4EAN

1.75A resolution structure of indole bound beta-glycosidase (W33G) from sulfolobus solfataricus


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.75 Å
  • R-Value Free: 0.191 
  • R-Value Work: 0.174 
  • R-Value Observed: 0.175 

wwPDB Validation   3D Report Full Report


This is version 1.3 of the entry. See complete history


Literature

Designing allosteric control into enzymes by chemical rescue of structure.

Deckert, K.Budiardjo, S.J.Brunner, L.C.Lovell, S.Karanicolas, J.

(2012) J Am Chem Soc 134: 10055-10060

  • DOI: https://doi.org/10.1021/ja301409g
  • Primary Citation of Related Structures:  
    4EAM, 4EAN

  • PubMed Abstract: 

    Ligand-dependent activity has been engineered into enzymes for purposes ranging from controlling cell morphology to reprogramming cellular signaling pathways. Where these successes have typically fused a naturally allosteric domain to the enzyme of interest, here we instead demonstrate an approach for designing a de novo allosteric effector site directly into the catalytic domain of an enzyme. This approach is distinct from traditional chemical rescue of enzymes in that it relies on disruption and restoration of structure, rather than active site chemistry, as a means to achieve modulate function. We present two examples, W33G in a β-glycosidase enzyme (β-gly) and W492G in a β-glucuronidase enzyme (β-gluc), in which we engineer indole-dependent activity into enzymes by removing a buried tryptophan side chain that serves as a buttress for the active site architecture. In both cases, we observe a loss of function, and in both cases we find that the subsequent addition of indole can be used to restore activity. Through a detailed analysis of β-gly W33G kinetics, we demonstrate that this rescued enzyme is fully functionally equivalent to the corresponding wild-type enzyme. We then present the apo and indole-bound crystal structures of β-gly W33G, which together establish the structural basis for enzyme inactivation and rescue. Finally, we use this designed switch to modulate β-glycosidase activity in living cells using indole. Disruption and recovery of protein structure may represent a general technique for introducing allosteric control into enzymes, and thus may serve as a starting point for building a variety of bioswitches and sensors.


  • Organizational Affiliation

    Department of Molecular Biosciences, University of Kansas, 1200 Sunnyside Avenue, Lawrence, Kansas 66045-7534, USA.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Beta-galactosidase
A, B
489Saccharolobus solfataricus P2Mutation(s): 1 
Gene Names: lacSSSO3019
EC: 3.2.1.23
UniProt
Find proteins for P22498 (Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2))
Explore P22498 
Go to UniProtKB:  P22498
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP22498
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Ligands 3 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
MPD
Query on MPD

Download Ideal Coordinates CCD File 
G [auth A],
L [auth B]
(4S)-2-METHYL-2,4-PENTANEDIOL
C6 H14 O2
SVTBMSDMJJWYQN-YFKPBYRVSA-N
IND
Query on IND

Download Ideal Coordinates CCD File 
F [auth A],
K [auth B]
INDOLE
C8 H7 N
SIKJAQJRHWYJAI-UHFFFAOYSA-N
CL
Query on CL

Download Ideal Coordinates CCD File 
C [auth A]
D [auth A]
E [auth A]
H [auth B]
I [auth B]
C [auth A],
D [auth A],
E [auth A],
H [auth B],
I [auth B],
J [auth B]
CHLORIDE ION
Cl
VEXZGXHMUGYJMC-UHFFFAOYSA-M
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.75 Å
  • R-Value Free: 0.191 
  • R-Value Work: 0.174 
  • R-Value Observed: 0.175 
  • Space Group: P 31 2 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 167.544α = 90
b = 167.544β = 90
c = 95.232γ = 120
Software Package:
Software NamePurpose
SCALAdata scaling
PHASERphasing
PHENIXrefinement
PDB_EXTRACTdata extraction
JDirectordata collection
XDSdata reduction

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2012-06-13
    Type: Initial release
  • Version 1.1: 2012-11-21
    Changes: Database references
  • Version 1.2: 2017-11-15
    Changes: Refinement description
  • Version 1.3: 2023-09-13
    Changes: Data collection, Database references, Derived calculations, Refinement description