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- PDB-9cx9: Structure of SH3 domain of Src in complex with beta-arrestin 1 -

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Entry
Database: PDB / ID: 9cx9
TitleStructure of SH3 domain of Src in complex with beta-arrestin 1
Components
  • Antibody fragment Fab30, heavy chain
  • Antibody fragment Fab30, light chain
  • Beta-arrestin-1
  • Proto-oncogene tyrosine-protein kinase Src
  • Vasopressin V2 receptor
KeywordsSIGNALING PROTEIN / GPCR signaling / arrestin / Src / SH3
Function / homology
Function and homology information


V2 vasopressin receptor binding / alpha-1A adrenergic receptor binding / follicle-stimulating hormone receptor binding / TGFBR3 regulates TGF-beta signaling / G alpha (s) signalling events / sensory perception of touch / follicle-stimulating hormone signaling pathway / alpha-1B adrenergic receptor binding / protein phosphorylated amino acid binding / renal water retention ...V2 vasopressin receptor binding / alpha-1A adrenergic receptor binding / follicle-stimulating hormone receptor binding / TGFBR3 regulates TGF-beta signaling / G alpha (s) signalling events / sensory perception of touch / follicle-stimulating hormone signaling pathway / alpha-1B adrenergic receptor binding / protein phosphorylated amino acid binding / renal water retention / Defective AVP does not bind AVPR2 and causes neurohypophyseal diabetes insipidus (NDI) / Lysosome Vesicle Biogenesis / regulation of systemic arterial blood pressure by vasopressin / Vasopressin-like receptors / renal water absorption / vasopressin receptor activity / Ub-specific processing proteases / angiotensin receptor binding / AP-2 adaptor complex binding / MAP2K and MAPK activation / Golgi Associated Vesicle Biogenesis / regulation of inositol trisphosphate biosynthetic process / telencephalon development / hemostasis / Cargo recognition for clathrin-mediated endocytosis / Clathrin-mediated endocytosis / negative regulation of GTPase activity / negative regulation of interleukin-8 production / clathrin-cargo adaptor activity / connexin binding / desensitization of G protein-coupled receptor signaling pathway / regulation of G protein-coupled receptor signaling pathway / arrestin family protein binding / G protein-coupled receptor internalization / mitogen-activated protein kinase kinase binding / stress fiber assembly / Thrombin signalling through proteinase activated receptors (PARs) / negative regulation of Notch signaling pathway / sensory perception / negative regulation of interleukin-6 production / response to morphine / clathrin binding / negative regulation of intrinsic apoptotic signaling pathway / positive regulation of Rho protein signal transduction / positive regulation of systemic arterial blood pressure / progesterone receptor signaling pathway / negative regulation of protein phosphorylation / pseudopodium / positive regulation of intracellular signal transduction / phototransduction / positive regulation of receptor internalization / positive regulation of vasoconstriction / endocytic vesicle / cysteine-type endopeptidase inhibitor activity / cellular response to hormone stimulus / activation of adenylate cyclase activity / immune system process / insulin-like growth factor receptor binding / negative regulation of protein ubiquitination / clathrin-coated pit / intracellular glucose homeostasis / negative regulation of extrinsic apoptotic signaling pathway / positive regulation of protein ubiquitination / nuclear estrogen receptor binding / positive regulation of insulin secretion involved in cellular response to glucose stimulus / GTPase activator activity / non-specific protein-tyrosine kinase / non-membrane spanning protein tyrosine kinase activity / clathrin-coated endocytic vesicle membrane / phosphoprotein binding / negative regulation of ERK1 and ERK2 cascade / positive regulation of protein phosphorylation / cell junction / endocytosis / G protein-coupled receptor binding / epidermal growth factor receptor signaling pathway / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / cell-cell junction / Vasopressin regulates renal water homeostasis via Aquaporins / Cargo recognition for clathrin-mediated endocytosis / negative regulation of neuron apoptotic process / protein transport / Clathrin-mediated endocytosis / adenylate cyclase-activating G protein-coupled receptor signaling pathway / protein tyrosine kinase activity / cytoplasmic vesicle / protein phosphatase binding / ubiquitin-dependent protein catabolic process / molecular adaptor activity / G alpha (s) signalling events / dendritic spine / basolateral plasma membrane / proteasome-mediated ubiquitin-dependent protein catabolic process / regulation of apoptotic process / cytoskeleton / positive regulation of MAPK cascade / transmembrane transporter binding / positive regulation of ERK1 and ERK2 cascade / regulation of cell cycle / cell differentiation
Similarity search - Function
Vasopressin V2 receptor / Vasopressin receptor / Arrestin, conserved site / Arrestins signature. / Arrestin / Arrestin, N-terminal / Arrestin-like, N-terminal / Arrestin C-terminal-like domain / Arrestin (or S-antigen), N-terminal domain / Arrestin (or S-antigen), C-terminal domain ...Vasopressin V2 receptor / Vasopressin receptor / Arrestin, conserved site / Arrestins signature. / Arrestin / Arrestin, N-terminal / Arrestin-like, N-terminal / Arrestin C-terminal-like domain / Arrestin (or S-antigen), N-terminal domain / Arrestin (or S-antigen), C-terminal domain / Arrestin (or S-antigen), C-terminal domain / Arrestin-like, C-terminal / : / 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. / G-protein coupled receptors family 1 signature. / Tyrosine-protein kinase, active site / Immunoglobulin E-set / 7 transmembrane receptor (rhodopsin family) / G protein-coupled receptor, rhodopsin-like / GPCR, rhodopsin-like, 7TM / G-protein coupled receptors family 1 profile. / 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
Proto-oncogene tyrosine-protein kinase Src / Beta-arrestin-1 / Vasopressin V2 receptor
Similarity search - Component
Biological speciesMus musculus (house mouse)
Rattus norvegicus (Norway rat)
Gallus gallus (chicken)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.34 Å
AuthorsPakharukova, N. / Bansia, H. / Lefkowitz, R.J. / des Georges, A.
Funding support United States, European Union, France, 5items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)HL016037-50 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM133598 United States
European Molecular Biology Organization (EMBO)ALTF 1071-2017European Union
Human Frontier Science Program (HFSP)LT000174/2018 France
Citation
Journal: Nat Commun / Year: 2026
Title: Mechanism of beta-arrestin 1 mediated Src activation via Src SH3 domain revealed by cryo-electron microscopy.
Authors: Natalia Pakharukova / Brittany N Thomas / Harsh Bansia / Linus Li / Dana K Bassford / Rinat R Abzalimov / Jihee Kim / Alem W Kahsai / Biswaranjan Pani / Kunhong Xiao / Roni Ochakovski / ...Authors: Natalia Pakharukova / Brittany N Thomas / Harsh Bansia / Linus Li / Dana K Bassford / Rinat R Abzalimov / Jihee Kim / Alem W Kahsai / Biswaranjan Pani / Kunhong Xiao / Roni Ochakovski / Shibo Liu / Xingdong Zhang / Seungkirl Ahn / Amedee des Georges / Robert J Lefkowitz /
Abstract: Beta-arrestins (βarrs) are key regulators and transducers of G-protein coupled receptor signaling; however, little is known of how βarrs communicate with their downstream effectors. Here, we ...Beta-arrestins (βarrs) are key regulators and transducers of G-protein coupled receptor signaling; however, little is known of how βarrs communicate with their downstream effectors. Here, we delineate structural mechanisms underlying βarr-mediated signal transduction. Using cryo-electron microscopy, we elucidate how βarr1 recruits and activates the non-receptor tyrosine kinase Src, a well-established signaling partner of βarrs. βarr1 engages Src SH3 through two distinct sites, each employing a different recognition mechanism: a polyproline motif in the N-domain and a non-proline-based interaction in the central crest region. At both sites βarr1 interacts with the aromatic surface of SH3, disrupting the autoinhibited conformation of Src and directly triggering its allosteric activation. This structural evidence establishes βarr1 as an active regulatory protein rather than a passive scaffold and suggests a potentially general mechanism for βarr-mediated signaling across diverse effectors.
#1: Journal: bioRxiv / Year: 2025
Title: Mechanism of beta-arrestin 1 mediated Src activation via Src SH3 domain revealed by cryo-electron microscopy.
Authors: Natalia Pakharukova / Brittany N Thomas / Harsh Bansia / Linus Li / Dana K Bassford / Rinat R Abzalimov / Jihee Kim / Alem W Kahsai / Biswaranjan Pani / Kunhong Xiao / Roni Ochakovski / ...Authors: Natalia Pakharukova / Brittany N Thomas / Harsh Bansia / Linus Li / Dana K Bassford / Rinat R Abzalimov / Jihee Kim / Alem W Kahsai / Biswaranjan Pani / Kunhong Xiao / Roni Ochakovski / Shibo Liu / Xingdong Zhang / Seungkirl Ahn / Amedee des Georges / Robert J Lefkowitz /
Abstract: Beta-arrestins (βarrs) are key regulators and transducers of G-protein coupled receptor signaling; however, little is known of how βarrs communicate with their downstream effectors. Here, we report ...Beta-arrestins (βarrs) are key regulators and transducers of G-protein coupled receptor signaling; however, little is known of how βarrs communicate with their downstream effectors. Here, we report the first structural insights into the fundamental mechanisms driving βarr-mediated signal transduction. Using cryo-electron microscopy, we elucidate how βarr1 recruits and activates the non-receptor tyrosine kinase Src, the first identified signaling partner of βarrs. βarr1 engages Src SH3 through two distinct sites, each employing a different recognition mechanism: a polyproline motif in the N-domain and a non-proline-based interaction in the central crest region. At both sites βarr1 interacts with the aromatic surface of SH3, disrupting the autoinhibited conformation of Src and directly triggering its allosteric activation. This structural evidence establishes βarr1 as an active regulatory protein rather than a passive scaffold and suggests a potentially general mechanism for βarr-mediated signaling across diverse effectors.
History
DepositionJul 31, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 13, 2024Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
H: Antibody fragment Fab30, heavy chain
L: Antibody fragment Fab30, light chain
V: Vasopressin V2 receptor
A: Beta-arrestin-1
B: Proto-oncogene tyrosine-protein kinase Src


Theoretical massNumber of molelcules
Total (without water)106,3045
Polymers106,3045
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Antibody Antibody fragment Fab30, heavy chain


Mass: 25512.354 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Escherichia coli (E. coli)
#2: Antibody Antibody fragment Fab30, light chain


Mass: 23435.064 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Escherichia coli (E. coli)
#3: Protein/peptide Vasopressin V2 receptor


Mass: 3550.936 Da / Num. of mol.: 1 / Fragment: UNP residues 343-371 / Source method: obtained synthetically
Details: Synthetic phosphopeptide mimicking the C-tail of vasopressin 2 receptor
Source: (synth.) Homo sapiens (human) / References: UniProt: P30518
#4: Protein Beta-arrestin-1 / Arrestin beta-1


Mass: 44049.160 Da / Num. of mol.: 1
Mutation: C59V, E92C, C125S, C140L, C150V, C242V, C251V, C269S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Arrb1
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P29066
#5: Protein Proto-oncogene tyrosine-protein kinase Src / Proto-oncogene c-Src / pp60c-src / p60-Src


Mass: 9756.567 Da / Num. of mol.: 1 / Mutation: R95C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken) / Gene: SRC
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P00523, non-specific protein-tyrosine kinase
Has ligand of interestN
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Beta-arrestin 1 bound to a G protein-coupled receptor phosphopeptide and antibody fragment Fab30 in complex with SH3 domain of Src
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 53.8 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
4cryoSPARC4.4.1CTF correction
7Coot0.9.8.3model fitting
8ISOLDEmodel fitting
10PHENIX1.20.1-4487model refinement
14cryoSPARC4.1.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.34 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 345529 / Symmetry type: POINT
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeInitial refinement model-IDChain-ID
14jqi

4jqi

4jqi1
24jqi

4jqi

V4jqi1V
34jqi

4jqi

L4jqi1L
44jqi

4jqi

H4jqi1H
52ptk

2ptk

B2ptk2B
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0024731
ELECTRON MICROSCOPYf_angle_d0.556440
ELECTRON MICROSCOPYf_dihedral_angle_d4.689673
ELECTRON MICROSCOPYf_chiral_restr0.043735
ELECTRON MICROSCOPYf_plane_restr0.003819

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