Nonhydrolyzable ATP Analog Research Articles

Phospholamban inhibits the cardiac calcium pump by interrupting an allosteric activation pathway

Phospholamban (PLB) is a transmembrane micropeptide that regulates the sarcoplasmic reticulum Ca2+-ATPase (SERCA) in cardiac muscle, but the physical mechanism of this regulation remains poorly understood. PLB reduces the Ca2+ sensitivity of active SERCA, increasing the Ca2+ concentration required for pump cycling. However, PLB does not decrease Ca2+ binding to SERCA when ATP is absent, suggesting PLB does not inhibit SERCA Ca2+ affinity. The prevailing explanation for these seemingly conflicting results is that PLB slows transitions in the SERCA enzymatic cycle associated with Ca2+ binding, altering transport Ca2+ dependence without actually affecting the equilibrium binding affinity of the Ca2+-coordinating sites. Here, we consider another hypothesis, that measurements of Ca2+ binding in the absence of ATP overlook important allosteric effects of nucleotide binding that increase SERCA Ca2+ binding affinity. We speculated that PLB inhibits SERCA by reversing this allostery. To test this, we used a fluorescent SERCA biosensor to quantify the Ca2+ affinity of non-cycling SERCA in the presence and absence of a non-hydrolyzable ATP-analog, AMPPCP. Nucleotide activation increased SERCA Ca2+ affinity, and this effect was reversed by co-expression of PLB. Interestingly, PLB had no effect on Ca2+ affinity in the absence of nucleotide. These results reconcile the previous conflicting observations from ATPase assays versus Ca2+ binding assays. Moreover, structural analysis of SERCA revealed a novel allosteric pathway connecting the ATP- and Ca2+-binding sites. We propose this pathway is disrupted by PLB binding. Thus, PLB reduces the equilibrium Ca2+ affinity of SERCA by interrupting allosteric activation of the pump byATP.

On the role of nucleotides and lipids in the polymerization of the actin homolog MreB from a Gram-positive bacterium.

In vivo, bacterial actin MreB assembles into dynamic membrane-associated filamentous structures that exhibit circumferential motion around the cell. Current knowledge of MreB biochemical and polymerization properties in vitro remains limited and is mostly based on MreB proteins from Gram-negative species. In this study, we report the first observation of organized protofilaments by electron microscopy and the first 3D-structure of MreB from a Gram-positive bacterium. We show that Geobacillus stearothermophilus MreB forms straight pairs of protofilaments on lipid surfaces in the presence of ATP or GTP, but not in the presence of ADP, GDP or non-hydrolysable ATP analogs. We demonstrate that membrane anchoring is mediated by two spatially close short hydrophobic sequences while electrostatic interactions also contribute to lipid binding, and show that the population of membrane-bound protofilament doublets is in steady-state. In solution, protofilament doublets were not detected in any condition tested. Instead, MreB formed large sheets regardless of the bound nucleotide, albeit at a higher critical concentration. Altogether, our results indicate that both lipids and ATP are facilitators of MreB polymerization, and are consistent with a dual effect of ATP hydrolysis, in promoting both membrane binding and filaments assembly/disassembly.

Biochemical and X-ray analyses of the players involved in the faRel2/aTfaRel2 toxin-antitoxin operon.

The aTfaRel2/faRel2 operon from Coprobacillus sp. D7 encodes a bicistronic type II toxin-antitoxin (TA) module. The FaRel2 toxin is a toxic small alarmone synthetase (toxSAS) that inhibits translation through the pyrophosphorylation of uncharged tRNAs at the 3'-CCA end. The toxin is neutralized by the antitoxin ATfaRel2 through the formation of an inactive TA complex. Here, the production, biophysical analysis and crystallization of ATfaRel2 and FaRel2 as well as of the ATfaRel2-FaRel2 complex are reported. ATfaRel2 is monomeric in solution. The antitoxin crystallized in space group P21212 with unit-cell parameters a = 53.3, b = 34.2, c = 37.6 Å, and the best crystal diffracted to a resolution of 1.24 Å. Crystals of FaRel2 in complex with APCPP, a nonhydrolysable ATP analogue, belonged to space group P21, with unit-cell parameters a = 31.5, b = 60.6, c = 177.2 Å, β = 90.6°, and diffracted to 2.6 Å resolution. The ATfaRel2-FaRel2Y128F complex forms a heterotetramer in solution composed of two toxins and two antitoxins. This complex crystallized in two space groups: F4132, with unit-cell parameters a = b = c = 227.1 Å, and P212121, with unit-cell parameters a = 51.7, b = 106.2, c = 135.1 Å. The crystals diffracted to 1.98 and 2.1 Å resolution, respectively.

ATP modulates the activity of the voltage-gated proton channel through direct binding interaction.

ATP is an important molecule implicated in diverse biochemical processes, including the modulation of ion channel and transporter activity. The voltage-gated proton channel (Hv1) controls proton flow through the transmembrane pathway in response to membrane potential, and various molecules regulate its activity. Although it is believed that ATP is not essential for Hv1 activity, a report has indicated that cytosolic ATP may modulate Hv1. However, the detailed molecular mechanism underlying the effect of ATP on Hv1 is unknown, and whether ATP is involved in the physiological regulation of Hv1 activity remains unclear. Here, we report that cytosolic ATP is required to maintain Hv1 activity. To gain insight into the underlying mechanism, we analysed the effects of ATP on the mouse Hv1 channel (mHv1) using electrophysiological and microscale thermophoresis (MST) methods. Intracellular ATP accelerated the activation kinetics of mHv1, thereby increasing the amplitude of the proton current within the physiological concentration range. The increase in proton current was reproduced with a non-hydrolysable ATP analogue, indicating that ATP directly influences Hv1 activity without an enzymatic reaction. The direct molecular interaction between the purified mHv1 protein and ATP was analysed and demonstrated through MST. In addition, ATP facilitation was observed for the endogenous proton current flowing through Hv1 in the physiological concentration range of ATP. These results suggest that ATP influences Hv1 activity via direct molecular interactions and is required for the physiological function of Hv1. KEY POINTS: We found that ATP is required to maintain the activity of voltage-gated proton channels (Hv1) and investigated the underlying molecular mechanism. Application of intracellular ATP increased the amplitude of the proton current flowing through Hv1, accompanied by an acceleration of activation kinetics. The direct interaction between purified Hv1 protein and ATP was quantitatively analysed using microscale thermophoresis. ATP enhanced endogenous proton currents in breast cancer cell lines. These results suggest that ATP influences Hv1 activity via direct molecular interactions and that its functional characteristics are required for the physiological activity of Hv1.

Unlicensed origin DNA melting by MCV and SV40 polyomavirus LT proteins is independent of ATP-dependent helicase activity

Cellular eukaryotic replication initiation helicases are first loaded as head-to-head double hexamers on double-stranded (ds) DNA origins and then initiate S-phase DNA melting during licensed (once per cell cycle) replication. Merkel cell polyomavirus (MCV) large T (LT) helicase oncoprotein similarly binds and melts its own 98-bp origin but replicates multiple times in a single cell cycle. To examine the actions of this unlicensed viral helicase, we quantitated multimerization of MCV LT molecules as they assembled on MCV DNA origins using real-time single-molecule microscopy. MCV LT formed highly stable double hexamers having 17-fold longer mean lifetime (τ, >1,500 s) on DNA than single hexamers. Unexpectedly, partial MCV LT assembly without double-hexamer formation was sufficient to melt origin dsDNA as measured by RAD51, RPA70, or S1 nuclease cobinding. DNA melting also occurred with truncated MCV LT proteins lacking the helicase domain, but was lost from a protein without the multimerization domain that could bind only as a monomer to DNA. SV40 polyomavirus LT also multimerized to the MCV origin without forming a functional hexamer but still melted origin DNA. MCV origin melting did not require ATP hydrolysis and occurred for both MCV and SV40 LT proteins using the nonhydrolyzable ATP analog, adenylyl-imidodiphosphate (AMP-PNP). LT double hexamers formed in AMP-PNP, and melted DNA, consistent with direct LT hexamer assembly around single-stranded (ss) DNA without the energy-dependent dsDNA-to-ssDNA melting and remodeling steps used by cellular helicases. These results indicate that LT multimerization rather than helicase activity is required for origin DNA melting during unlicensed virus replication.

Architecture of a complete Bce-type antimicrobial peptide resistance module

Gram-positive bacteria synthesize and secrete antimicrobial peptides that target the essential process of peptidoglycan synthesis. These antimicrobial peptides not only regulate the dynamics of microbial communities but are also of clinical importance as exemplified by peptides such as bacitracin, vancomycin, and daptomycin. Many gram-positive species have evolved specialized antimicrobial peptide sensing and resistance machinery known as Bce modules. These modules are membrane protein complexes formed by an unusual Bce-type ABC transporter interacting with a two-component system sensor histidine kinase. In this work, we provide the first structural insight into how the membrane protein components of these modules assemble into a functional complex. A cryo-EM structure of an entire Bce module revealed an unexpected mechanism of complex assembly, and extensive structural flexibility in the sensor histidine kinase. Structures of the complex in the presence of a non-hydrolysable ATP analog reveal how nucleotide binding primes the complex for subsequent activation. Accompanying biochemical data demonstrate how the individual membrane protein components of the complex exert functional control over one another to create a tightly regulated enzymatic system.

Despite their structural similarities, the cytosolic isoforms of human Hsp90 show different behaviour in thermal unfolding due to their conformation: An FTIR study

Heat shock proteins 90 (Hsp90) are chaperones that promote the proper folding of other proteins under high temperature stress situations. Hsp90s are highly conserved and ubiquitous proteins, and in mammalian cells, they are localized in the cytoplasm, endoplasmic reticulum, and mitochondria. Cytoplasmic Hsp90 are named Hsp90α and Hsp90β and differ mainly in their expression pattern: Hsp90α is expressed under stress conditions, while Hsp90β is a constitutive protein. Structurally, both share the same characteristics by presenting three well-conserved domains, one of which, the N-terminal domain, has a binding site for ATP to which various drugs targeting this protein, including radicicol, can bind. The protein is mainly found in dimeric form and adopts different conformations depending on the presence of ligands, co-chaperones and client proteins. In this study, some aspects of structure and thermal unfolding of cytoplasmic human Hsp90 were analysed by infrared spectroscopy. The effect on Hsp90β of binding with a non-hydrolysable ATP analogue and radicicol was also examined. The results obtained showed that despite the high similarity in secondary structure the two isoforms exhibit substantial differences in their behaviour during thermal unfolding, as Hsp90α exhibits higher thermal stability, slower denaturation process and different event sequence during unfolding. Ligand binding strongly stabilizes Hsp90β and slightly modifies the secondary structure of the protein as well. Most likely, these structural and thermostability characteristics are closely related to the conformational cycling of the chaperone and its propensity to exist in monomer or dimer form.

Crystal structure of the motor domain of centromere-associated protein E in complex with a non-hydrolysable ATP analogue.

Centromere-associated protein E (CENP-E) is a kinesin motor protein essential for mitosis and a new target for anticancer agents with less side effects. To rationally design anticancer drug candidates based on structure, it is important to determine the three-dimensional structure of the CENP-E motor domain bound to its inhibitor. Here, we report the first crystal structure of the CENP-E motor domain in complex with a non-hydrolysable ATP analogue, adenylyl-imidodiphosphate (AMPPNP). Furthermore, the structure is compared with the ADP-bound form of the CENP-E motor domain as well as the AMPPNP-bound forms of other kinesins. This study indicates that helix α4 of CENP-E participates in the slow binding of CENP-E to microtubules. These results will contribute to the development of anticancer drugs targeting CENP-E.

Phospholamban inhibits the cardiac calcium pump by reversing allosteric effects of ATP on calcium affinity.

Phospholamban (PLB) is a transmembrane micropeptide that regulates the sarcoplasmic reticulum Ca2+ ATPase (SERCA) in cardiac muscle. PLB reduces the apparent Ca2+ sensitivity of SERCA, increasing the Ca2+ concentration required for pump cycling. However, PLB does not decrease Ca2+ binding to SERCA when ATP is absent, and the pump is not cycling. One prevailing explanation for these seemingly conflicting results is PLB may slow the Ca2+ binding step in the SERCA enzymatic cycle, altering the Ca2+ dependence of cycling without affecting true Ca2+ affinity. Alternatively, we speculated that equilibrium measurements of Ca2+ binding to non-cycling SERCA may overlook important allosteric effects of bound nucleotide. Specifically, ATP is known to have an allosteric effect on SERCA that is distinct from its role as a catalytic substrate and source of energy. When ATP binds, it increases the affinity of SERCA for Ca2+ - exactly the opposite of the PLB effect. We speculated that PLB may inhibit SERCA by reversing pre-activation of SERCA Ca2+ affinity by ATP. To test this hypothesis, we used a 2-color SERCA biosensor expressed in microsomal membranes to measure Ca2+ binding to the pump reported by changes in intramolecular FRET. We quantified the Ca2+ affinity of non-cycling SERCA in the absence of ATP, then mimicked ATP binding while preventing enzymatic cycling using the non-hydrolyzable ATP-analog AMPPCP. Interestingly, nucleotide activation by AMPPCP increased the Ca2+ affinity of 2-color SERCA, but this effect was reversed by co-expression of PLB. PLB had no effect on Ca2+ affinity in the absence of nucleotide. These results may reconcile the seemingly paradoxical effects of PLB. We propose that PLB reduces the true Ca2+ affinity of SERCA by reversing allosteric activation of the pump by ATP.

Stabilization of DNA fork junctions by Smc5/6 complexes revealed by single-molecule imaging.

SMC complexes play key roles in genome maintenance, where they ensure efficient genome replication and segregation. The SMC complex Smc5/6 is a crucial player in DNA replication and repair, yet many molecular features that determine its roles are unclear. Here, we use single-molecule microscopy to investigate Smc5/6's interaction with DNA. We find that Smc5/6 forms oligomers that dynamically redistribute on dsDNA by 1D diffusion and statically bind to ssDNA. Using combined force manipulation and single-molecule microscopy, we generate ssDNA-dsDNA junctions that mimic structures present in DNA repair intermediates or replication forks. We show that Smc5/6 accumulates at these junction sites, stabilizes the fork, and promotes the retention of RPA. Our observations provide a model for the complex's enrichment at sites of replication stress and DNA lesions from where it coordinates the recruitment and activation of downstream repair proteins.

Recombinant Human Cytomegalovirus Expressing an Analog-Sensitive Kinase pUL97 as Novel Tool for Functional Analyses.

The human cytomegalovirus (HCMV) is a member of the beta-herpesvirus family and inflicts life-long latent infections in its hosts. HCMV has been shown to manipulate and dysregulate many cellular processes. One major interactor with the cellular host is the viral kinase pUL97. The UL97 gene is essential for viral replication, and kinase-deficient mutants of pUL97 display a severe replication defect. Recently, another group established an analog-sensitive version of the pUL97 protein. This mutant kinase can be treated with a non-hydrolysable ATP analog, thereby inhibiting its kinase function. This process is reversible by removing the ATP analog by media change. We introduced this mutant version of the pUL97 protein into the laboratory strain Ad169 of HCMV, BADwt, creating a BAD-UL97-as1 viral mutant. This mutant virus replicated normally in infected cells in the absence of the ATP analog and maintained its ability to phosphorylate its cellular substrates. However, when treated with the ATP analog, BAD-UL97-as1 displayed a defect in the production of intra- and extracellular viral DNA and in the production of viral progeny. Furthermore, in the presence of 3MB-PP1, a well-established substrate of pUL97 was no longer hyperphosphorylated. This effect was detectable as early as 4 h post treatment, which allows for studies on pUL97 without the complication of low viral titers. Nevertheless, we observed off-target effects of 3MB-PP1 on several cellular processes, which should be considered with this approach.

Association of adenosine signaling gene signature with estrogen receptor-positive breast and prostate cancer bone metastasis.

Bone metastasis is a common and devastating consequence of several major cancer types, including breast and prostate. Osteocytes are the predominant bone cell, and through connexin (Cx) 43 hemichannels release ATP to the bone microenvironment that can be hydrolyzed to adenosine. Here, we investigated how genes related to ATP paracrine signaling are involved in two common bone-metastasizing malignancies, estrogen receptor positive (ER+) breast and prostate cancers. Compared to other sites, bone metastases of both cancer types expressed higher levels of ENTPD1 and NT5E, which encode CD39 and CD73, respectively, and hydrolyze ATP to adenosine. ADORA3, encoding the adenosine A3 receptor, had a similar expression pattern. In primary ER+ breast cancer, high levels of the triplet ENTPD1/NT5E/ADORA3 expression signature was correlated with lower overall, distant metastasis-free, and progression-free survival. In ER+ bone metastasis biopsies, this expression signature is associated with lower survival. This expression signature was also higher in bone-metastasizing primary prostate cancers than in those that caused other tumor events or did not lead to progressive disease. In 3D culture, a non-hydrolyzable ATP analog inhibited the growth of breast and prostate cancer cell lines more than ATP did. A3 inhibition also reduced spheroid growth. Large-scale screens by the Drug Repurposing Hub found ER+ breast cancer cell lines were uniquely sensitive to adenosine receptor antagonists. Together, these data suggest a vital role for extracellular ATP degradation and adenosine receptor signaling in cancer bone metastasis, and this study provides potential diagnostic means for bone metastasis and specific targets for treatment and prevention.

Structural and functional studies of Arabidopsis thaliana triphosphate tunnel metalloenzymes reveal roles for additional domains

Triphosphate tunnel metalloenzymes (TTMs) are found in all biological kingdoms and have been characterized in microorganisms and animals. Members of the TTM family have divergent biological functions and act on a range of triphosphorylated substrates (RNA, thiamine triphosphate, and inorganic polyphosphate). TTMs in plants have received considerably less attention and are unique in that some homologs harbor additional domains including a P-loop kinase and transmembrane domain. Here, we report on structural and functional aspects of the multimodular TTM1 and TTM2 of Arabidopsis thaliana. Our tissue and cellular microscopy studies show that both AtTTM1 and AtTTM2 are expressed in actively dividing (meristem) tissue and are tail-anchored proteins at the outer mitochondrial membrane, mediated by the single C-terminal transmembrane domain, supporting earlier studies. In addition, we reveal from crystal structures of AtTTM1 in the presence and absence of a nonhydrolyzable ATP analog a catalytically incompetent TTM tunnel domain tightly interacting with the P-loop kinase domain that is locked in an inactive conformation. Our structural comparison indicates that a helical hairpin may facilitate movement of the TTM domain, thereby activating the kinase. Furthermore, we conducted genetic studies to show that AtTTM2 is important for the developmental transition from the vegetative to the reproductive phase in Arabidopsis, whereas its closest paralog AtTTM1 is not. We demonstrate through rational design of mutations based on the 3D structure that both the P-loop kinase and TTM tunnel modules of AtTTM2 are required for the developmental switch. Together, our results provide insight into the structure and function of plant TTM domains.

Non-Nitrogen-Containing Bisphosphonates Prevent Pyrophosphorylation of Exocytosis Proteins.

Clodronate, a non-nitrogen-containing bisphosphonate (non-NBP), is intracellularly converted into non-hydrolyzable ATP analogs. Clodronate and its analogs impair normal cell functions, including the exocytosis process. However, how this occurs in mast cells is still not well characterized. To summarize the possible mechanisms of clodronate-mediated exocytosis inhibition in mast cells. Non-NBPs display several possible mechanisms of exocytosis inhibition in various cell types, including vesicular nucleotide transporter (VNUT) and purinergic receptor inhibition. Inhibition of purinergic receptors has been shown in mast cells, but VNUT inhibition remains to be confirmed. Inhibition of protein prenylation by non-NBPs has also been shown; however, direct evidence of non-NBPs in prenylated exocytosis proteins is still contradictory. Finally, non-NBPs may inhibit mast cell exocytosis via impairment of protein pyrophosphorylation. This mechanism is less studied, and direct evidence of the involvement of pyrophosphorylated proteins in exocytosis is still lacking. Non-NBPs may affect mast cell exocytosis by interacting with purinergic receptors or VNUT or by preventing post-translational modifications of exocytosis protein(s), i.e., prenylation and pyrophosphorylation. The latter needs further investigation to provide direct evidence of a role for non- NBPs.

The 26S Proteasome Switches between ATP-Dependent and -Independent Mechanisms in Response to Substrate Ubiquitination.

The ubiquitin–proteasome system is responsible for the bulk of protein degradation in eukaryotic cells. Proteins are generally targeted to the 26S proteasome through the attachment of polyubiquitin chains. Several proteins also contain ubiquitin-independent degrons (UbIDs) that allow for proteasomal targeting without the need for ubiquitination. Our laboratory previously showed that UbID substrates are less processively degraded than ubiquitinated substrates, but the mechanism underlying this difference remains unclear. We therefore designed two model substrates containing both a ubiquitination site and a UbID for a more direct comparison. We found UbID degradation to be overall less robust, with complete degradation only occurring with loosely folded substrates. UbID degradation was unaffected by the nonhydrolyzable ATP analog ATPγS, indicating that UbID degradation proceeds in an ATP-independent manner. Stabilizing substrates halted UbID degradation, indicating that the proteasome can only capture UbID substrates if they are already at least transiently unfolded, as confirmed using native-state proteolysis. The 26S proteasome therefore switches between ATP-independent weak degradation and ATP-dependent robust unfolding and degradation depending on whether or not the substrate is ubiquitinated.

The 26S Proteasome Switches Between ATP‐Dependent and Independent Mechanisms in Response to Substrate Ubiquitination

The Ubiquitin‐Proteasome System is responsible for the bulk of protein degradation in eukaryotic cells. Proteins are generally targeted to the 26S proteasome by the attachment of polyubiquitin chains. A number of proteins also contain ubiquitin‐independent degrons (UbID) that allow for proteasomal targeting without the need for ubiquitination. UbID substrates are degraded less processively than ubiquitinated substrates, but the mechanism underlying this difference remains unclear. We therefore designed two model substrates containing both a ubiquitination site and a UbID for a more direct comparison. We found UbID degradation to be overall less robust with complete degradation only occurring with relatively unstable substrates (those that were at least transiently unfolded as determined by protease sensitivity). Surprisingly, UbID degradation was unaffected by the non‐hydrolyzable ATP analog ATPγS, which halts ATP‐dependent engagement and translocation of substrates. Furthermore, ubiquitin‐dependent degradation proceeded in a strikingly similar fashion to UbID degradation in the presence of ATPγS, suggesting the 26S proteasome may have a “default” ATP‐ and ubiquitin‐independent mechanism that is capable of unfolding and degrading less‐stable proteins.

Remnants of the Balbiani body are required for formation of RNA transport granules in Xenopus oocytes

SummaryThe Balbiani body (Bb), an organelle comprised of mitochondria, ER, and RNA, is found in the oocytes of most organisms. In Xenopus, the structure is initially positioned immediately adjacent to the nucleus, extends toward the vegetal pole, and eventually disperses, leaving behind a region highly enriched in mitochondria. This area is later transversed by RNP complexes that are being localized to the vegetal cortex. Inhibition of mitochondrial ATP synthesis prevents perinuclear formation of the transport complexes that can be reversed by a nonhydrolyzable ATP analog, indicating the nucleotide is acting as a hydrotrope. The protein composition, sensitivity to hexanediol, and coalescence in the absence of transport provide evidence that the transport RNP complexes are biocondensates. The breakdown of the Bb engenders regions of clustered mitochondria that are used not to meet extraordinary energy demands, but rather to promote a liquid-liquid phase separation.

Cryo-EM structure of AMP-PNP-bound human mitochondrial ATP-binding cassette transporter ABCB7

ATP-binding cassette subfamily B member 7 (ABCB7) is localized in the inner membrane of mitochondria, playing a critical role in iron metabolism. Here, we determined the structure of the nonhydrolyzable ATP analog adenosine-5′-(β-γ-imido) triphosphate (AMP-PNP) bound human ABCB7 at 3.3 Å by single-particle electron cryo-microscopy (cryo-EM). The AMP-PNP-bound human ABCB7 shows an inverted V-shaped homodimeric architecture with an inward-facing open conformation. One AMP-PNP molecule and Mg2+ were identified in each nucleotide-binding domain (NBD) of the hABCB7 monomer. Moreover, four disease-causing missense mutations of human ABCB7 have been mapped to the structure, creating a hotspot map for X-linked sideroblastic anemia and ataxia disease. Our results provide a structural basis for further understanding the transport mechanism of the mitochondrial ABC transporter.

Cryo-EM reconstructions of inhibitor-bound SMG1 kinase reveal an autoinhibitory state dependent on SMG8.

The PI3K-related kinase (PIKK) SMG1 monitors the progression of metazoan nonsense-mediated mRNA decay (NMD) by phosphorylating the RNA helicase UPF1. Previous work has shown that the activity of SMG1 is impaired by small molecule inhibitors, is reduced by the SMG1 interactors SMG8 and SMG9, and is downregulated by the so-called SMG1 insertion domain. However, the molecular basis for this complex regulatory network has remained elusive. Here, we present cryo-electron microscopy reconstructions of human SMG1-9 and SMG1-8-9 complexes bound to either a SMG1 inhibitor or a non-hydrolyzable ATP analog at overall resolutions ranging from 2.8 to 3.6 Å. These structures reveal the basis with which a small molecule inhibitor preferentially targets SMG1 over other PIKKs. By comparison with our previously reported substrate-bound structure (Langer et al.,2020), we show that the SMG1 insertion domain can exert an autoinhibitory function by directly blocking the substrate-binding path as well as overall access to the SMG1 kinase active site. Together with biochemical analysis, our data indicate that SMG1 autoinhibition is stabilized by the presence of SMG8. Our results explain the specific inhibition of SMG1 by an ATP-competitive small molecule, provide insights into regulation of its kinase activity within the NMD pathway, and expand the understanding of PIKK regulatory mechanisms in general.

The CopA2-Type P1B-Type ATPase CcoI Serves as Central Hub for cbb 3-Type Cytochrome Oxidase Biogenesis.

Copper (Cu)-transporting P1B-type ATPases are ubiquitous metal transporters and crucial for maintaining Cu homeostasis in all domains of life. In bacteria, the P1B-type ATPase CopA is required for Cu-detoxification and exports excess Cu(I) in an ATP-dependent reaction from the cytosol into the periplasm. CopA is a member of the CopA1-type ATPase family and has been biochemically and structurally characterized in detail. In contrast, less is known about members of the CopA2-type ATPase family, which are predicted to transport Cu(I) into the periplasm for cuproprotein maturation. One example is CcoI, which is required for the maturation of cbb3-type cytochrome oxidase (cbb3-Cox) in different species. Here, we reconstituted purified CcoI of Rhodobacter capsulatus into liposomes and determined Cu transport using solid-supported membrane electrophysiology. The data demonstrate ATP-dependent Cu(I) translocation by CcoI, while no transport is observed in the presence of a non-hydrolysable ATP analog. CcoI contains two cytosolically exposed N-terminal metal binding sites (N-MBSs), which are both important, but not essential for Cu delivery to cbb3-Cox. CcoI and cbb3-Cox activity assays in the presence of different Cu concentrations suggest that the glutaredoxin-like N-MBS1 is primarily involved in regulating the ATPase activity of CcoI, while the CopZ-like N-MBS2 is involved in Cu(I) acquisition. The interaction of CcoI with periplasmic Cu chaperones was analyzed by genetically fusing CcoI to the chaperone SenC. The CcoI-SenC fusion protein was fully functional in vivo and sufficient to provide Cu for cbb3-Cox maturation. In summary, our data demonstrate that CcoI provides the link between the cytosolic and periplasmic Cu chaperone networks during cbb3-Cox assembly.