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- PDB-7rx9: Structure of autoinhibited P-Rex1 -

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Basic information

Entry
Database: PDB / ID: 7rx9
TitleStructure of autoinhibited P-Rex1
ComponentsPhosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 protein, Endolysin chimera
KeywordsSIGNALING PROTEIN / P-Rex1 / P-Rex2 / GEF / cell growth / Rac1 / Cdc42
Function / homology
Function and homology information


regulation of dendrite development / regulation of actin filament polymerization / neutrophil activation / negative regulation of TOR signaling / regulation of small GTPase mediated signal transduction / RHOB GTPase cycle / NRAGE signals death through JNK / superoxide metabolic process / RHOC GTPase cycle / RHOJ GTPase cycle ...regulation of dendrite development / regulation of actin filament polymerization / neutrophil activation / negative regulation of TOR signaling / regulation of small GTPase mediated signal transduction / RHOB GTPase cycle / NRAGE signals death through JNK / superoxide metabolic process / RHOC GTPase cycle / RHOJ GTPase cycle / RHOQ GTPase cycle / CDC42 GTPase cycle / T cell differentiation / RHOG GTPase cycle / protein serine/threonine kinase inhibitor activity / RHOA GTPase cycle / RAC2 GTPase cycle / RAC3 GTPase cycle / viral release from host cell by cytolysis / actin filament polymerization / peptidoglycan catabolic process / neutrophil chemotaxis / RAC1 GTPase cycle / positive regulation of substrate adhesion-dependent cell spreading / GTPase activator activity / guanyl-nucleotide exchange factor activity / dendritic shaft / phospholipid binding / cell wall macromolecule catabolic process / lysozyme / lysozyme activity / G alpha (12/13) signalling events / growth cone / host cell cytoplasm / intracellular signal transduction / defense response to bacterium / positive regulation of cell migration / G protein-coupled receptor signaling pathway / perinuclear region of cytoplasm / enzyme binding / plasma membrane / cytosol
Similarity search - Function
Dbl Homology Domain; Chain A / Dbl homology (DH) domain / : / Guanine-nucleotide dissociation stimulator, CDC24, conserved site / Dbl homology (DH) domain signature. / Domain found in Dishevelled, Egl-10, and Pleckstrin (DEP) / DEP domain profile. / Domain found in Dishevelled, Egl-10, and Pleckstrin / DEP domain / : ...Dbl Homology Domain; Chain A / Dbl homology (DH) domain / : / Guanine-nucleotide dissociation stimulator, CDC24, conserved site / Dbl homology (DH) domain signature. / Domain found in Dishevelled, Egl-10, and Pleckstrin (DEP) / DEP domain profile. / Domain found in Dishevelled, Egl-10, and Pleckstrin / DEP domain / : / SOS1/NGEF-like PH domain / Dbl homology (DH) domain superfamily / RhoGEF domain / Guanine nucleotide exchange factor for Rho/Rac/Cdc42-like GTPases / Dbl homology (DH) domain / Dbl homology (DH) domain profile. / Endolysin T4 type / T4-type lysozyme / : / Glycoside hydrolase, family 24 / Phage lysozyme / Lysozyme domain superfamily / PH domain profile. / Pleckstrin homology domain. / Pleckstrin homology domain / PDZ domain profile. / Domain present in PSD-95, Dlg, and ZO-1/2. / PDZ domain / PDZ superfamily / Lysozyme-like domain superfamily / PH-like domain superfamily / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily / Up-down Bundle / Mainly Alpha
Similarity search - Domain/homology
Endolysin / Phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 protein
Similarity search - Component
Biological speciesHomo sapiens (human)
Enterobacteria phage T4 (virus)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.22 Å
AuthorsEllisdon, A.M. / Chang, Y.
Funding support Australia, 2items
OrganizationGrant numberCountry
National Health and Medical Research Council (NHMRC, Australia)APP1128120 Australia
National Health and Medical Research Council (NHMRC, Australia)APP1146578 Australia
CitationJournal: Nat Struct Mol Biol / Year: 2022
Title: Structure of the metastatic factor P-Rex1 reveals a two-layered autoinhibitory mechanism.
Authors: Yong-Gang Chang / Christopher J Lupton / Charles Bayly-Jones / Alastair C Keen / Laura D'Andrea / Christina M Lucato / Joel R Steele / Hari Venugopal / Ralf B Schittenhelm / James C ...Authors: Yong-Gang Chang / Christopher J Lupton / Charles Bayly-Jones / Alastair C Keen / Laura D'Andrea / Christina M Lucato / Joel R Steele / Hari Venugopal / Ralf B Schittenhelm / James C Whisstock / Michelle L Halls / Andrew M Ellisdon /
Abstract: P-Rex (PI(3,4,5)P-dependent Rac exchanger) guanine nucleotide exchange factors potently activate Rho GTPases. P-Rex guanine nucleotide exchange factors are autoinhibited, synergistically activated by ...P-Rex (PI(3,4,5)P-dependent Rac exchanger) guanine nucleotide exchange factors potently activate Rho GTPases. P-Rex guanine nucleotide exchange factors are autoinhibited, synergistically activated by Gβγ and PI(3,4,5)P binding and dysregulated in cancer. Here, we use X-ray crystallography, cryogenic electron microscopy and crosslinking mass spectrometry to determine the structural basis of human P-Rex1 autoinhibition. P-Rex1 has a bipartite structure of N- and C-terminal modules connected by a C-terminal four-helix bundle that binds the N-terminal Pleckstrin homology (PH) domain. In the N-terminal module, the Dbl homology (DH) domain catalytic surface is occluded by the compact arrangement of the DH-PH-DEP1 domains. Structural analysis reveals a remarkable conformational transition to release autoinhibition, requiring a 126° opening of the DH domain hinge helix. The off-axis position of Gβγ and PI(3,4,5)P binding sites further suggests a counter-rotation of the P-Rex1 halves by 90° facilitates PH domain uncoupling from the four-helix bundle, releasing the autoinhibited DH domain to drive Rho GTPase signaling.
History
DepositionAug 22, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 10, 2022Provider: repository / Type: Initial release
Revision 1.1Aug 24, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.2Oct 18, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 protein, Endolysin chimera
hetero molecules


Theoretical massNumber of molelcules
Total (without water)70,6037
Polymers70,0271
Non-polymers5766
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, cross-linking
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)151.256, 151.256, 94.287
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number91
Space group name H-MP4122
Space group name HallP4w2c
Symmetry operation#1: x,y,z
#2: -y,x,z+1/4
#3: y,-x,z+3/4
#4: x,-y,-z+1/2
#5: -x,y,-z
#6: -x,-y,z+1/2
#7: y,x,-z+3/4
#8: -y,-x,-z+1/4

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Components

#1: Protein Phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 protein, Endolysin chimera / P-Rex1 / PtdIns(3 / 4 / 5)-dependent Rac exchanger 1 / Lysis protein / Lysozyme / Muramidase / P- ...P-Rex1 / PtdIns(3 / 4 / 5)-dependent Rac exchanger 1 / Lysis protein / Lysozyme / Muramidase / P-Rex1 / PtdIns(3 / 4 / 5)-dependent Rac exchanger 1


Mass: 70026.695 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Enterobacteria phage T4 (virus)
Gene: PREX1, KIAA1415 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q8TCU6, UniProt: P00720, lysozyme
#2: Chemical
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: SO4
Has ligand of interestN
Sequence detailsT4-Lysozyme is spliced/inserted into the P-Rex1 structure in a loop region to enable ...T4-Lysozyme is spliced/inserted into the P-Rex1 structure in a loop region to enable crystallisation. However, the T4L could not be built into the PDB model as it was too flexible. Density was present but too poor to build/model the T4L.

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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: 3.86 Å3/Da / Density % sol: 68.1 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / Details: 1.8 M (NH4)2SO4, 0.05 MES pH 6.0

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Australian Synchrotron / Beamline: MX2 / Wavelength: 0.95373 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Jul 7, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.95373 Å / Relative weight: 1
ReflectionResolution: 3.22→47.83 Å / Num. obs: 18039 / % possible obs: 99.1 % / Redundancy: 6.4 % / Biso Wilson estimate: 119.41 Å2 / CC1/2: 0.998 / Net I/σ(I): 7.8
Reflection shellResolution: 3.22→3.48 Å / Num. unique obs: 3597 / CC1/2: 0.358

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Processing

Software
NameVersionClassification
PHENIX1.19.2_4158refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4YON

4yon


Resolution: 3.22→47.83 Å / SU ML: 0.4288 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 30.4388
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2621 902 5.02 %
Rwork0.2361 17056 -
obs0.2375 17958 98.68 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 127.08 Å2
Refinement stepCycle: LAST / Resolution: 3.22→47.83 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3525 0 30 0 3555
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00283611
X-RAY DIFFRACTIONf_angle_d0.5444860
X-RAY DIFFRACTIONf_chiral_restr0.0397534
X-RAY DIFFRACTIONf_plane_restr0.0062616
X-RAY DIFFRACTIONf_dihedral_angle_d12.01971369
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.22-3.430.3561560.33342683X-RAY DIFFRACTION96.2
3.43-3.690.32521570.27312827X-RAY DIFFRACTION99.87
3.69-4.060.24791280.23462845X-RAY DIFFRACTION99.6
4.06-4.650.24171510.20242853X-RAY DIFFRACTION99.87
4.65-5.860.24071450.22782904X-RAY DIFFRACTION99.93
5.86-47.830.25931650.23412944X-RAY DIFFRACTION96.67
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)
16.319221748621.45844547389-0.4208351206477.089408693040.8450906177133.873908829180.3171542004620.495716202430.726087944639-0.403349615566-0.286056714240.10575526379-0.531212359294-0.475524296656-0.03303172612410.7664411859970.1181561910540.1730961923210.825416170129-0.02663561537690.69850648586361.402515429917.07601955843.87165416488
23.74416782722-3.62916752933-0.6946778427664.528148100740.9272561320862.039218173770.2271652171540.1433123427340.122978688938-0.138408816579-0.1666210876590.0166229964835-0.05345303141840.0307795370657-0.05507863004260.72765368066-0.04957605551940.0478250081690.810393805277-0.1696128883420.87081588873751.22948600445.5187679516821.8717963149
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 42 through 236 )42 - 2361 - 189
22chain 'A' and (resid 237 through 501 )237 - 501190 - 432

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