homology-directed DNA repair.

The tumor-suppressor breast cancer 1 (BRCA1) in complex with BRCA1-associated really interesting new gene (RING) domain 1 (BARD1) is a RING-type ubiquitin E3 ligase that modifies nucleosomal histone and other substrates. The importance of BRCA1-BARD1 E3 activity in tumor suppression remains highly controversial, mainly stemming from studying mutant ligase-deficient BRCA1-BARD1 species that we show here still retain significant ligase activity. Using full-length BRCA1-BARD1, we establish robust BRCA1-BARD1-mediated ubiquitylation with specificity, uncover multiple modes of activity modulation, and construct a truly ligase-null variant and a variant specifically impaired in targeting nucleosomal histones. Cells expressing either of these BRCA1-BARD1 separation-of-function alleles are hypersensitive to DNA-damaging agents. Furthermore, we demonstrate that BRCA1-BARD1 ligase is not only required for DNA resection during homology-directed repair (HDR) but also contributes to later stages for HDR completion. Altogether, our findings reveal crucial, previously unrecognized roles of BRCA1-BARD1 ligase activity in genome repair via HDR, settle prior controversies regarding BRCA1-BARD1 ligase functions, and catalyze new efforts to uncover substrates related to tumor suppression.

Structural and functional asymmetry of RING trimerization controls priming and extension events in TRIM5α autoubiquitylation.

TRIM5α is an E3 ubiquitin ligase of the TRIM family that binds to the capsids of primate immunodeficiency viruses and blocks viral replication after cell entry. Here we investigate how synthesis of K63-linked polyubiquitin is upregulated by transient proximity of three RING domains in honeycomb-like assemblies formed by TRIM5α on the surface of the retroviral capsid. Proximity of three RINGs creates an asymmetric arrangement, in which two RINGs form a catalytic dimer that activates E2-ubiquitin conjugates and the disordered N-terminus of the third RING acts as the substrate for N-terminal autoubiquitylation. RING dimerization is required for activation of the E2s that contribute to the antiviral function of TRIM5α, UBE2W and heterodimeric UBE2N/V2, whereas the proximity of the third RING enhances the rate of each of the two distinct steps in the autoubiquitylation process: the initial N-terminal monoubiquitylation (priming) of TRIM5α by UBE2W and the subsequent extension of the K63-linked polyubiquitin chain by UBE2N/V2. The mechanism we describe explains how recognition of infection-associated epitope patterns by TRIM proteins initiates polyubiquitin-mediated downstream events in innate immunity.

Structural and functional characterization explains loss of dNTPase activity of the cancer-specific R366C/H mutant SAMHD1 proteins.

Elevated intracellular levels of dNTPs have been shown to be a biochemical marker of cancer cells. Recently, a series of mutations in the multifunctional dNTP triphosphohydrolase (dNTPase), sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1), have been reported in various cancers. Here, we investigated the structure and functions of SAMHD1 R366C/H mutants, found in colon cancer and leukemia. Unlike many other cancer-specific mutations, the SAMHD1 R366 mutations do not alter cellular protein levels of the enzyme. However, R366C/H mutant proteins exhibit a loss of dNTPase activity, and their X-ray structures demonstrate the absence of dGTP substrate in their active site, likely because of a loss of interaction with the γ-phosphate of the substrate. The R366C/H mutants failed to reduce intracellular dNTP levels and restrict HIV-1 replication, functions of SAMHD1 that are dependent on the ability of the enzyme to hydrolyze dNTPs. However, these mutants retain dNTPase-independent functions, including mediating dsDNA break repair, interacting with CtIP and cyclin A2, and suppressing innate immune responses. Finally, SAMHD1 degradation in human primary-activated/dividing CD4+ T cells further elevates cellular dNTP levels. This study suggests that the loss of SAMHD1 dNTPase activity induced by R366 mutations can mechanistically contribute to the elevated dNTP levels commonly found in cancer cells.

Nucleic acid binding by SAMHD1 contributes to the antiretroviral activity and is enhanced by the GpsN modification.

SAMHD1 impedes infection of myeloid cells and resting T lymphocytes by retroviruses, and the enzymatic activity of the protein-dephosphorylation of deoxynucleotide triphosphates (dNTPs)-implicates enzymatic dNTP depletion in innate antiviral immunity. Here we show that the allosteric binding sites of the enzyme are plastic and can accommodate oligonucleotides in place of the allosteric activators, GTP and dNTP. SAMHD1 displays a preference for oligonucleotides containing phosphorothioate bonds in the Rp configuration located 3' to G nucleotides (GpsN), the modification pattern that occurs in a mechanism of antiviral defense in prokaryotes. In the presence of GTP and dNTPs, binding of GpsN-containing oligonucleotides promotes formation of a distinct tetramer with mixed occupancy of the allosteric sites. Mutations that impair formation of the mixed-occupancy complex abolish the antiretroviral activity of SAMHD1, but not its ability to deplete dNTPs. The findings link nucleic acid binding to the antiretroviral activity of SAMHD1, shed light on the immunomodulatory effects of synthetic phosphorothioated oligonucleotides and raise questions about the role of nucleic acid phosphorothioation in human innate immunity.

Nanobodies against the metal binding domains of ATP7B as tools to study copper transport in the cell.

Nanobodies are genetically engineered single domain antibodies derived from the unusual heavy-chain only antibodies found in llamas and camels. The small size of the nanobodies and flexible selection schemes make them uniquely versatile tools for protein biochemistry and cell biology. We have developed a panel of nanobodies against the metal binding domains of the human copper transporter ATP7B, a multidomain membrane protein with a complex regulation of enzymatic activity and intracellular localization. To enable the use of the nanobodies as tools to investigate copper transport in the cell, we characterized their binding sites and affinity by isothermal titration calorimetry and NMR. We have identified nanobodies against each of the first four metal binding domains of ATP7B, with a wide affinity range, as evidenced by dissociation constants from below 10-9 to 10-6 M. We found both the inhibitory and activating nanobodies among those tested. The diverse properties of the nanobodies make the panel useful for the structural studies of ATP7B, immunoaffinity purification of the protein, modulation of its activity in the cell, protein dynamics studies, and as mimics of copper chaperone ATOX1, the natural interaction partner of ATP7B.

The Structure of Metal Binding Domain 1 of the Copper Transporter ATP7B Reveals Mechanism of a Singular Wilson Disease Mutation.

Copper-transporter ATP7B maintains copper homeostasis in the human cells and delivers copper to the biosynthetic pathways for incorporation into the newly synthesized copper-containing proteins. ATP7B is a target of several hundred mutations that lead to Wilson disease, a chronic copper toxicosis. ATP7B contains a chain of six cytosolic metal-binding domains (MBDs), the first four of which (MBD1-4) are believed to be regulatory, and the last two (MBD5-6) are required for enzyme activity. We report the NMR structure of MBD1, the last unsolved metal-binding domain of ATP7B. The structure reveals the disruptive mechanism of G85V mutation, one of the very few disease causing missense mutations in the MBD1-4 region of ATP7B.

The metal chaperone Atox1 regulates the activity of the human copper transporter ATP7B by modulating domain dynamics.

The human transporter ATP7B delivers copper to the biosynthetic pathways and maintains copper homeostasis in the liver. Mutations in ATP7B cause the potentially fatal hepatoneurological disorder Wilson disease. The activity and intracellular localization of ATP7B are regulated by copper, but the molecular mechanism of this regulation is largely unknown. We show that the copper chaperone Atox1, which delivers copper to ATP7B, and the group of the first three metal-binding domains (MBD1-3) are central to the activity regulation of ATP7B. Atox1-Cu binding to ATP7B changes domain dynamics and interactions within the MBD1-3 group and activates ATP hydrolysis. To understand the mechanism linking Atox1-MBD interactions and enzyme activity, we have determined the MBD1-3 conformational space using small angle X-ray scattering and identified changes in MBD dynamics caused by apo-Atox1 and Atox1-Cu by solution NMR. The results show that copper transfer from Atox1 decreases domain interactions within the MBD1-3 group and increases the mobility of the individual domains. The N-terminal segment of MBD1-3 was found to interact with the nucleotide-binding domain of ATP7B, thus physically coupling the domains involved in copper binding and those involved in ATP hydrolysis. Taken together, the data suggest a regulatory mechanism in which Atox1-mediated copper transfer activates ATP7B by releasing inhibitory constraints through increased freedom of MBD1-3 motions.

Binding of Copper and Cisplatin to Atox1 Is Mediated by Glutathione through the Formation of Metal-Sulfur Clusters.

Copper is an essential nutrient required for many biological processes involved in primary metabolism, but free copper is toxic due to its ability to catalyze formation of free radicals. To prevent toxic effects, in the cell copper is bound to proteins and low molecular weight compounds, such as glutathione, at all times. The widely used chemotherapy agent cisplatin is known to bind to copper-transporting proteins, including copper chaperone Atox1. Cisplatin interactions with Atox1 and other copper transporters are linked to cancer resistance to platinum-based chemotherapy. Here we analyze the binding of copper and cisplatin to Atox1 in the presence of glutathione under redox conditions that mimic intracellular environment. We show that copper(I) and glutathione form large polymers with a molecular mass of approximately 8 kDa, which can transfer copper to Atox1. Cisplatin also can form polymers with glutathione, albeit at a slower rate. Analysis of simultaneous binding of copper and cisplatin to Atox1 under physiological conditions shows that both metals are bound to the protein through copper-sulfur-platinum bridges.

Dynamics of the metal binding domains and regulation of the human copper transporters ATP7B and ATP7A.

Copper transporters ATP7A and ATP7B regulate copper levels in the human cells and deliver copper to the biosynthetic pathways. ATP7A and ATP7B belong to the P-type ATPases and share much of the domain architecture and the mechanism of ATP hydrolysis with the other, well-studied, enzymes of this type. A unique structural feature of the copper ATPases is the chain of six cytosolic metal-binding domains (MBDs), which are believed to be involved in copper-dependent regulation of the activity and intracellular localization of these enzymes. Although the structures of all the MBDs have been solved, the mechanism of copper-dependent regulation of ATP7B and ATP7A, the roles of individual MBDs, and the relationship between the regulatory and catalytic copper binding are still unknown. We describe the structure and dynamics of the MBDs, review the current knowledge about their functional roles and propose a mechanism of regulation of ATP7B by copper-dependent changes in the dynamics and conformation of the MBD chain. Transient interactions between the MBDs, rather than transitions between distinct static conformations are likely to form the structural basis of regulation of the ATP-dependent copper transporters in human cells. © 2016 IUBMB Life, 69(4):226-235, 2017.

Interactions between metal-binding domains modulate intracellular targeting of Cu(I)-ATPase ATP7B, as revealed by nanobody binding.

The biologically and clinically important membrane transporters are challenging proteins to study because of their low level of expression, multidomain structure, and complex molecular dynamics that underlies their activity. ATP7B is a copper transporter that traffics between the intracellular compartments in response to copper elevation. The N-terminal domain of ATP7B (N-ATP7B) is involved in binding copper, but the role of this domain in trafficking is controversial. To clarify the role of N-ATP7B, we generated nanobodies that interact with ATP7B in vitro and in cells. In solution NMR studies, nanobodies revealed the spatial organization of N-ATP7B by detecting transient functionally relevant interactions between metal-binding domains 1-3. Modulation of these interactions by nanobodies in cells enhanced relocalization of the endogenous ATP7B toward the plasma membrane linking molecular and cellular dynamics of the transporter. Stimulation of ATP7B trafficking by nanobodies in the absence of elevated copper provides direct evidence for the important role of N-ATP7B structural dynamics in regulation of ATP7B localization in a cell.

Copper chaperone Atox1 interacts with the metal-binding domain of Wilson's disease protein in cisplatin detoxification.

Human copper transporters ATP7B (Wilson's disease protein) and ATP7A (Menkes' disease protein) have been implicated in tumour resistance to cisplatin, a widely used anticancer drug. Cisplatin binds to the copper-binding sites in the N-terminal domain of ATP7B, and this binding may be an essential step of cisplatin detoxification involving copper ATPases. In the present study, we demonstrate that cisplatin and a related platinum drug carboplatin produce the same adduct following reaction with MBD2 [metal-binding domain (repeat) 2], where platinum is bound to the side chains of the cysteine residues in the CxxC copper-binding motif. This suggests the same mechanism for detoxification of both drugs by ATP7B. Platinum can also be transferred to MBD2 from copper chaperone Atox1, which was shown previously to bind cisplatin. Binding of the free cisplatin and reaction with the cisplatin-loaded Atox1 produce the same protein-bound platinum intermediate. Transfer of platinum along the copper-transport pathways in the cell may serve as a mechanism of drug delivery to its target in the cell nucleus, and explain tumour-cell resistance to cisplatin associated with the overexpression of copper transporters ATP7B and ATP7A.

Molecular events initiating exit of a copper-transporting ATPase ATP7B from the trans-Golgi network.

The copper-transporting ATPase ATP7B has a dual intracellular localization: the trans-Golgi network (TGN) and cytosolic vesicles. Changes in copper levels, kinase-mediated phosphorylation, and mutations associated with Wilson disease alter the steady-state distribution of ATP7B between these compartments. To identify a primary molecular event that triggers ATP7B exit from the TGN, we characterized the folding, activity, and trafficking of the ATP7B variants with mutations within the regulatory N-terminal domain (N-ATP7B). We found that structural changes disrupting the inter-domain contacts facilitate ATP7B exit from the TGN. Mutating Ser-340/341 in the N-ATP7B individually or together to Ala, Gly, Thr, or Asp produced active protein and shifted the steady-state localization of ATP7B to vesicles, independently of copper levels. The Ser340/341G mutant had a lower kinase-mediated phosphorylation under basal conditions and no copper-dependent phosphorylation. Thus, negative charges introduced by copper-dependent phosphorylation are not obligatory for ATP7B trafficking from the TGN. The Ser340/341A mutation did not alter the overall fold of N-ATP7B, but significantly decreased interactions with the nucleotide-binding domain, mimicking consequences of copper binding to N-ATP7B. We propose that structural changes that specifically alter the inter-domain contacts initiate exit of ATP7B from the TGN, whereas increased phosphorylation may be needed to maintain an open interface between the domains.

Structural characterization of partially disordered human Chibby: insights into its function in the Wnt-signaling pathway.

The Wnt/ß-catenin signaling pathway is critical to embryonic development as well as adult tissue regeneration. Dysregulation of this pathway can lead to a variety of human diseases, in particular cancers. Chibby (Cby), a small and highly conserved protein, plays an antagonistic role in Wnt signaling by inhibiting the binding of ß-catenin to Tcf/Lef family proteins, a protein interaction that is essential for the transcriptional activation of Wnt target genes. Cby is also involved in regulating intracellular distribution of ß-catenin. Phosphorylated Cby forms a ternary complex with 14-3-3 protein and ß-catenin, facilitating the export of ß-catenin from the nucleus. On the other hand, the antagonistic function of Cby is inhibited upon binding to thyroid cancer-1 (TC-1). To dissect the structure-function relationship of Cby, we have used NMR spectroscopy, ESI-MS, CD, and DLS to extensively characterize the structure of human Cby. Our results show that the 126-residue Cby is partially disordered under nondenaturing conditions. While the N-terminal portion of the protein is predominantly unstructured in solution, the C-terminal half of Cby adopts a coiled-coil structure through self-association. Initial data for the binding studies of Cby to 14-3-3ζ (one of the isoforms in the 14-3-3 family) and TC-1 via these two distinct structural modules have also been obtained. It is noteworthy that in a recent large-scale analysis of the intrinsically disordered proteome of mouse, a substantial number of disordered proteins are predicted to have coiled-coil motif presence in their sequences. The combination of these two molecular recognition features could facilitate disordered Cby in assembling protein complexes via different modes of interaction.