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- PDB-7w6y: Crystal structure of Kangiella koreensis RseP orthologue in compl... -

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Basic information

Entry
Database: PDB / ID: 7w6y
TitleCrystal structure of Kangiella koreensis RseP orthologue in complex with batimastat in space group P1
ComponentsAnti sigma-E protein, RseA
KeywordsHYDROLASE / Intramembrane protease
Function / homology
Function and homology information


Hydrolases; Acting on peptide bonds (peptidases); Metalloendopeptidases / metalloendopeptidase activity / proteolysis / membrane / metal ion binding
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
Similarity search - Domain/homology
Chem-BAT / Zinc metalloprotease
Similarity search - Component
Biological speciesKangiella koreensis DSM 16069 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 3.1 Å
AuthorsImaizumi, Y. / Takanuki, K. / 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Anti sigma-E protein, RseA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)50,9863
Polymers50,4431
Non-polymers5432
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area950 Å2
ΔGint-42 kcal/mol
Surface area26040 Å2
MethodPISA
Unit cell
Length a, b, c (Å)44.560, 49.780, 76.080
Angle α, β, γ (deg.)86.897, 79.181, 82.189
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 Anti sigma-E protein, RseA


Mass: 50443.270 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Kangiella koreensis DSM 16069 (bacteria)
Strain: DSM 16069 / KCTC 12182 / SW-125 / Gene: Kkor_0748 / Production host: Escherichia coli (E. coli) / References: UniProt: C7R5Z1
#2: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#3: Chemical ChemComp-BAT / 4-(N-HYDROXYAMINO)-2R-ISOBUTYL-2S-(2-THIENYLTHIOMETHYL)SUCCINYL-L-PHENYLALANINE-N-METHYLAMIDE / BATIMASTAT / BB94 / Batimastat


Mass: 477.640 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C23H31N3O4S2 / Comment: inhibitor*YM
Has ligand of interestN

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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: 3.26 Å3/Da / Density % sol: 62.24 %
Description: The entry contains friedel pairs in F_plus/minus columns and I_plus/minus columns
Crystal growTemperature: 293 K / Method: lipidic cubic phase
Details: 28-30% (vol./vol.) Polyethylene glycol 500 monomethyl ether, 100 mM NaCl, 100 mM CaCl2, and 100 mM HEPES-Na (pH7.0)

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SPring-8 / Beamline: BL32XU / Wavelength: 0.97 Å
DetectorType: DECTRIS EIGER X 9M / Detector: PIXEL / Date: Nov 2, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97 Å / Relative weight: 1
ReflectionResolution: 3.1→49.3 Å / Num. obs: 22986 / % possible obs: 99.8 % / Redundancy: 135.8 % / Biso Wilson estimate: 97.24 Å2 / CC1/2: 0.999 / Rpim(I) all: 0.038 / Net I/σ(I): 20
Reflection shellResolution: 3.1→3.21 Å / Mean I/σ(I) obs: 1.4 / Num. unique obs: 1130 / CC1/2: 0.821 / Rpim(I) all: 1.601

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Processing

Software
NameVersionClassification
PHENIX1.19.2_4158refinement
XDSdata reduction
XSCALEdata scaling
AutoSolphasing
RefinementMethod to determine structure: SAD / Resolution: 3.1→40.91 Å / SU ML: 0.5177 / Cross valid method: FREE R-VALUE / σ(F): 1.45 / Phase error: 34.5898
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Details: The entry contains friedel pairs in F_plus/minus columns and I_plus/minus columns
RfactorNum. reflection% reflection
Rfree0.2987 1075 4.68 %
Rwork0.2564 21871 -
obs0.2583 22946 99.64 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 92.3 Å2
Refinement stepCycle: LAST / Resolution: 3.1→40.91 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3197 0 33 0 3230
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00213303
X-RAY DIFFRACTIONf_angle_d0.50294481
X-RAY DIFFRACTIONf_chiral_restr0.0419512
X-RAY DIFFRACTIONf_plane_restr0.0043566
X-RAY DIFFRACTIONf_dihedral_angle_d12.42881170
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.1-3.240.47871280.41592699X-RAY DIFFRACTION99.54
3.24-3.410.36561430.31512764X-RAY DIFFRACTION99.97
3.41-3.630.31751020.30322732X-RAY DIFFRACTION99.93
3.63-3.90.32241380.27812762X-RAY DIFFRACTION100
3.91-4.30.30281480.24962706X-RAY DIFFRACTION99.79
4.3-4.920.32431110.25192779X-RAY DIFFRACTION98.43
4.92-6.190.30331750.25482696X-RAY DIFFRACTION99.97
6.2-40.910.23341300.21312733X-RAY DIFFRACTION99.55

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