5EYP

TUBULIN-DARPIN COMPLEX


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.90 Å
  • R-Value Free: 0.208 
  • R-Value Work: 0.173 
  • R-Value Observed: 0.175 

Starting Model: experimental
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wwPDB Validation   3D Report Full Report


Ligand Structure Quality Assessment 


This is version 1.2 of the entry. See complete history


Literature

Destabilizing an interacting motif strengthens the association of a designed ankyrin repeat protein with tubulin.

Ahmad, S.Pecqueur, L.Dreier, B.Hamdane, D.Aumont-Nicaise, M.Pluckthun, A.Knossow, M.Gigant, B.

(2016) Sci Rep 6: 28922-28922

  • DOI: https://doi.org/10.1038/srep28922
  • Primary Citation of Related Structures:  
    5EYL, 5EYP

  • PubMed Abstract: 

    Affinity maturation by random mutagenesis and selection is an established technique to make binding molecules more suitable for applications in biomedical research, diagnostics and therapy. Here we identified an unexpected novel mechanism of affinity increase upon in vitro evolution of a tubulin-specific designed ankyrin repeat protein (DARPin). Structural analysis indicated that in the progenitor DARPin the C-terminal capping repeat (C-cap) undergoes a 25° rotation to avoid a clash with tubulin upon binding. Additionally, the C-cap appears to be involved in electrostatic repulsion with tubulin. Biochemical and structural characterizations demonstrated that the evolved mutants achieved a gain in affinity through destabilization of the C-cap, which relieves the need of a DARPin conformational change upon tubulin binding and removes unfavorable interactions in the complex. Therefore, this specific case of an order-to-disorder transition led to a 100-fold tighter complex with a subnanomolar equilibrium dissociation constant, remarkably associated with a 30% decrease of the binding surface.


  • Organizational Affiliation

    Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ Paris-Sud, Université Paris-Saclay, 91198, Gif-sur-Yvette cedex, France.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Tubulin alpha chain451Ovis ariesMutation(s): 2 
UniProt
Find proteins for D0VWZ0 (Ovis aries)
Explore D0VWZ0 
Go to UniProtKB:  D0VWZ0
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupD0VWZ0
Sequence Annotations
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  • Reference Sequence
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Entity ID: 2
MoleculeChains Sequence LengthOrganismDetailsImage
Tubulin beta chain445Ovis ariesMutation(s): 2 
UniProt
Find proteins for D0VWY9 (Ovis aries)
Explore D0VWY9 
Go to UniProtKB:  D0VWY9
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupD0VWY9
Sequence Annotations
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  • Reference Sequence
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Entity ID: 3
MoleculeChains Sequence LengthOrganismDetailsImage
DESIGNED ANKYRIN REPEAT PROTEIN (DARPIN)C [auth F]169synthetic constructMutation(s): 0 
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
Sequence Annotations
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  • Reference Sequence
Small Molecules
Ligands 6 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
GTP
Query on GTP

Download Ideal Coordinates CCD File 
D [auth A]GUANOSINE-5'-TRIPHOSPHATE
C10 H16 N5 O14 P3
XKMLYUALXHKNFT-UUOKFMHZSA-N
GDP
Query on GDP

Download Ideal Coordinates CCD File 
K [auth B]GUANOSINE-5'-DIPHOSPHATE
C10 H15 N5 O11 P2
QGWNDRXFNXRZMB-UUOKFMHZSA-N
LOC
Query on LOC

Download Ideal Coordinates CCD File 
L [auth B]N-[(7S)-1,2,3,10-tetramethoxy-9-oxo-6,7-dihydro-5H-benzo[d]heptalen-7-yl]ethanamide
C22 H25 N O6
IAKHMKGGTNLKSZ-INIZCTEOSA-N
SO4
Query on SO4

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F [auth A],
G [auth A],
M [auth B]
SULFATE ION
O4 S
QAOWNCQODCNURD-UHFFFAOYSA-L
GOL
Query on GOL

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H [auth A]
I [auth A]
J [auth A]
N [auth B]
O [auth B]
H [auth A],
I [auth A],
J [auth A],
N [auth B],
O [auth B],
P [auth F],
Q [auth F],
R [auth F]
GLYCEROL
C3 H8 O3
PEDCQBHIVMGVHV-UHFFFAOYSA-N
MG
Query on MG

Download Ideal Coordinates CCD File 
E [auth A]MAGNESIUM ION
Mg
JLVVSXFLKOJNIY-UHFFFAOYSA-N
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.90 Å
  • R-Value Free: 0.208 
  • R-Value Work: 0.173 
  • R-Value Observed: 0.175 
  • Space Group: P 1 21 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 87.57α = 90
b = 71.76β = 99.85
c = 93.05γ = 90
Software Package:
Software NamePurpose
BUSTERrefinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing

Structure Validation

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Ligand Structure Quality Assessment 


Entry History & Funding Information

Deposition Data


Funding OrganizationLocationGrant Number
French National Research AgencyFranceANR-12-BSV8-0002-01

Revision History  (Full details and data files)

  • Version 1.0: 2016-07-20
    Type: Initial release
  • Version 1.1: 2017-09-06
    Changes: Author supporting evidence
  • Version 1.2: 2024-01-10
    Changes: Data collection, Database references, Derived calculations, Refinement description