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- EMDB-29366: Co-structure of the Human Metapneunomovirus RNA-dependent RNA pol... -

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Basic information

Entry
Database: EMDB / ID: EMD-29366
TitleCo-structure of the Human Metapneunomovirus RNA-dependent RNA polymerase with MRK-1
Map dataSharpened map used for model refinement
Sample
  • Complex: HUMAN METAPNEUMOVIRUS POLYMERASE (L) PROTEIN BOUND BY THE TETRAMERIC PHOSPHOPROTEIN (P) AND COMPLEXED WITH MRK-1
    • Protein or peptide: Phosphoprotein
  • Protein or peptide: RNA-directed RNA polymerase L
  • Ligand: 4-(2-aminopropan-2-yl)-N'-[4-(cyclopropyloxy)-3-methoxybenzoyl]-6-(4-fluorophenyl)pyridine-2-carbohydrazide
KeywordsRNA-BINDING PROTEIN / HMPV / RDRP / RNA-DEPENDENT RNA POLYMERASE / PRNTASE / POLYRIBONUCLEOTIDYL TRANSFERASE / RNA CAPPING / VIRAL REPLICATION / VIRAL PROTEIN / REPLICATION
Function / homology
Function and homology information


NNS virus cap methyltransferase / GDP polyribonucleotidyltransferase / Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides / virion component / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / host cell cytoplasm / RNA-directed RNA polymerase / RNA-dependent RNA polymerase activity / GTPase activity / ATP binding / metal ion binding
Similarity search - Function
Phosphoprotein, pneumoviral / Pneumovirus phosphoprotein / RNA-directed RNA polymerase L methyltransferase domain, rhabdovirus / Virus-capping methyltransferase, MT domain / Mononegavirales RNA-directed RNA polymerase catalytic domain / Mononegavirus L protein 2-O-ribose methyltransferase / Mononegavirales mRNA-capping domain V / RNA-directed RNA polymerase L, C-terminal / Mononegavirales RNA dependent RNA polymerase / Mononegavirales mRNA-capping region V ...Phosphoprotein, pneumoviral / Pneumovirus phosphoprotein / RNA-directed RNA polymerase L methyltransferase domain, rhabdovirus / Virus-capping methyltransferase, MT domain / Mononegavirales RNA-directed RNA polymerase catalytic domain / Mononegavirus L protein 2-O-ribose methyltransferase / Mononegavirales mRNA-capping domain V / RNA-directed RNA polymerase L, C-terminal / Mononegavirales RNA dependent RNA polymerase / Mononegavirales mRNA-capping region V / RdRp of negative ssRNA viruses with non-segmented genomes catalytic domain profile. / Mononegavirus L protein 2'-O-ribose methyltransferase domain profile.
Similarity search - Domain/homology
RNA-directed RNA polymerase L / Phosphoprotein
Similarity search - Component
Biological speciesHuman metapneumovirus CAN97-83 / Human metapneumovirus
Methodsingle particle reconstruction / cryo EM / Resolution: 2.74 Å
AuthorsFischmann TO
Funding support United States, 1 items
OrganizationGrant numberCountry
Other private United States
CitationJournal: Commun Biol / Year: 2023
Title: Conserved allosteric inhibitory site on the respiratory syncytial virus and human metapneumovirus RNA-dependent RNA polymerases.
Authors: Victoria A Kleiner / Thierry O Fischmann / John A Howe / Douglas C Beshore / Michael J Eddins / Yan Hou / Todd Mayhood / Daniel Klein / Debbie D Nahas / Bob J Lucas / He Xi / Edward Murray / ...Authors: Victoria A Kleiner / Thierry O Fischmann / John A Howe / Douglas C Beshore / Michael J Eddins / Yan Hou / Todd Mayhood / Daniel Klein / Debbie D Nahas / Bob J Lucas / He Xi / Edward Murray / Daphne Y Ma / Krista Getty / Rachel Fearns /
Abstract: Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) are related RNA viruses responsible for severe respiratory infections and resulting disease in infants, elderly, and ...Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) are related RNA viruses responsible for severe respiratory infections and resulting disease in infants, elderly, and immunocompromised adults. Therapeutic small molecule inhibitors that bind to the RSV polymerase and inhibit viral replication are being developed, but their binding sites and molecular mechanisms of action remain largely unknown. Here we report a conserved allosteric inhibitory site identified on the L polymerase proteins of RSV and HMPV that can be targeted by a dual-specificity, non-nucleoside inhibitor, termed MRK-1. Cryo-EM structures of the inhibitor in complexes with truncated RSV and full-length HMPV polymerase proteins provide a structural understanding of how MRK-1 is active against both viruses. Functional analyses indicate that MRK-1 inhibits conformational changes necessary for the polymerase to engage in RNA synthesis initiation and to transition into an elongation mode. Competition studies reveal that the MRK-1 binding pocket is distinct from that of a capping inhibitor with an overlapping resistance profile, suggesting that the polymerase conformation bound by MRK-1 may be distinct from that involved in mRNA capping. These findings should facilitate optimization of dual RSV and HMPV replication inhibitors and provide insights into the molecular mechanisms underlying their polymerase activities.
History
DepositionJan 4, 2023-
Header (metadata) releaseJun 14, 2023-
Map releaseJun 14, 2023-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29366.png

Downloads & links

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Map

FileDownload / File: emd_29366.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map used for model refinement
Voxel sizeX=Y=Z: 0.84 Å
Density
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-1.2681677 - 1.9156141
Average (Standard dev.)0.001608864 (±0.05163311)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 251.99998 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Unsharpened half-map

Fileemd_29366_half_map_1.map
AnnotationUnsharpened half-map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unsharpened half-map

Fileemd_29366_half_map_2.map
AnnotationUnsharpened half-map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : HUMAN METAPNEUMOVIRUS POLYMERASE (L) PROTEIN BOUND BY THE TETRAME...

EntireName: HUMAN METAPNEUMOVIRUS POLYMERASE (L) PROTEIN BOUND BY THE TETRAMERIC PHOSPHOPROTEIN (P) AND COMPLEXED WITH MRK-1
Components
  • Complex: HUMAN METAPNEUMOVIRUS POLYMERASE (L) PROTEIN BOUND BY THE TETRAMERIC PHOSPHOPROTEIN (P) AND COMPLEXED WITH MRK-1
    • Protein or peptide: Phosphoprotein
  • Protein or peptide: RNA-directed RNA polymerase L
  • Ligand: 4-(2-aminopropan-2-yl)-N'-[4-(cyclopropyloxy)-3-methoxybenzoyl]-6-(4-fluorophenyl)pyridine-2-carbohydrazide

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Supramolecule #1: HUMAN METAPNEUMOVIRUS POLYMERASE (L) PROTEIN BOUND BY THE TETRAME...

SupramoleculeName: HUMAN METAPNEUMOVIRUS POLYMERASE (L) PROTEIN BOUND BY THE TETRAMERIC PHOSPHOPROTEIN (P) AND COMPLEXED WITH MRK-1
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #2
Source (natural)Organism: Human metapneumovirus CAN97-83

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Macromolecule #1: RNA-directed RNA polymerase L

MacromoleculeName: RNA-directed RNA polymerase L / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: RNA-directed RNA polymerase
Source (natural)Organism: Human metapneumovirus
Molecular weightTheoretical: 235.226891 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MGSDYKDHDG DYKDHDIDYK DDDDKGSGSL EVLFQGPMDP LNESTVNVYL PDSYLKGVIS FSETNAIGSC LLKRPYLKND NTAKVAIEN PVIEHVRLKN AVNSKMKISD YKVVEPVNMQ HEIMKNVHSC ELTLLKQFLT RSKNISTLKL NMICDWLQLK S TSDDTSIL ...String:
MGSDYKDHDG DYKDHDIDYK DDDDKGSGSL EVLFQGPMDP LNESTVNVYL PDSYLKGVIS FSETNAIGSC LLKRPYLKND NTAKVAIEN PVIEHVRLKN AVNSKMKISD YKVVEPVNMQ HEIMKNVHSC ELTLLKQFLT RSKNISTLKL NMICDWLQLK S TSDDTSIL SFIDVEFIPS WVSNWFSNWY NLNKLILEFR REEVIRTGSI LCRSLGKLVF IVSSYGCIVK SNKSKRVSFF TY NQLLTWK DVMLSRFNAN FCIWVSNSLN ENQEGLGLRS NLQGMLTNKL YETVDYMLSL CCNEGFSLVK EFEGFIMSEI LRI TEHAQF STRFRNTLLN GLTDQLTKLK NKNRLRVHST VLENNDYPMY EVVLKLLGDT LRCIKLLINK NLENAAELYY IFRI FGHPM VDERDAMDAV KLNNEITKIL RLESLTELRG AFILRIIKGF VDNNKRWPKI KNLKVLSKRW TMYFKAKNYP SQLEL SEQD FLELAAIQFE QEFSVPEKTN LEMVLNDKAI SPPKRLIWSV YPKNYLPETI KNRYLEETFN ASDSLKTRRV LEYYLK DNK FDQKELKSYV VRQEYLNDKE HIVSLTGKER ELSVGRMFAM QPGKQRQIQI LAEKLLADNI VPFFPETLTK YGDLDLQ RI MEIKSELSSI KTRRNDSYNN YIARASIVTD LSKFNQAFRY ETTAICADVA DELHGTQSLF CWLHLIVPMT TMICAYRH A PPETKGEYDI DKIEEQSGLY RYHMGGIEGW CQKLWTMEAI SLLDVVSVKT RCQMTSLLNG DNQSIDVSKP VKLSEGLDE VKADYRLAVK MLKEIRDAYR NIGHKLKEGE TYISRDLQFI SKVIQSEGVM HPTPIKKVLR VGPWINTILD DIKTSAESIG SLCQELEFR GESIIVSLIL RNFWLYNLYM HESKQHPLAG KQLFKQLNKT LTSVQRFFEI KRENEVVDLW MNIPMQFGGG D PVVFYRSF YRRTPDFLTE AISHVDILLK ISANIKNETK VSFFKALLSI EKNERATLTT LMRDPQAVGS ERQAKVTSDI NR TAVTSIL SLSPNQLFSD SAIHYSRNEE EVGIIAENIT PVYPHGLRVL YESLPFHKAE KVVNMISGTK SITNLLQRTS AIN GEDIDR AVSMMLENLG LLSRILSVVV DSIEIPIKSN GRLICCQISR TLRETSWNNM EIVGVTSPSI TTCMDVIYAT SSHL KGIII EKFSTDRTTR GQRGPKSPWV GSSTQEKKLV PVYNRQILSK QQREQLEAIG KMRWVYKGTP GLRRLLNKIC LGSLG ISYK CVKPLLPRFM SVNFLHRLSV SSRPMEFPAS VPAYRTTNYH FDTSPINQAL SERFGNEDIN LVFQNAISCG ISIMSV VEQ LTGRSPKQLV LIPQLEEIDI MPPPVFQGKF NYKLVDKITS DQHIFSPDKI DMLTLGKMLM PTIKGQKTDQ FLNKREN YF HGNNLIESLS AALACHWCGI LTEQCIENNI FKKDWGDGFI SDHAFMDFKI FLCVFKTKLL CSWGSQGKNI KDEDIVDE S IDKLLRIDNT FWRMFSKVMF EPKVKKRIML YDVKFLSLVG YIGFKNWFIE QLRSAELHEI PWIVNAEGDL VEIKSIKIY LQLIEQSLFL RITVLNYTDM AHALTRLIRK KLMCDNALLT PISSPMVNLT QVIDPTTQLD YFPKITFERL KNYDTSSNYA KGKLTRNYM ILLPWQHVNR YNFVFSSTGC KVSLKTCIGK LMKDLNPKVL YFIGEGAGNW MARTACEYPD IKFVYRSLKD D LDHHYPLE YQRVIGELSR IIDSGEGLSM ETTDATQKTH WDLIHRVSKD ALLITLCDAE FKDRDDFFKM VILWRKHVLS CR ICTTYGT DLYLFAKYHA KDCNVKLPFF VRSVATFIMQ GSKLSGSECY ILLTLGHHNS LPCHGEIQNS KMKIAVCNDF YAA KKLDNK SIEANCKSLL SGLRIPINKK ELDRQRRLLT LQSNHSSVAT VGGSKIIESK WLTNKASTII DWLEHILNSP KGEL NYDFF EALENTYPNM IKLIDNLGNA EIKKLIKVTG YMLVSKK

UniProtKB: RNA-directed RNA polymerase L

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Macromolecule #2: Phosphoprotein

MacromoleculeName: Phosphoprotein / type: protein_or_peptide / ID: 2 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Human metapneumovirus
Molecular weightTheoretical: 34.814141 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MSFPEGKDIL FMGNEAAKLA EAFQKSLRKP SHKRSQSIIG EKVNTVSETL ELPTISRPTK PTILSEPKLA WTDKGGAIKT EAKQTIKVM DPIEEEEFTE KRVLPSSDGK TPAEKKLKPS TNTKKKVSFT PNEPGKYTKL EKDALDLLSD NEEEDAESSI L TFEERDTS ...String:
MSFPEGKDIL FMGNEAAKLA EAFQKSLRKP SHKRSQSIIG EKVNTVSETL ELPTISRPTK PTILSEPKLA WTDKGGAIKT EAKQTIKVM DPIEEEEFTE KRVLPSSDGK TPAEKKLKPS TNTKKKVSFT PNEPGKYTKL EKDALDLLSD NEEEDAESSI L TFEERDTS SLSIEARLES IEEKLSMILG LLRTLNIATA GPTAARDGIR DAMIGIREEL IADIIKEAKG KAAEMMEEEM NQ RTKIGNG SVKLTEKAKE LNKIVEDEST SGESEEEEEL KDTQENNQED DIYQLIMKGE NKYFQGHHHH HHH

UniProtKB: Phosphoprotein

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Macromolecule #3: 4-(2-aminopropan-2-yl)-N'-[4-(cyclopropyloxy)-3-methoxybenzoyl]-6...

MacromoleculeName: 4-(2-aminopropan-2-yl)-N'-[4-(cyclopropyloxy)-3-methoxybenzoyl]-6-(4-fluorophenyl)pyridine-2-carbohydrazide
type: ligand / ID: 3 / Number of copies: 1 / Formula: Y6L
Molecular weightTheoretical: 478.515 Da
Chemical component information

ChemComp-Y6L:
4-(2-aminopropan-2-yl)-N'-[4-(cyclopropyloxy)-3-methoxybenzoyl]-6-(4-fluorophenyl)pyridine-2-carbohydrazide

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.6 mg/mL
BufferpH: 7.4
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Detector mode: COUNTING / Average electron dose: 45.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6u5o

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.74 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 524654
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL
Output model

PDB-8fpj:
Co-structure of the Human Metapneunomovirus RNA-dependent RNA polymerase with MRK-1

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