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- PDB-9zpq: Crystal structure of B. burgdorferi HtrA protease domain Ser226Al... -

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Basic information

Entry
Database: PDB / ID: 9zpq
TitleCrystal structure of B. burgdorferi HtrA protease domain Ser226Ala mutant
ComponentsPeriplasmic serine protease DO
KeywordsHYDROLASE / HtrA / serine protease domain / chaperone / calcium binding / trimer
Function / homology
Function and homology information


serine-type endopeptidase activity / proteolysis
Similarity search - Function
Peptidase S1C, Do / PDZ domain / Peptidase S1C / Trypsin-like peptidase domain / PDZ domain profile. / Domain present in PSD-95, Dlg, and ZO-1/2. / PDZ domain / PDZ superfamily / Peptidase S1, PA clan
Similarity search - Domain/homology
Periplasmic serine protease DO
Similarity search - Component
Biological speciesBorreliella burgdorferi B31 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.1 Å
AuthorsShakya, A.K. / Herzberg, O.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI080615 United States
CitationJournal: J Mol Biol / Year: 2026
Title: Assembly and Flexibility of Borrelia burgdorferi Periplasmic HtrA Hexamers.
Authors: Anil Kumar Shakya / D Travis Gallager / Vipin Singh Rana / Suruchi Singh / S Saif Hasan / Utpal Pal / Osnat Herzberg /
Abstract: The chaperone/protease HtrA from the Lyme disease pathogen Borrelia burgdorferi (BbHtrA) functions in the maturation of the periplasmic protein BB0323 involved in pathogen infectivity and in the ...The chaperone/protease HtrA from the Lyme disease pathogen Borrelia burgdorferi (BbHtrA) functions in the maturation of the periplasmic protein BB0323 involved in pathogen infectivity and in the degradation of several other key host and spirochete proteins. Hence, BbHtrA is considered an anti-borrelial drug target candidate. BbHtrA contains a N-terminal serine protease domain followed by two PDZ domains (PDZ1-2). Here, we report a 3.4 Å resolution single particle cryo-EM reconstruction of hexameric BbHtrA whose active site serine was replaced by an alanine, and the 2.0 Å and 1.5 Å resolution crystal structures of the recombinant protease catalytic domain and PDZ1-2 fragment, respectively. The BbHtrA cryo-EM structure forms an asymmetric dimer of trimers unlike the symmetric oligomers of other HtrA family members. The different conformations of the six linkers between the protease domains and their respective PDZ1-2 break the symmetry, nevertheless, pairs of PDZ2 domains mediate trimer-trimer interactions through identical interfaces unique to BbHtrA. Features associated with the cryo-EM electron potential map at each protease active site were modeled as a nine-residue peptide of unknown sequence, which could originate from the expression host organism. The crystal structures recapitulate at higher resolution trimer interactions via the catalytic domains and trimer-trimer association via PDZ2-PDZ2 interactions. The serine protease oxyanion hole that stabilizes the substrate transition state is malformed in the crystal structure and fully formed in the peptide-bound BbHtrA cryo-EM structure, suggesting a regulatory mechanism intended to avoid undesired cleavage.
History
DepositionDec 17, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 17, 2026Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: Periplasmic serine protease DO
hetero molecules


Theoretical massNumber of molelcules
Total (without water)25,5995
Polymers25,2831
Non-polymers3164
Water1,36976
1
A: Periplasmic serine protease DO
hetero molecules

A: Periplasmic serine protease DO
hetero molecules

A: Periplasmic serine protease DO
hetero molecules


Theoretical massNumber of molelcules
Total (without water)76,79715
Polymers75,8483
Non-polymers94912
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-y,x-y,z1
crystal symmetry operation3_555-x+y,-x,z1
Buried area6170 Å2
ΔGint-32 kcal/mol
Surface area24010 Å2
MethodPISA
Unit cell
Length a, b, c (Å)114.537, 114.537, 101.504
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number155
Space group name H-MH32
Components on special symmetry positions
IDModelComponents
11A-301-

CA

21A-443-

HOH

31A-471-

HOH

41A-474-

HOH

51A-475-

HOH

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Components

#1: Protein Periplasmic serine protease DO


Mass: 25282.684 Da / Num. of mol.: 1 / Mutation: S226A
Source method: isolated from a genetically manipulated source
Details: mutated catalytic serine residue S226A; fusion protein that was cleaved with precision protease leaving a GPLGS pentapeptide at the N-terminus.
Source: (gene. exp.) Borreliella burgdorferi B31 (bacteria) / Strain: B31 / Gene: BB_0104 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: O51131
#2: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C3H8O3
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 76 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationN

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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: 2.45 Å3/Da / Density % sol: 49.89 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 8
Details: 2 M sodium chloride and 10% Polyethylene glycol 6000

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: NSLS / Beamline: X17B1 / Wavelength: 0.9201 Å
DetectorType: DECTRIS EIGER X 9M / Detector: PIXEL / Date: Jun 23, 2024
RadiationMonochromator: double crystal Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9201 Å / Relative weight: 1
ReflectionResolution: 2.1→30.2 Å / Num. obs: 14557 / % possible obs: 96.6 % / Redundancy: 2 % / CC1/2: 0.999 / Net I/σ(I): 10.6
Reflection shellResolution: 2.1→2.2 Å / Num. unique obs: 1469 / CC1/2: 0.416

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Processing

Software
NameVersionClassification
PHENIX(1.20.1_4487: ???)refinement
PDB_EXTRACTdata extraction
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.1→30.16 Å / SU ML: 0.24 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 28.12 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2186 681 4.68 %
Rwork0.1785 --
obs0.1803 14552 96.61 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.1→30.16 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1442 0 19 76 1537
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0081508
X-RAY DIFFRACTIONf_angle_d0.9242037
X-RAY DIFFRACTIONf_dihedral_angle_d7.381206
X-RAY DIFFRACTIONf_chiral_restr0.064239
X-RAY DIFFRACTIONf_plane_restr0.007257
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.1-2.260.36051220.30062420X-RAY DIFFRACTION86
2.26-2.490.29751300.24362850X-RAY DIFFRACTION100
2.49-2.850.28691330.21172857X-RAY DIFFRACTION100
2.85-3.590.2211420.18872870X-RAY DIFFRACTION100
3.59-30.160.18241540.14842874X-RAY DIFFRACTION98

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