MDA5 disease variant M854K prevents ATP-dependent structural discrimination of viral and cellular RNA

AbstractOur innate immune responses to viral RNA are vital defenses. Long cytosolic double-stranded RNA (dsRNA) is recognized by MDA5. The ATPase activity of MDA5 contributes to its dsRNA binding selectivity. Mutations that reduce RNA selectivity can cause autoinflammatory disease. Here, we show how the disease-associated MDA5 variant M854K perturbs MDA5-dsRNA recognition. M854K MDA5 constitutively activates interferon signaling in the absence of exogenous RNA. M854K MDA5 lacks ATPase activity and binds more stably to synthetic Alu:Alu dsRNA. CryoEM structures of MDA5-dsRNA filaments at different stages of ATP hydrolysis show that the K854 sidechain forms polar bonds that constrain the conformation of MDA5 subdomains, disrupting key steps in the ATPase cycle- RNA footprint expansion and helical twist modulation. The M854K mutation inhibits ATP-dependent RNA proofreading via an allosteric mechanism, allowing MDA5 to form signaling complexes on endogenous RNAs. This work provides insights on how MDA5 recognizes dsRNA in health and disease.

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MDA5 autoimmune disease variant M854K prevents ATP-dependent structural discrimination of viral and cellular RNA

10.1101/2021.02.01.429133 ◽

2021 ◽

Author(s):

Qin Yu ◽

Alba Herrero del Valle ◽

Rahul Singh ◽

Yorgo Modis

Keyword(s):

Autoimmune Disease ◽

Atpase Activity ◽

Atp Hydrolysis ◽

Side Chain ◽

Innate Immune Responses ◽

Dsrna Binding ◽

Exogenous Rna ◽

Health And Disease ◽

Disease Variant ◽

Key Steps

AbstractOur innate immune responses to viral RNA are vital defenses. Long cytosolic double-stranded RNA (dsRNA) is recognized by MDA5. The ATPase activity of MDA5 contributes to its dsRNA binding selectivity. Mutations that reduce RNA selectivity can cause autoimmune disease. Here, we show how the disease-associated MDA5 variant M854K perturbs MDA5-dsRNA recognition. M854K MDA5 constitutively activates interferon signaling in the absence of exogenous RNA. M854K MDA5 lacks ATPase activity and binds more tightly to synthetic Alu:Alu dsRNA. CryoEM structures MDA5-dsRNA filaments at different stages of ATP hydrolysis show that the K854 side-chain forms polar bonds that constrain the conformation of MDA5 subdomains, disrupting key steps in the ATPase cycle-RNA footprint expansion and helical twist modulation. The M854K mutation inhibits ATP-dependent RNA proofreading via a novel allosteric mechanism, allowing MDA5 to form signaling complexes on endogenous RNAs. This work provides new insights on how MDA5 recognizes dsRNA in health and disease.

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CryoEM structures of MDA5-dsRNA filaments at different stages of ATP hydrolysis

10.1101/376319 ◽

2018 ◽

Author(s):

Qin Yu ◽

Kun Qu ◽

Yorgo Modis

Keyword(s):

Rna Binding ◽

Atp Hydrolysis ◽

Type I ◽

List Type ◽

Base Pairs ◽

Nucleotide Analogs ◽

Double Stranded Rna ◽

Transition State Analogs ◽

Helical Twist ◽

Dsrna Binding

SummaryDouble-stranded RNA (dsRNA) is a potent proinflammatory signature of viral infection. Long cytosolic dsRNA is recognized by MDA5. The cooperative assembly of MDA5 into helical filaments on dsRNA nucleates the assembly of a multiprotein type-I-interferon signaling platform. Here, we determined cryoEM structures of MDA5-dsRNA filaments with different helical twists and bound nucleotide analogs, at resolutions sufficient to build and refine atomic models. The structures identify the filament forming interfaces, which encode the dsRNA binding cooperativity and length specificity of MDA5. The predominantly hydrophobic interface contacts confer flexibility, reflected in the variable helical twist within filaments. Mutation of filament-forming residues can result in loss or gain of signaling activity. Each MDA5 molecule spans 14 or 15 RNA base pairs, depending on the twist. Variations in twist also correlate with variations in the occupancy and type of nucleotide in the active site, providing insights on how ATP hydrolysis contributes to MDA5-dsRNA recognition.eTOCStructures of MDA5 bound to double-stranded RNA reveal a flexible, predominantly hydrophobic filament forming interface. The filaments have variable helical twist. Structures determined with ATP and transition state analogs show how the ATPase cycle is coupled to changes in helical twist, the mode of RNA binding and the length of the RNA footprint of MDA5.HighlightsCryoEM structures of MDA5-dsRNA filaments determined for three catalytic statesFilament forming interfaces are flexible and predominantly hydrophobicMutation of filament-forming residues can cause loss or gain of IFN-β signalingATPase cycle is coupled to changes in filament twist and size of the RNA footprint

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The extracellular gate shapes the energy profile of an ABC exporter

10.1101/421073 ◽

2018 ◽

Cited By ~ 1

Author(s):

Cedric A.J. Hutter ◽

M. Hadi Timachi ◽

Lea M. Hürlimann ◽

Iwan Zimmermann ◽

Pascal Egloff ◽

...

Keyword(s):

Atpase Activity ◽

Drug Transport ◽

Atp Hydrolysis ◽

Underlying Mechanism ◽

Single Domain Antibody ◽

Gate Opening ◽

Transport Cycle ◽

Double Electron ◽

Double Electron Electron Resonance ◽

Key Steps

ABSTRACTABC exporters harness the energy of ATP to pump substrates across membranes. Extracellular gate opening and closure are key steps of the transport cycle, but the underlying mechanism is poorly understood. Here, we generated a synthetic single domain antibody (sybody) that recognizes the heterodimeric ABC exporter TM287/288 exclusively in the presence of ATP, which was essential to solve a 3.2 Å crystal structure of the outward-facing transporter. The sybody binds to an extracellular wing and strongly inhibits ATPase activity by shifting the transporter’s conformational equilibrium towards the outward-facing state, as shown by double electron-electron resonance (DEER). Mutations that facilitate extracellular gate opening resulted in a comparable equilibrium shift and strongly reduced ATPase activity and drug transport. Using the sybody as conformational probe, we demonstrate that efficient extracellular gate closure is required to dissociate the NBD dimer after ATP hydrolysis to reset the transporter back to its inward-facing state.

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Faculty Opinions recommendation of Roles of caspase-8 and caspase-10 in innate immune responses to double-stranded RNA.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1014308.371940 ◽

2006 ◽

Author(s):

Michael Gale

Keyword(s):

Immune Responses ◽

Innate Immune ◽

Caspase 8 ◽

Innate Immune Responses ◽

Double Stranded Rna

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Tonoplast and plasma membrane ATPases from maize lines of high or low vigour

Seed Science Research ◽

10.1017/s0960258500001203 ◽

1992 ◽

Vol 2 (2) ◽

pp. 105-111 ◽

Cited By ~ 6

Author(s):

S. Sánchez-Nieto ◽

R. Rodríguez-Sotres ◽

P. González-Romo ◽

I. Bernal-Lugo ◽

M. Gavilanes-Ruíz

Keyword(s):

Zea Mays ◽

Plasma Membrane ◽

Acid Phosphatase ◽

Atpase Activity ◽

Kinetic Analysis ◽

Atp Hydrolysis ◽

Membrane Atpases ◽

Substrate Concentrations ◽

Tonoplast Atpase ◽

Specific Inhibitors

AbstractThe effectiveness of ATPase in germinated seed may play an important role in the vigour of germination. The activities of tonoplast and plasma membrane ATPases in two maize (Zea mays L.) lines with different vigour of germination were determined. ATP hydrolysis was measured in microsomal fractions from coleoptiles along with the responses to specific inhibitors for the plasma membrane, tonoplast and mitochondrial ATPases as well as for acid phosphatase. Nitrate-sensitive ATPase activity was 1.5–3.0 times lower in the low-vigour line than in the high-vigour line. Kinetic analysis of ATP hydrolysis at different substrate concentrations revealed the existence of two enzymes in the microsomal fractions of the two lines. The Vmax of enzyme 1 in the low-vigour line was a third of that in the high-vigour line. This enzyme was identified as the nitrate-sensitive or tonoplast ATPase on the basis of measurements of ATP hydrolysis in the presence of specific inhibitors at high (8.12mm) and low (0.77mm) ATP concentrations.

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Requirement of PKR Dimerization Mediated by Specific Hydrophobic Residues for Its Activation by Double-Stranded RNA and Its Antigrowth Effects in Yeast

Molecular and Cellular Biology ◽

10.1128/mcb.18.12.7009 ◽

1998 ◽

Vol 18 (12) ◽

pp. 7009-7019 ◽

Cited By ~ 40

Author(s):

Rekha C. Patel ◽

Ganes C. Sen

Keyword(s):

Helical Structure ◽

Wild Type ◽

Dimerization Domain ◽

Double Stranded Rna ◽

Mutant Proteins ◽

Hydrophobic Residues ◽

Dsrna Binding ◽

Dependent Protein ◽

Substitution Mutations

ABSTRACT The roles of protein dimerization and double-stranded RNA (dsRNA) binding in the biochemical and cellular activities of PKR, the dsRNA-dependent protein kinase, were investigated. We have previously shown that both properties of the protein are mediated by the same domain. Here we show that dimerization is mediated by hydrophobic residues present on one side of an amphipathic α-helical structure within this domain. Appropriate substitution mutations of residues on that side produced mutants with increased or decreased dimerization activities. Using these mutants, we demonstrated that dimerization is not essential for dsRNA binding. However, enhancing dimerization artificially, by providing an extraneous dimerization domain, increased dsRNA binding of both wild-type and mutant proteins. In vitro, the dimerization-defective mutants could not be activated by dsRNA but were activated normally by heparin. In Saccharomyces cerevisiae, unlike wild-type PKR, these mutants could not inhibit cell growth and the dsRNA-binding domain of the dimerization-defective mutants could not prevent the antigrowth effect of wild-type PKR. These results demonstrate the biological importance of the dimerization properties of PKR.

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Activation of the double-stranded RNA (dsRNA)-activated human protein kinase in vivo in the absence of its dsRNA binding domain.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.91.22.10551 ◽

1994 ◽

Vol 91 (22) ◽

pp. 10551-10555 ◽

Cited By ~ 31

Author(s):

S. B. Lee ◽

S. R. Green ◽

M. B. Mathews ◽

M. Esteban

Keyword(s):

Protein Kinase ◽

Binding Domain ◽

Human Protein ◽

Double Stranded Rna ◽

Human Protein Kinase ◽

Dsrna Binding

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Identification of Rab6 as an N-ethylmaleimide-sensitive fusion protein-binding protein

Biochemical Journal ◽

10.1042/bj3520165 ◽

2000 ◽

Vol 352 (1) ◽

pp. 165-173 ◽

Cited By ~ 14

Author(s):

Sang Yeul HAN ◽

Dong Yoon PARK ◽

Sang Dai PARK ◽

Seung Hwan HONG

Keyword(s):

Fusion Protein ◽

Atpase Activity ◽

Atp Hydrolysis ◽

Polyclonal Antibodies ◽

Binding Protein ◽

Vesicular Transport ◽

Brain Extract ◽

Terminal Domain ◽

Protein Receptor ◽

And Function

In this study we show the interaction of N-ethylmaleimide-sensitive fusion protein (NSF) with a small GTP-binding protein, Rab6. NSF is an ATPase involved in the vesicular transport within eukaryotic cells. Using the yeast two-hybrid system, we have isolated new NSF-binding proteins from the rat lung cDNA library. One of them was Rab6, which is involved in the vesicular transport within the Golgi and trans-Golgi network as a Ras-like GTPase. We demonstrated that the N-terminal domain of NSF interacted with the C-terminal domain of Rab6, and these proteins were co-immunoprecipitated from the rat brain extract. This interaction was maintained preferentially in the presence of hydrolysable ATP. Recombinant NSF-His6 can also bind to C-terminal Rab6–glutathione S-transferase under the conditions to allow the ATP hydrolysis. Surprisingly, Rab6 stimulates the ATPase activity of NSF by approx. 2-fold as does α-soluble NSF attachment protein receptor. Anti-Rab6 polyclonal antibodies significantly inhibited the Rab6-stimulated ATPase activity of NSF. Furthermore, we found that Rab3 and Rab4 can also associate with NSF and stimulate its ATPase activity. Taken together, we propose a model in which Rab can form an ATP hydrolysis-regulated complex with NSF, and function as a signalling molecule to deliver the signal of vesicle fusion through the interaction with NSF.

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Stimulation of the human mitochondrial transporter ABCB10 by zinc-mesoporphrin

PLoS ONE ◽

10.1371/journal.pone.0238754 ◽

2020 ◽

Vol 15 (11) ◽

pp. e0238754

Author(s):

Melissa Martinez ◽

Gregory A. Fendley ◽

Alexandra D. Saxberg ◽

Maria E. Zoghbi

Keyword(s):

Oxidative Damage ◽

Atpase Activity ◽

Animal Studies ◽

Atp Hydrolysis ◽

Aminolevulinic Acid ◽

Negative Control ◽

Hydrolysis Rate ◽

Inner Mitochondrial Membrane ◽

Experimental Conditions ◽

Mitochondrial Transporter

Heme biosynthesis occurs through a series of reactions that take place within the cytoplasm and mitochondria, so intermediates need to move across these cellular compartments. However, the specific membrane transport mechanisms involved in the process are not yet identified. The ATP-binding cassette protein ABCB10 is essential for normal heme production, as knocking down this transporter in mice is embryonically lethal and accompanied by severe anemia plus oxidative damage. The role of ABCB10 is unknown, but given its location in the inner mitochondrial membrane, it has been proposed as a candidate to export either an early heme precursor or heme. Alternatively, ABCB10 might transport a molecule important for protection against oxidative damage. To help discern between these possibilities, we decided to study the effect of heme analogs, precursors, and antioxidant peptides on purified human ABCB10. Since substrate binding increases the ATP hydrolysis rate of ABC transporters, we have determined the ability of these molecules to activate purified ABCB10 reconstituted in lipid nanodiscs using ATPase measurements. Under our experimental conditions, we found that the only heme analog increasing ABCB10 ATPase activity was Zinc-mesoporphyrin. This activation of almost seventy percent was specific for ABCB10, as the ATPase activity of a negative control bacterial ABC transporter was not affected. The activation was also observed in cysteine-less ABCB10, suggesting that Zinc-mesoporphyrin’s effect did not require binding to typical heme regulatory motifs. Furthermore, our data indicate that ABCB10 was not directly activated by neither the early heme precursor delta-aminolevulinic acid nor glutathione, downsizing their relevance as putative substrates for this transporter. Although additional studies are needed to determine the physiological substrate of ABCB10, our findings reveal Zinc-mesoporphyrin as the first tool compound to directly modulate ABCB10 activity and raise the possibility that some actions of Zinc-mesoporphyrin in cellular and animal studies could be mediated by ABCB10.

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