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- PDB-5nx3: Combinatorial Engineering of Proteolytically Resistant APPI Varia... -

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Basic information

Entry
Database: PDB / ID: 5nx3
TitleCombinatorial Engineering of Proteolytically Resistant APPI Variants that Selectively Inhibit Human Kallikrein 6 for Cancer Therapy
Components
  • (Amyloid-beta A4 protein) x 3
  • Kallikrein-6KLK6
KeywordsHYDROLASE / Serine_protease / inhibitor / complex / Hydrolase-Inhibitor complex
Function / homology
Function and homology information


tissue regeneration / positive regulation of G protein-coupled receptor signaling pathway / cornified envelope / regulation of epidermal growth factor-activated receptor activity / signaling receptor activator activity / collateral sprouting in absence of injury / cytosolic mRNA polyadenylation / microglia development / hormone metabolic process / regulation of synapse structure or activity ...tissue regeneration / positive regulation of G protein-coupled receptor signaling pathway / cornified envelope / regulation of epidermal growth factor-activated receptor activity / signaling receptor activator activity / collateral sprouting in absence of injury / cytosolic mRNA polyadenylation / microglia development / hormone metabolic process / regulation of synapse structure or activity / Formyl peptide receptors bind formyl peptides and many other ligands / axo-dendritic transport / synaptic assembly at neuromuscular junction / smooth endoplasmic reticulum calcium ion homeostasis / axon midline choice point recognition / amyloid precursor protein metabolic process / astrocyte activation involved in immune response / regulation of spontaneous synaptic transmission / regulation of Wnt signaling pathway / mating behavior / ciliary rootlet / Lysosome Vesicle Biogenesis / PTB domain binding / Golgi-associated vesicle / positive regulation of amyloid fibril formation / neuron remodeling / Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / : / Deregulated CDK5 triggers multiple neurodegenerative pathways in Alzheimer's disease models / intercellular bridge / presynaptic active zone / nuclear envelope lumen / modulation of excitatory postsynaptic potential / suckling behavior / COPII-coated ER to Golgi transport vesicle / regulation of neuron projection development / dendrite development / regulation of cell differentiation / smooth endoplasmic reticulum / regulation of NMDA receptor activity / TRAF6 mediated NF-kB activation / Advanced glycosylation endproduct receptor signaling / neuromuscular process controlling balance / regulation of presynapse assembly / The NLRP3 inflammasome / protein autoprocessing / intracellular copper ion homeostasis / transition metal ion binding / regulation of multicellular organism growth / negative regulation of long-term synaptic potentiation / Hydrolases; Acting on peptide bonds (peptidases); Serine endopeptidases / negative regulation of neuron differentiation / collagen catabolic process / ECM proteoglycans / spindle midzone / positive regulation of T cell migration / Purinergic signaling in leishmaniasis infection / positive regulation of calcium-mediated signaling / forebrain development / regulation of peptidyl-tyrosine phosphorylation / positive regulation of chemokine production / clathrin-coated pit / Notch signaling pathway / positive regulation of G2/M transition of mitotic cell cycle / myelination / ionotropic glutamate receptor signaling pathway / positive regulation of protein metabolic process / neuron projection maintenance / cholesterol metabolic process / extracellular matrix organization / positive regulation of glycolytic process / positive regulation of mitotic cell cycle / response to interleukin-1 / axonogenesis / adult locomotory behavior / trans-Golgi network membrane / dendritic shaft / locomotory behavior / platelet alpha granule lumen / positive regulation of peptidyl-threonine phosphorylation / secretory granule / learning / central nervous system development / positive regulation of interleukin-1 beta production / positive regulation of long-term synaptic potentiation / astrocyte activation / endosome lumen / synapse organization / Post-translational protein phosphorylation / regulation of long-term neuronal synaptic plasticity / positive regulation of JNK cascade / microglial cell activation / TAK1-dependent IKK and NF-kappa-B activation / visual learning / neuromuscular junction / serine-type endopeptidase inhibitor activity / recycling endosome / cognition / response to wounding / positive regulation of inflammatory response
Similarity search - Function
Pancreatic trypsin inhibitor Kunitz domain / Factor Xa Inhibitor / Amyloidogenic glycoprotein, copper-binding / Amyloidogenic glycoprotein, copper-binding domain conserved site / Amyloidogenic glycoprotein, copper-binding domain superfamily / Copper-binding of amyloid precursor, CuBD / Amyloid precursor protein (APP) copper-binding (CuBD) domain signature. / Amyloidogenic glycoprotein, amyloid-beta peptide superfamily / Beta-amyloid peptide (beta-APP) / Amyloidogenic glycoprotein, amyloid-beta peptide ...Pancreatic trypsin inhibitor Kunitz domain / Factor Xa Inhibitor / Amyloidogenic glycoprotein, copper-binding / Amyloidogenic glycoprotein, copper-binding domain conserved site / Amyloidogenic glycoprotein, copper-binding domain superfamily / Copper-binding of amyloid precursor, CuBD / Amyloid precursor protein (APP) copper-binding (CuBD) domain signature. / Amyloidogenic glycoprotein, amyloid-beta peptide superfamily / Beta-amyloid peptide (beta-APP) / Amyloidogenic glycoprotein, amyloid-beta peptide / Beta-amyloid precursor protein C-terminal / Amyloidogenic glycoprotein, intracellular domain, conserved site / Beta-amyloid precursor protein C-terminus / Amyloid precursor protein (APP) intracellular domain signature. / Amyloid precursor protein (APP) E1 domain profile. / Amyloid precursor protein (APP) E2 domain profile. / Amyloidogenic glycoprotein, extracellular / Amyloidogenic glycoprotein, heparin-binding / Amyloidogenic glycoprotein, E2 domain / E2 domain superfamily / Amyloidogenic glycoprotein, heparin-binding domain superfamily / Amyloid A4 N-terminal heparin-binding / E2 domain of amyloid precursor protein / amyloid A4 / Amyloidogenic glycoprotein / Proteinase inhibitor I2, Kunitz, conserved site / Pancreatic trypsin inhibitor (Kunitz) family signature. / BPTI/Kunitz family of serine protease inhibitors. / Pancreatic trypsin inhibitor Kunitz domain / Kunitz/Bovine pancreatic trypsin inhibitor domain / Pancreatic trypsin inhibitor (Kunitz) family profile. / Pancreatic trypsin inhibitor Kunitz domain superfamily / Few Secondary Structures / Irregular / Serine proteases, trypsin family, histidine active site / Serine proteases, trypsin family, serine active site / Peptidase S1A, chymotrypsin family / Serine proteases, trypsin family, histidine active site. / Serine proteases, trypsin domain profile. / Serine proteases, trypsin family, serine active site. / Trypsin-like serine protease / Serine proteases, trypsin domain / Trypsin / Trypsin-like serine proteases / PH-like domain superfamily / Thrombin, subunit H / Peptidase S1, PA clan, chymotrypsin-like fold / Peptidase S1, PA clan / Beta Barrel / Mainly Beta
Similarity search - Domain/homology
Amyloid-beta precursor protein / Kallikrein-6
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.296 Å
AuthorsShahar, A. / Sananes, A. / Radisky, E.S. / Papo, N.
CitationJournal: J.Biol.Chem. / Year: 2018
Title: A potent, proteolysis-resistant inhibitor of kallikrein-related peptidase 6 (KLK6) for cancer therapy, developed by combinatorial engineering.
Authors: Sananes, A. / Cohen, I. / Shahar, A. / Hockla, A. / De Vita, E. / Miller, A.K. / Radisky, E.S. / Papo, N.
History
DepositionMay 9, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 30, 2018Provider: repository / Type: Initial release
Revision 1.1Jun 12, 2019Group: Data collection / Database references / Category: citation / citation_author / pdbx_database_proc
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Jan 17, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Kallikrein-6
B: Amyloid-beta A4 protein
D: Amyloid-beta A4 protein
C: Amyloid-beta A4 protein


Theoretical massNumber of molelcules
Total (without water)42,9384
Polymers42,9384
Non-polymers00
Water2,558142
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3560 Å2
ΔGint-27 kcal/mol
Surface area15260 Å2
MethodPISA
Unit cell
Length a, b, c (Å)58.058, 77.752, 91.130
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein Kallikrein-6 / KLK6 / Neurosin / Protease M / SP59 / Serine protease 18 / Serine protease 9 / Zyme


Mass: 24417.695 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: KLK6, PRSS18, PRSS9 / Cell line (production host): Hi5 / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: Q92876, Hydrolases; Acting on peptide bonds (peptidases); Serine endopeptidases
#2: Protein/peptide Amyloid-beta A4 protein / ABPP / APPI / APP / Alzheimer disease amyloid protein / Amyloid precursor protein / Amyloid-beta ...ABPP / APPI / APP / Alzheimer disease amyloid protein / Amyloid precursor protein / Amyloid-beta precursor protein / Cerebral vascular amyloid peptide / CVAP / PreA4 / Protease nexin-II / PN-II


Mass: 2945.086 Da / Num. of mol.: 1 / Fragment: Inhibitor domain, UNP Residues 294-346
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: APP, A4, AD1 / Production host: Komagataella phaffii GS115 (fungus) / References: UniProt: P05067
#3: Protein Amyloid-beta A4 protein / ABPP / APPI / APP / Alzheimer disease amyloid protein / Amyloid precursor protein / Amyloid-beta ...ABPP / APPI / APP / Alzheimer disease amyloid protein / Amyloid precursor protein / Amyloid-beta precursor protein / Cerebral vascular amyloid peptide / CVAP / PreA4 / Protease nexin-II / PN-II


Mass: 6324.029 Da / Num. of mol.: 1 / Fragment: Inhibitor domain, UNP Residues 306-346
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: APP, A4, AD1 / Production host: Komagataella phaffii GS115 (fungus) / References: UniProt: P05067
#4: Protein Amyloid-beta A4 protein / ABPP / APPI / APP / Alzheimer disease amyloid protein / Amyloid precursor protein / Amyloid-beta ...ABPP / APPI / APP / Alzheimer disease amyloid protein / Amyloid precursor protein / Amyloid-beta precursor protein / Cerebral vascular amyloid peptide / CVAP / PreA4 / Protease nexin-II / PN-II


Mass: 9251.103 Da / Num. of mol.: 1 / Fragment: Inhibitor domain, UNP Residues 289-346
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: APP, A4, AD1 / Production host: Komagataella phaffii GS115 (fungus) / References: UniProt: P05067
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 142 / Source method: isolated from a natural source / Formula: H2O

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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: 2.4 Å3/Da / Density % sol: 48.67 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop
Details: 0.1M tri-Na Citrate pH 5.6, 20% 2-propanol , 20% Polyethylene Glycol 4000

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID23-1 / Wavelength: 0.976 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Sep 21, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.976 Å / Relative weight: 1
ReflectionResolution: 2.296→48.965 Å / Num. obs: 18865 / % possible obs: 99.14 % / Redundancy: 2 % / Rpim(I) all: 0.053 / Net I/σ(I): 8.51
Reflection shellResolution: 2.296→2.378 Å / Redundancy: 2 % / Mean I/σ(I) obs: 2.13 / Num. unique all: 1770 / CC1/2: 0.76 / Rpim(I) all: 0.3521 / % possible all: 95.57

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Processing

Software
NameVersionClassification
PHENIX(1.11.1_2575: ???)refinement
XDSdata reduction
XDSdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1AAP & 1LO6

1aap

1lo6


Resolution: 2.296→48.965 Å / SU ML: 0.24 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 21.79
RfactorNum. reflection% reflection
Rfree0.2261 945 5.01 %
Rwork0.1749 --
obs0.1774 18857 99.15 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 2.296→48.965 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2539 0 0 142 2681
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0072632
X-RAY DIFFRACTIONf_angle_d0.9123577
X-RAY DIFFRACTIONf_dihedral_angle_d7.5622020
X-RAY DIFFRACTIONf_chiral_restr0.053371
X-RAY DIFFRACTIONf_plane_restr0.005471
LS refinement shell

Refine-ID: X-RAY DIFFRACTION

Resolution (Å)Highest resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
2.2961-2.41710.30541470.2401243997
2.4171-2.56850.26221450.21822501100
2.5685-2.76690.27361380.22570100
2.7669-3.04530.24121240.20192551100
3.0453-3.48580.24931140.17392594100
3.4858-4.39130.19681350.1515253597
4.39130.1821420.14572722100
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
12.0621-0.5483-0.88772.807-1.44313.60040.07740.196-0.1561-0.022-0.0240.16210.0866-0.1924-0.05190.18350.0072-0.01030.1641-0.02040.1381-1.336753.6752-34.7693
20.1710.24880.40573.48761.12643.46750.0260.43340.116-0.07020.1448-0.07260.33240.527-0.17220.20560.0520.00130.2211-0.04360.2429.119953.9454-37.7388
31.98791.33980.8574.8521.06774.26030.05480.21850.1762-0.3165-0.07310.0591-0.06110.0773-0.05010.140.10640.02920.1775-0.02840.12925.324556.435-36.1147
46.411-0.659-0.5813.21-0.1974.09410.20550.5541-0.1948-0.2716-0.04490.1279-0.2501-0.3676-0.18230.26750.0586-0.02830.12130.02130.1739-2.56160.5488-42.7492
55.56720.91470.32766.5786-0.10972.9438-0.1547-0.01410.56410.10750.35451.0415-0.5771-0.588-0.16180.52920.02880.01340.2775-0.00710.1805-6.93665.9591-23.7952
64.32561.03160.7791.42830.07575.673-0.0428-0.2062-0.42070.1580.08620.05260.4938-0.1126-0.01710.2781-0.00490.01820.16050.04510.1904-3.535948.5534-24.1231
77.45261.70541.57829.1156-2.38141.7253-0.1427-0.90550.93811.09910.20940.1998-1.05730.0654-0.08260.48730.0851-0.07670.381-0.1210.34366.08665.4608-12.5772
83.52190.31380.01193.5341-0.42114.6709-0.0747-0.38680.04880.43970.09710.05370.0443-0.34850.01520.26530.05410.00710.19270.00730.1885-1.786757.4689-21.9237
93.1571-2.42521.87736.3872-0.12071.6738-0.0606-0.73440.14580.26010.1319-0.1481-0.2391-0.2769-0.01220.46790.12740.01280.2763-0.00190.18833.21751.7783-12.8904
103.28940.5474-1.38674.02361.35992.1337-0.03460.15620.5539-0.00690.1484-0.2394-0.8692-0.2878-0.11150.3610.0196-0.01980.15450.00280.27894.013469.8288-31.5296
114.51624.1623-1.98734.71590.21788.9584-0.0317-0.11460.13980.3296-0.48480.49070.1944-0.53140.36130.24120.0044-0.06680.2914-0.08740.2887-21.407270.6032-34.5477
125.61650.1735-2.11247.5115-0.08817.5679-0.1639-0.17040.25490.0165-0.0205-0.2094-0.1507-0.01050.21390.21710.0017-0.07590.20480.00910.1724-15.617272.5751-38.3169
138.366-3.07510.34918.074.25292.85830.19620.18840.2827-1.0193-0.4676-0.1378-1.013-0.51110.11570.4619-0.0449-0.01990.3320.06450.2472-13.454379.6693-44.8398
146.5728-5.00331.39686.7612-1.04584.660.2024-0.07980.2910.1137-0.4406-0.57270.46650.38830.21620.2203-0.029-0.05350.29180.05850.28817.595644.347-22.9008
156.6992-3.97551.02422.39420.02768.54760.43370.35180.2548-0.4143-0.4841-0.55170.39260.56530.01830.23290.0547-0.01580.29220.02830.292920.414542.3502-24.7418
165.58462.10831.76547.9656-3.66293.26690.74981.18970.3779-1.0072-0.7753-0.2466-0.78470.6787-0.03620.57590.24440.14910.70550.0560.453128.049539.5169-29.8768
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1chain 'A' and (resid 16 through 54 )
2X-RAY DIFFRACTION2chain 'A' and (resid 55 through 67 )
3X-RAY DIFFRACTION3chain 'A' and (resid 69 through 103 )
4X-RAY DIFFRACTION4chain 'A' and (resid 104 through 123 )
5X-RAY DIFFRACTION5chain 'A' and (resid 124 through 140 )
6X-RAY DIFFRACTION6chain 'A' and (resid 141 through 164 )
7X-RAY DIFFRACTION7chain 'A' and (resid 165 through 179 )
8X-RAY DIFFRACTION8chain 'A' and (resid 180 through 215 )
9X-RAY DIFFRACTION9chain 'A' and (resid 216 through 225 )
10X-RAY DIFFRACTION10chain 'A' and (resid 226 through 244 )
11X-RAY DIFFRACTION11chain 'B' and (resid 3 through 15 )
12X-RAY DIFFRACTION12chain 'D' and (resid 18 through 47 )
13X-RAY DIFFRACTION13chain 'D' and (resid 48 through 57 )
14X-RAY DIFFRACTION14chain 'C' and (resid 3 through 24 )
15X-RAY DIFFRACTION15chain 'C' and (resid 25 through 47 )
16X-RAY DIFFRACTION16chain 'C' and (resid 48 through 55 )

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