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- PDB-7aa4: Structure of ClpC1-NTD bound to a CymA analogue -

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Basic information

Entry
Database: PDB / ID: 7aa4
TitleStructure of ClpC1-NTD bound to a CymA analogue
Components
  • Negative regulator of genetic competence ClpC/mecB
  • polymer Cyclomarin A analogue
KeywordsCHAPERONE / helical receptor domain / cyclomarin A inhibtor
Function / homology
Function and homology information


cellular response to heat / ATP hydrolysis activity / ATP binding / cytoplasm
Similarity search - Function
: / Clp amino terminal domain, pathogenicity island component / Clp repeat (R) domain profile. / Clp, repeat (R) domain / Clp, N-terminal domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Negative regulator of genetic competence ClpC/mecB
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria)
Streptomyces (bacteria)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 1.68 Å
AuthorsMeinhart, A. / Morreale, F.E. / Kaiser, M. / Clausen, T.
Funding support Austria, 1items
OrganizationGrant numberCountry
Vienna Science and Technology Fund (WWTF)WWTF // LS17-29 Austria
CitationJournal: Cell / Year: 2022
Title: BacPROTACs mediate targeted protein degradation in bacteria.
Authors: Francesca E Morreale / Stefan Kleine / Julia Leodolter / Sabryna Junker / David M Hoi / Stepan Ovchinnikov / Anastasia Okun / Juliane Kley / Robert Kurzbauer / Lukas Junk / Somraj Guha / ...Authors: Francesca E Morreale / Stefan Kleine / Julia Leodolter / Sabryna Junker / David M Hoi / Stepan Ovchinnikov / Anastasia Okun / Juliane Kley / Robert Kurzbauer / Lukas Junk / Somraj Guha / David Podlesainski / Uli Kazmaier / Guido Boehmelt / Harald Weinstabl / Klaus Rumpel / Volker M Schmiedel / Markus Hartl / David Haselbach / Anton Meinhart / Markus Kaiser / Tim Clausen /
Abstract: Hijacking the cellular protein degradation system offers unique opportunities for drug discovery, as exemplified by proteolysis-targeting chimeras. Despite their great promise for medical chemistry, ...Hijacking the cellular protein degradation system offers unique opportunities for drug discovery, as exemplified by proteolysis-targeting chimeras. Despite their great promise for medical chemistry, so far, it has not been possible to reprogram the bacterial degradation machinery to interfere with microbial infections. Here, we develop small-molecule degraders, so-called BacPROTACs, that bind to the substrate receptor of the ClpC:ClpP protease, priming neo-substrates for degradation. In addition to their targeting function, BacPROTACs activate ClpC, transforming the resting unfoldase into its functional state. The induced higher-order oligomer was visualized by cryo-EM analysis, providing a structural snapshot of activated ClpC unfolding a protein substrate. Finally, drug susceptibility and degradation assays performed in mycobacteria demonstrate in vivo activity of BacPROTACs, allowing selective targeting of endogenous proteins via fusion to an established degron. In addition to guiding antibiotic discovery, the BacPROTAC technology presents a versatile research tool enabling the inducible degradation of bacterial proteins.
History
DepositionSep 3, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 11, 2021Provider: repository / Type: Initial release
Revision 1.1Jun 15, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.journal_id_CSD ..._citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Jul 6, 2022Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first
Revision 1.3Jan 31, 2024Group: Data collection / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model / struct_conn
Item: _struct_conn.pdbx_leaving_atom_flag

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Negative regulator of genetic competence ClpC/mecB
B: polymer Cyclomarin A analogue


Theoretical massNumber of molelcules
Total (without water)18,3232
Polymers18,3232
Non-polymers00
Water3,459192
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1060 Å2
ΔGint-10 kcal/mol
Surface area7840 Å2
MethodPISA
Unit cell
Length a, b, c (Å)31.350, 33.680, 35.810
Angle α, β, γ (deg.)86.18, 94.22, 103.18
Int Tables number1
Space group name H-MP1
Symmetry operation#1: x,y,z

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Components

#1: Protein Negative regulator of genetic competence ClpC/mecB


Mass: 17387.918 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: clpC_1, ERS007741_03095 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A655JDN0
#2: Protein/peptide polymer Cyclomarin A analogue


Mass: 935.182 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptomyces (bacteria)
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 192 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.11 Å3/Da / Density % sol: 41.67 %
Crystal growTemperature: 294 K / Method: vapor diffusion, hanging drop / pH: 6.5
Details: 100 mM MES/imidazole pH 6.5, 6% (w/v) PEG 20K, 2% (v/v) PEG MME 550, 120 mM 1,6-hexanediol, 120 mM (RS)-1,2-propanediol, 120 mM 2-propanol, 120 mM 1,4-butanediol

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU MICROMAX-007 HF / Wavelength: 1.5406 Å
DetectorType: MARRESEARCH / Detector: IMAGE PLATE / Date: May 3, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5406 Å / Relative weight: 1
ReflectionResolution: 1.68→25 Å / Num. obs: 15176 / % possible obs: 93.8 % / Redundancy: 5.8 % / CC1/2: 0.998 / Rrim(I) all: 0.062 / Net I/σ(I): 22.6
Reflection shellResolution: 1.68→1.72 Å / Redundancy: 5.4 % / Mean I/σ(I) obs: 12.5 / Num. unique obs: 1015 / CC1/2: 0.992 / Rrim(I) all: 0.112 / % possible all: 86.5

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Processing

Software
NameVersionClassification
PHENIX(1.17.1_3660: ???)refinement
MAR345dtbdata collection
XDSdata reduction
XSCALEdata scaling
PHASERphasing
Cootmodel building
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3WDB

3wdb


Resolution: 1.68→24.77 Å / SU ML: 0.19 / Cross valid method: FREE R-VALUE / σ(F): 1.99 / Phase error: 21.62 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.2033 757 4.99 %Random selection
Rwork0.1696 ---
obs0.1714 15165 93.81 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 1.68→24.77 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1291 0 0 192 1483
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0061344
X-RAY DIFFRACTIONf_angle_d0.7891816
X-RAY DIFFRACTIONf_dihedral_angle_d26.756186
X-RAY DIFFRACTIONf_chiral_restr0.049205
X-RAY DIFFRACTIONf_plane_restr0.004236
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.68-1.810.24361450.1742774X-RAY DIFFRACTION91
1.81-1.990.19841510.15672863X-RAY DIFFRACTION93
1.99-2.280.20531510.15392875X-RAY DIFFRACTION94
2.28-2.870.19271550.17812934X-RAY DIFFRACTION95
2.87-24.770.2011550.17512962X-RAY DIFFRACTION97

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