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- PDB-8gmb: Crystal structure of the full-length Bruton's tyrosine kinase (PH... -

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Basic information

Entry
Database: PDB / ID: 8gmb
TitleCrystal structure of the full-length Bruton's tyrosine kinase (PH-TH domain not visible)
ComponentsTyrosine-protein kinase BTK
KeywordsTRANSFERASE / ATP-binding / Lipid-binding / transcription regulation / immunity
Function / homology
Function and homology information


G beta:gamma signalling through BTK / RHO GTPases Activate WASPs and WAVEs / G alpha (12/13) signalling events / FCERI mediated Ca+2 mobilization / G alpha (q) signalling events / Antigen activates B Cell Receptor (BCR) leading to generation of second messengers / monocyte proliferation / positive regulation of interleukin-17A production / Regulation of actin dynamics for phagocytic cup formation / eosinophil homeostasis ...G beta:gamma signalling through BTK / RHO GTPases Activate WASPs and WAVEs / G alpha (12/13) signalling events / FCERI mediated Ca+2 mobilization / G alpha (q) signalling events / Antigen activates B Cell Receptor (BCR) leading to generation of second messengers / monocyte proliferation / positive regulation of interleukin-17A production / Regulation of actin dynamics for phagocytic cup formation / eosinophil homeostasis / proteoglycan catabolic process / positive regulation of type III hypersensitivity / B cell affinity maturation / positive regulation of synoviocyte proliferation / histamine secretion by mast cell / neutrophil homeostasis / positive regulation of cGAS/STING signaling pathway / cellular response to molecule of fungal origin / positive regulation of type I hypersensitivity / cellular response to interleukin-7 / DAP12 signaling / positive regulation of immunoglobulin production / negative regulation of cytokine production / phospholipase activator activity / negative regulation of interleukin-10 production / negative regulation of B cell proliferation / positive regulation of NLRP3 inflammasome complex assembly / phosphatidylinositol-3,4,5-trisphosphate binding / phospholipase binding / cell maturation / positive regulation of B cell proliferation / positive regulation of phagocytosis / cellular response to reactive oxygen species / B cell receptor signaling pathway / non-membrane spanning protein tyrosine kinase activity / non-specific protein-tyrosine kinase / positive regulation of interleukin-6 production / positive regulation of tumor necrosis factor production / cytoplasmic vesicle / protein tyrosine kinase activity / response to lipopolysaccharide / intracellular signal transduction / membrane raft / innate immune response / apoptotic process / perinuclear region of cytoplasm / zinc ion binding / ATP binding / identical protein binding / nucleus / plasma membrane / cytoplasm / cytosol
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 ...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 / Serine-threonine/tyrosine-protein kinase, catalytic domain / Protein tyrosine and serine/threonine kinase / 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: 3.4 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 24, 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)77,0652
Polymers76,4001
Non-polymers6651
Water00
1
A: Tyrosine-protein kinase BTK
hetero molecules

A: Tyrosine-protein kinase BTK
hetero molecules


Theoretical massNumber of molelcules
Total (without water)154,1304
Polymers152,8012
Non-polymers1,3302
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation4_555y,x,-z1
Buried area6140 Å2
ΔGint-63 kcal/mol
Surface area42680 Å2
MethodPISA
Unit cell
Length a, b, c (Å)125.718, 125.718, 110.091
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number152
Space group name H-MP3121
Space group name HallP312"
Symmetry operation#1: x,y,z
#2: -y,x-y,z+1/3
#3: -x+y,-x,z+2/3
#4: x-y,-y,-z+2/3
#5: -x,-x+y,-z+1/3
#6: y,x,-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: 76400.344 Da / Num. of mol.: 1
Mutation: E298A, K300A, E301A, A384P, S386P, T387P, A388P, L390F, 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
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: 3.29 Å3/Da / Density % sol: 66.06 %
Description: Monomer proteins at 18 mg/ml in 20 mM TRIS/HCl, pH 8.0, 150 mM NaCl, 10% glycerol 5 mM DTT were mixed with an equal volume of the precipitant solution, and then suspend over 0.25 ml of ...Description: Monomer proteins at 18 mg/ml in 20 mM TRIS/HCl, pH 8.0, 150 mM NaCl, 10% glycerol 5 mM DTT were mixed with an equal volume of the precipitant solution, and then suspend over 0.25 ml of precipitant solution. Leaf-shaped hexagonal crystals grew to up to 0.4 mm on a side after a period of two weeks. Crystals were frozen by transferring them, in six sequential steps, to a solution containing 0.1M Bis-Tris Propane, pH 7.5, 0.2M potassium thiocyanate, 20% PEG 3350 and 20% glycerol.
Crystal growTemperature: 277.15 K / Method: vapor diffusion / pH: 7.5
Details: 20% PEG 3350, 0.1M Bis-Tris Propane, 0.2M potassium thiocyanate
PH range: 7.2-7.6

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 23-ID-B / Wavelength: 1.033167 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Oct 23, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.033167 Å / Relative weight: 1
ReflectionResolution: 3.194→108.875 Å / Num. obs: 11550 / % possible obs: 79.61 % / Redundancy: 88.5 % / CC1/2: 0.998 / Rmerge(I) obs: 0.268 / Rpim(I) all: 0.029 / Rrim(I) all: 0.269 / Net I/σ(I): 17.8
Reflection shellResolution: 3.194→3.533 Å / Redundancy: 82.2 % / Rmerge(I) obs: 9.606 / Mean I/σ(I) obs: 2 / Num. unique obs: 577 / CC1/2: 0.743 / Rpim(I) all: 1.059 / Rrim(I) all: 9.665 / % possible all: 13.2

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Processing

Software
NameVersionClassification
autoPROCdata processing
PHENIX1.20.1-4487-000refinement
PHASERphasing
Cootmodel building
XDSdata scaling
autoPROCdata reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 3.4→19.94 Å / SU ML: 0.3309 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 34.8727
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2891 624 5.57 %
Rwork0.2719 10584 -
obs0.2727 11208 79.61 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 157.69 Å2
Refinement stepCycle: LAST / Resolution: 3.4→19.94 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3119 0 49 0 3168
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00163257
X-RAY DIFFRACTIONf_angle_d0.42264465
X-RAY DIFFRACTIONf_chiral_restr0.0389483
X-RAY DIFFRACTIONf_plane_restr0.0028578
X-RAY DIFFRACTIONf_dihedral_angle_d10.16811049
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.4-3.740.4045810.3635999X-RAY DIFFRACTION31.38
3.74-4.280.33321900.30032806X-RAY DIFFRACTION85.75
4.28-5.370.27441510.29213355X-RAY DIFFRACTION100
5.37-19.940.27392020.2493424X-RAY DIFFRACTION100
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)
11.055923246110.08270664148161.537352275620.642432527329-0.6465814860763.38029924153-0.430308715046-0.324906552889-0.7816491045720.09577589194850.1721786470250.04653247494610.1254633467580.5008334440660.08306970647260.9967139899250.678950084796-0.2218169169681.67411534650.1583058564320.462203987166-38.8230255061-49.7086850088-16.4130196467
26.16564574471.41483972383-0.2491408107826.97131073828-2.683885370197.2554802551-0.263984488232-0.1929301777690.7953038202830.4714329245120.2033119475780.255843896575-0.6439161184240.3988906364310.05759003336060.6260143398160.0617451785767-0.06794524995860.674058723557-0.05264600412570.5616332176154.80456893342-51.006184099232.2002474626
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 216 through 382 )216 - 3823 - 161
22chain 'A' and (resid 383 through 659 )383 - 659162 - 438

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