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- PDB-7w6x: Crystal structure of E. coli RseP in complex with batimastat -

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Basic information

Entry
Database: PDB / ID: 7w6x
TitleCrystal structure of E. coli RseP in complex with batimastat
ComponentsRegulator of sigma-E protease RseP
KeywordsHYDROLASE / Intramembrane protease
Function / homology
Function and homology information


anti-sigma factor antagonist activity / Hydrolases; Acting on peptide bonds (peptidases); Metalloendopeptidases / cellular response to cell envelope stress / metalloendopeptidase activity / positive regulation of DNA-templated transcription / proteolysis / metal ion binding / plasma membrane
Similarity search - Function
Peptidase M50, putative membrane-associated zinc metallopeptidase / Peptidase M50 / Peptidase family M50 / PDZ domain 6 / PDZ domain / PDZ domain profile. / Domain present in PSD-95, Dlg, and ZO-1/2. / PDZ domain / PDZ superfamily / Neutral zinc metallopeptidases, zinc-binding region signature.
Similarity search - Domain/homology
Chem-BAT / Regulator of sigma-E protease RseP
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.2 Å
AuthorsTakanuki, K. / Imaizumi, Y. / Nogi, T.
Funding support Japan, 4items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)19H03170 Japan
Japan Society for the Promotion of Science (JSPS)26291016 Japan
Japan Society for the Promotion of Science (JSPS)22370039 Japan
Japan Society for the Promotion of Science (JSPS)19687004 Japan
CitationJournal: Sci Adv / Year: 2022
Title: Mechanistic insights into intramembrane proteolysis by site-2 protease homolog RseP.
Authors: Yuki Imaizumi / Kazunori Takanuki / Takuya Miyake / Mizuki Takemoto / Kunio Hirata / Mika Hirose / Rika Oi / Tatsuya Kobayashi / Kenichi Miyoshi / Rie Aruga / Tatsuhiko Yokoyama / Shizuka ...Authors: Yuki Imaizumi / Kazunori Takanuki / Takuya Miyake / Mizuki Takemoto / Kunio Hirata / Mika Hirose / Rika Oi / Tatsuya Kobayashi / Kenichi Miyoshi / Rie Aruga / Tatsuhiko Yokoyama / Shizuka Katagiri / Hiroaki Matsuura / Kenji Iwasaki / Takayuki Kato / Mika K Kaneko / Yukinari Kato / Michiko Tajiri / Satoko Akashi / Osamu Nureki / Yohei Hizukuri / Yoshinori Akiyama / Terukazu Nogi /
Abstract: Site-2 proteases are a conserved family of intramembrane proteases that cleave transmembrane substrates to regulate signal transduction and maintain proteostasis. Here, we elucidated crystal ...Site-2 proteases are a conserved family of intramembrane proteases that cleave transmembrane substrates to regulate signal transduction and maintain proteostasis. Here, we elucidated crystal structures of inhibitor-bound forms of bacterial site-2 proteases including RseP. Structure-based chemical modification and cross-linking experiments indicated that the RseP domains surrounding the active center undergo conformational changes to expose the substrate-binding site, suggesting that RseP has a gating mechanism to regulate substrate entry. Furthermore, mutational analysis suggests that a conserved electrostatic linkage between the transmembrane and peripheral membrane-associated domains mediates the conformational changes. In vivo cleavage assays also support that the substrate transmembrane helix is unwound by strand addition to the intramembrane β sheet of RseP and is clamped by a conserved asparagine residue at the active center for efficient cleavage. This mechanism underlying the substrate binding, i.e., unwinding and clamping, appears common across distinct families of intramembrane proteases that cleave transmembrane segments.
History
DepositionDec 2, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 7, 2022Provider: repository / Type: Initial release
Revision 1.1Nov 13, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / pdbx_entry_details / pdbx_modification_feature
Item: _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Regulator of sigma-E protease RseP
hetero molecules


Theoretical massNumber of molelcules
Total (without water)50,7124
Polymers50,1031
Non-polymers6083
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1200 Å2
ΔGint-63 kcal/mol
Surface area24990 Å2
MethodPISA
Unit cell
Length a, b, c (Å)47.340, 56.120, 69.670
Angle α, β, γ (deg.)68.167, 74.591, 69.346
Int Tables number1
Space group name H-MP1
Space group name HallP1
Symmetry operation#1: x,y,z

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Components

#1: Protein Regulator of sigma-E protease RseP / S2P endopeptidase / Site-2 protease RseP / S2P protease RseP / Site-2-type intramembrane protease


Mass: 50103.047 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: K12 / Gene: rseP, ecfE, yaeL, b0176, JW0171 / Production host: Escherichia coli (E. coli)
References: UniProt: P0AEH1, Hydrolases; Acting on peptide bonds (peptidases); Metalloendopeptidases
#2: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-BAT / 4-(N-HYDROXYAMINO)-2R-ISOBUTYL-2S-(2-THIENYLTHIOMETHYL)SUCCINYL-L-PHENYLALANINE-N-METHYLAMIDE / BATIMASTAT / BB94


Mass: 477.640 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C23H31N3O4S2 / Feature type: SUBJECT OF INVESTIGATION / Comment: inhibitor*YM
Has ligand of interestY
Has protein modificationY

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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: 3.17 Å3/Da / Density % sol: 61.2 %
Crystal growTemperature: 293 K / Method: lipidic cubic phase / pH: 7
Details: 30% (vol./vol.) polyethylene glycol 500 dimethyl ether, 100 mM NaCl, 100 mM MgCl2, 100 mM HEPES-Na (pH 7.0)

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SPring-8 / Beamline: BL32XU / Wavelength: 1 Å
DetectorType: DECTRIS EIGER X 9M / Detector: PIXEL / Date: Dec 14, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 3.2→43.77 Å / Num. obs: 10129 / % possible obs: 99.7 % / Redundancy: 8.4 % / Biso Wilson estimate: 103.16 Å2 / CC1/2: 0.995 / Rpim(I) all: 0.098 / Net I/σ(I): 7.5
Reflection shellResolution: 3.2→3.31 Å / Mean I/σ(I) obs: 1.1 / Num. unique obs: 1028 / CC1/2: 0.417 / Rpim(I) all: 1.347

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Processing

Software
NameVersionClassification
PHENIX1.19.2_4158refinement
XDSdata reduction
XSCALEdata scaling
MOLREPphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 3.2→43.76 Å / SU ML: 0.521 / Cross valid method: FREE R-VALUE / σ(F): 1.93 / Phase error: 33.9463
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.3053 471 4.65 %
Rwork0.246 9655 -
obs0.2486 10126 99.69 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 94.52 Å2
Refinement stepCycle: LAST / Resolution: 3.2→43.76 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3444 0 34 0 3478
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00193557
X-RAY DIFFRACTIONf_angle_d0.47744837
X-RAY DIFFRACTIONf_chiral_restr0.0419555
X-RAY DIFFRACTIONf_plane_restr0.0041620
X-RAY DIFFRACTIONf_dihedral_angle_d11.49131289
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.2-3.660.3321530.29523237X-RAY DIFFRACTION99.94
3.66-4.610.32041560.26863198X-RAY DIFFRACTION99.32
4.61-43.760.28881620.223220X-RAY DIFFRACTION99.82

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