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- PDB-8s93: Crystal structure of the PH-TH/kinase complex of Bruton's tyrosin... -

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Basic information

Entry
Database: PDB / ID: 8s93
TitleCrystal structure of the PH-TH/kinase complex of Bruton's tyrosine kinase
ComponentsTyrosine-protein kinase BTK
KeywordsTRANSFERASE / Bruton's tyrosine kinase / Non-receptor tyrosine kinase / complex / lipid-binding / ATP-binding
Function / homology
Function and homology information


negative regulation of B cell activation / G beta:gamma signalling through BTK / RHO GTPases Activate WASPs and WAVEs / FCERI mediated Ca+2 mobilization / G alpha (q) signalling events / G alpha (12/13) signalling events / Antigen activates B Cell Receptor (BCR) leading to generation of second messengers / Regulation of actin dynamics for phagocytic cup formation / proteoglycan catabolic process / monocyte proliferation ...negative regulation of B cell activation / G beta:gamma signalling through BTK / RHO GTPases Activate WASPs and WAVEs / FCERI mediated Ca+2 mobilization / G alpha (q) signalling events / G alpha (12/13) signalling events / Antigen activates B Cell Receptor (BCR) leading to generation of second messengers / Regulation of actin dynamics for phagocytic cup formation / proteoglycan catabolic process / monocyte proliferation / positive regulation of interleukin-17A production / eosinophil homeostasis / positive regulation of type III hypersensitivity / B cell affinity maturation / histamine secretion by mast cell / positive regulation of synoviocyte proliferation / negative regulation of leukocyte proliferation / neutrophil homeostasis / cellular response to molecule of fungal origin / positive regulation of type I hypersensitivity / cellular response to interleukin-7 / DAP12 signaling / negative regulation of cytokine production / positive regulation of NLRP3 inflammasome complex assembly / phospholipase activator activity / negative regulation of interleukin-10 production / negative regulation of B cell proliferation / phospholipase binding / phosphatidylinositol-3,4,5-trisphosphate binding / positive regulation of immunoglobulin production / positive regulation of phagocytosis / positive regulation of B cell proliferation / cell maturation / response to organic substance / B cell receptor signaling pathway / non-specific protein-tyrosine kinase / non-membrane spanning protein tyrosine kinase activity / cellular response to reactive oxygen species / positive regulation of interleukin-6 production / positive regulation of tumor necrosis factor production / T cell receptor signaling pathway / cytoplasmic vesicle / protein tyrosine kinase activity / response to lipopolysaccharide / adaptive immune response / intracellular signal transduction / membrane raft / phosphorylation / innate immune response / apoptotic process / perinuclear region of cytoplasm / ATP binding / identical protein binding / metal ion binding / nucleus / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Tyrosine-protein kinase BTK, SH3 domain / Zinc finger, Btk motif / BTK motif / Zinc finger Btk-type profile. / Bruton's tyrosine kinase Cys-rich motif / PH domain / PH domain profile. / Pleckstrin homology domain. / Pleckstrin homology domain / SH3 domain ...Tyrosine-protein kinase BTK, SH3 domain / Zinc finger, Btk motif / BTK motif / Zinc finger Btk-type profile. / Bruton's tyrosine kinase Cys-rich motif / PH domain / PH domain profile. / Pleckstrin homology domain. / Pleckstrin homology domain / SH3 domain / SH2 domain / Src homology 2 (SH2) domain profile. / Src homology 2 domains / SH2 domain / Src homology 3 domains / SH2 domain superfamily / SH3-like domain superfamily / Src homology 3 (SH3) domain profile. / SH3 domain / Tyrosine-protein kinase, catalytic domain / Tyrosine kinase, catalytic domain / Tyrosine protein kinases specific active-site signature. / PH-like domain superfamily / Tyrosine-protein kinase, active site / Protein tyrosine and serine/threonine kinase / Serine-threonine/tyrosine-protein kinase, catalytic domain / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
Chem-9AJ / Tyrosine-protein kinase BTK
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.1 Å
AuthorsLin, D.Y. / Andreotti, A.H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID) United States
CitationJournal: Elife / Year: 2024
Title: Conformational heterogeneity of the BTK PHTH domain drives multiple regulatory states.
Authors: David Yin-Wei Lin / Lauren E Kueffer / Puneet Juneja / Thomas E Wales / John R Engen / Amy H Andreotti /
Abstract: Full-length Bruton's tyrosine kinase (BTK) has been refractory to structural analysis. The nearest full-length structure of BTK to date consists of the autoinhibited SH3-SH2-kinase core. Precisely ...Full-length Bruton's tyrosine kinase (BTK) has been refractory to structural analysis. The nearest full-length structure of BTK to date consists of the autoinhibited SH3-SH2-kinase core. Precisely how the BTK N-terminal domains (the Pleckstrin homology/Tec homology [PHTH] domain and proline-rich regions [PRR] contain linker) contribute to BTK regulation remains unclear. We have produced crystals of full-length BTK for the first time but despite efforts to stabilize the autoinhibited state, the diffraction data still reveal only the SH3-SH2-kinase core with no electron density visible for the PHTH-PRR segment. Cryo-electron microscopy (cryoEM) data of full-length BTK, on the other hand, provide the first view of the PHTH domain within full-length BTK. CryoEM reconstructions support conformational heterogeneity in the PHTH-PRR region wherein the globular PHTH domain adopts a range of states arrayed around the autoinhibited SH3-SH2-kinase core. On the way to activation, disassembly of the SH3-SH2-kinase core opens a new autoinhibitory site on the kinase domain for PHTH domain binding that is ultimately released upon interaction of PHTH with phosphatidylinositol (3,4,5)-trisphosphate. Membrane-induced dimerization activates BTK and we present here a crystal structure of an activation loop swapped BTK kinase domain dimer that likely represents the conformational state leading to trans-autophosphorylation. Together, these data provide the first structural elucidation of full-length BTK and allow a deeper understanding of allosteric control over the BTK kinase domain during distinct stages of activation.
History
DepositionMar 27, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 16, 2023Provider: repository / Type: Initial release
Revision 1.1Jan 24, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2Jan 31, 2024Group: Database references / Category: citation / citation_author / Item: _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Tyrosine-protein kinase BTK
hetero molecules


Theoretical massNumber of molelcules
Total (without water)53,1434
Polymers52,3211
Non-polymers8223
Water3,207178
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)38.376, 77.383, 82.323
Angle α, β, γ (deg.)90.000, 97.980, 90.000
Int Tables number4
Space group name H-MP1211
Space group name HallP2yb
Symmetry operation#1: x,y,z
#2: -x,y+1/2,-z

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Components

#1: Protein Tyrosine-protein kinase BTK / Agammaglobulinemia tyrosine kinase / ATK / B-cell progenitor kinase / BPK / Bruton tyrosine kinase ...Agammaglobulinemia tyrosine kinase / ATK / B-cell progenitor kinase / BPK / Bruton tyrosine kinase / Kinase EMB


Mass: 52320.707 Da / Num. of mol.: 1
Fragment: PHTH domain residues 1-171 and Kinase domain residues 396-659
Mutation: Q91A, I92A, I94A, I95A, K430R, L542M S543T, V555T, R562K, S564A, P565S, Y617P
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Btk, Bpk / Production host: Escherichia coli (E. coli)
References: UniProt: P35991, non-specific protein-tyrosine kinase
#2: Chemical ChemComp-9AJ / 2-[3'-(hydroxymethyl)-1-methyl-5-({5-[(2S)-2-methyl-4-(oxetan-3-yl)piperazin-1-yl]pyridin-2-yl}amino)-6-oxo[1,6-dihydro[3,4'-bipyridine]]-2'-yl]-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one


Mass: 664.796 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C37H44N8O4 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O3
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 178 / 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: 2.31 Å3/Da / Density % sol: 46.84 % / Description: clusters of thin plates and needles
Crystal growTemperature: 277 K / Method: vapor diffusion / pH: 5.6
Details: 20%PEG3350, 0.1M Bis-Tris, pH5.6, 0.2M magnesium chloride
PH range: 5.4-5.8

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 23-ID-D / Wavelength: 1.03322 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Oct 17, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.03322 Å / Relative weight: 1
ReflectionResolution: 2.001→81.527 Å / Num. obs: 21684 / % possible obs: 67.2 % / Observed criterion σ(I): 1.2 / Redundancy: 3.7 % / Biso Wilson estimate: 28.03 Å2 / CC1/2: 0.992 / Rmerge(I) obs: 0.134 / Rpim(I) all: 0.079 / Rrim(I) all: 0.156 / Net I/σ(I): 7.1
Reflection shellResolution: 2.001→2.197 Å / Redundancy: 3.3 % / Rmerge(I) obs: 0.803 / Mean I/σ(I) obs: 1.6 / Num. unique obs: 1084 / CC1/2: 0.54 / Rpim(I) all: 0.514 / % possible all: 13.9

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
PHASERphasing
autoPROCdata processing
Cootmodel building
autoPROCdata scaling
autoPROCdata reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.1→19.71 Å / SU ML: 0.2613 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 28.1129
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2465 1098 5.13 %
Rwork0.1982 20294 -
obs0.2006 21392 76.76 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 39.89 Å2
Refinement stepCycle: LAST / Resolution: 2.1→19.71 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3469 0 56 178 3703
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00183676
X-RAY DIFFRACTIONf_angle_d0.48874974
X-RAY DIFFRACTIONf_chiral_restr0.0393517
X-RAY DIFFRACTIONf_plane_restr0.0028633
X-RAY DIFFRACTIONf_dihedral_angle_d17.4347537
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.1-2.20.3194430.2769768X-RAY DIFFRACTION23.7
2.2-2.310.3428720.26861154X-RAY DIFFRACTION35.11
2.31-2.460.29941230.24672216X-RAY DIFFRACTION67.25
2.46-2.650.32011580.25822929X-RAY DIFFRACTION89.14
2.65-2.910.27941840.2453228X-RAY DIFFRACTION98.56
2.91-3.330.28871860.20323311X-RAY DIFFRACTION99.83
3.33-4.190.1931600.163326X-RAY DIFFRACTION99.6
4.19-19.710.20331720.1743362X-RAY DIFFRACTION99.63
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)
11.654864266460.28050552994-0.1709775049431.37886371792-0.02102749122881.93248600465-0.007518468255770.3043222823230.0685410289873-0.23121407919-0.06288646897210.2072907095120.260439062928-0.093396413451-8.60288845229E-50.4548172780910.00531856389122-0.04164330616250.5227664798540.07764179206510.3594369137087.6907326892211.9267453656-9.45682665911
20.185995513746-0.04827757293870.07660289716330.109820784267-0.1034693369890.09515496003920.07986032605350.4870789909210.0538993346371-0.258039969852-0.178534240530.02905775907850.278919031415-0.1872713351690.0005569150960110.4960835244240.09902546623170.2091080193621.079983316330.02446313588530.53020081461525.48232500866.95339350772-16.3400016745
31.33537274573-0.555657738222-0.4658118430711.20194964059-0.5910813807321.87912403177-0.113505961980.24358930163-0.238727868529-0.0896276924778-0.0738914999111-0.1614130532340.5524500881720.659283382310.0004085828263930.4564674301310.03831200862350.0490852623360.492828841350.02857166340740.33957495259213.92225188023.21831203488-2.39105463131
41.288046127550.464960351148-0.1973716292510.730871667873-0.8186476835071.26366244309-0.1433279545820.0413341271712-0.141229966159-0.5342035300310.2340219112230.1016660898610.2367336835950.5991627945050.000357875698660.4746818009350.05006681262670.03971883020210.4159165820470.04204765904440.34170642700612.68398686262.698722083956.93012889641
51.501259066560.929205036569-0.09609805189611.24982666492-0.1935936558990.80961542294-0.06521265665510.106084710830.180483896143-0.1946678210870.1010213408010.05283745155610.001122156595960.06270390980677.97265290245E-50.1734233262810.00282651508543-0.02419905475550.171356443115-0.01903148882920.196923258378.59798771366.5644040166624.8101250477
60.083276233433-0.126027391728-0.1627766231540.559380470982-0.08244747422170.533187880274-0.01948295298280.2240020222090.0873360777804-0.2710958974710.1202136482420.1690382295690.414532942402-0.0687116416780.01007322909110.209077347132-0.000377907935486-0.0045844614970.1898585621290.005806324512770.207561319913.63999380074-12.179762827725.1961153641
71.718343850780.178062666042-0.8731776793661.934711053940.2696409038490.5106523868410.0092220667195-0.05969496112320.0182999328892-0.00587901047375-0.0193710503149-0.257018892971-0.1123455344910.199445508741-0.002152556033160.220447193964-0.02195401596920.001347862670590.16367804505-0.01268862445480.20161654747414.1503393529-3.8812886825435.1060314689
81.277051094660.6450647819740.4656393472361.43211498811-0.6151463846571.30268807001-0.0154253662027-0.3157567888590.004563791017860.2785651751350.046242159005-0.0219976288957-0.148670247702-0.02415377150541.07849832515E-50.1544787541550.0223425929704-0.01749567435850.161712717919-0.006430230133070.16505058455.33846859547-12.263064074843.9566842789
90.9588056776950.224699937641-1.248025593751.607685033290.299118604282.321783710580.02113388863270.130768265979-0.536654409505-0.1351313388290.0308028253203-0.00114293721660.161986111838-0.1419180441870.005269258739880.220024219182-0.01289243671730.01412109074190.127894358628-0.001361795547030.2850860544625.18274008816-21.859285314232.9545876577
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION / Auth asym-ID: A / Label asym-ID: A

IDRefine TLS-IDSelection detailsAuth seq-IDLabel seq-ID
11chain 'A' and (resid 2 through 66 )2 - 662 - 64
22chain 'A' and (resid 67 through 99 )67 - 9965 - 86
33chain 'A' and (resid 100 through 152 )100 - 15287 - 139
44chain 'A' and (resid 153 through 407 )153 - 407140 - 177
55chain 'A' and (resid 408 through 494 )408 - 494178 - 264
66chain 'A' and (resid 495 through 515 )495 - 515265 - 285
77chain 'A' and (resid 516 through 575 )516 - 575286 - 345
88chain 'A' and (resid 576 through 623 )576 - 623346 - 393
99chain 'A' and (resid 624 through 659 )624 - 659394 - 429

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