3IXG

X-ray crystal structure of the extended-spectrum AmpC T70I mutant beta-lactamase with and without benzo(b)thiophene-2-boronic acid bound at 2.14 Angstrom resolution


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.14 Å
  • R-Value Free: 0.245 
  • R-Value Work: 0.197 
  • R-Value Observed: 0.199 

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Ligand Structure Quality Assessment 

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Literature

Structural bases for stability-function tradeoffs in antibiotic resistance.

Thomas, V.L.McReynolds, A.C.Shoichet, B.K.

(2010) J Mol Biol 396: 47-59

  • DOI: https://doi.org/10.1016/j.jmb.2009.11.005
  • Primary Citation of Related Structures:  
    3IWI, 3IWO, 3IWQ, 3IXB, 3IXD, 3IXG, 3IXH

  • PubMed Abstract: 

    Preorganization of enzyme active sites for substrate recognition typically comes at a cost to the stability of the folded form of the protein; consequently, enzymes can be dramatically stabilized by substitutions that attenuate the size and preorganization "strain" of the active site. How this stability-activity tradeoff constrains enzyme evolution has remained less certain, and it is unclear whether one should expect major stability insults as enzymes mutate towards new activities or how these new activities manifest structurally. These questions are both germane and easy to study in beta-lactamases, which are evolving on the timescale of years to confer resistance to an ever-broader spectrum of beta-lactam antibiotics. To explore whether stability is a substantial constraint on this antibiotic resistance evolution, we investigated extended-spectrum mutants of class C beta-lactamases, which had evolved new activity versus third-generation cephalosporins. Five mutant enzymes had between 100-fold and 200-fold increased activity against the antibiotic cefotaxime in enzyme assays, and the mutant enzymes all lost thermodynamic stability (from 1.7 kcal mol(-)(1) to 4.1 kcal mol(-)(1)), consistent with the stability-function hypothesis. Intriguingly, several of the substitutions were 10-20 A from the catalytic serine; the question of how they conferred extended-spectrum activity arose. Eight structures, including complexes with inhibitors and extended-spectrum antibiotics, were determined by X-ray crystallography. Distinct mechanisms of action, including changes in the flexibility and ground-state structures of the enzyme, are revealed for each mutant. These results explain the structural bases for the antibiotic resistance conferred by these substitutions and their corresponding decrease in protein stability, which will constrain the evolution of new antibiotic resistance.


  • Organizational Affiliation

    Graduate Program in Pharmaceutical Sciences and Pharmacogenomics, University of California San Francisco, San Francisco, CA 94158-2518, USA.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Beta-lactamase
A, B
358Escherichia coli K-12Mutation(s): 1 
Gene Names: ampAampCb4150JW4111
EC: 3.5.2.6
UniProt
Find proteins for P00811 (Escherichia coli (strain K12))
Explore P00811 
Go to UniProtKB:  P00811
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP00811
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Ligands 1 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
BZB
Query on BZB

Download Ideal Coordinates CCD File 
C [auth B]BENZO[B]THIOPHENE-2-BORONIC ACID
C8 H7 B O2 S
YNCYPMUJDDXIRH-UHFFFAOYSA-N
Binding Affinity Annotations 
IDSourceBinding Affinity
BZB PDBBind:  3IXG Ki: 27 (nM) from 1 assay(s)
BindingDB:  3IXG Ki: 27 (nM) from 1 assay(s)
IC50: min: 150, max: 300 (nM) from 3 assay(s)
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.14 Å
  • R-Value Free: 0.245 
  • R-Value Work: 0.197 
  • R-Value Observed: 0.199 
  • Space Group: C 1 2 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 114.162α = 90
b = 77.361β = 122.12
c = 91.747γ = 90
Software Package:
Software NamePurpose
SCALAdata scaling
MOLREPphasing
REFMACrefinement
PDB_EXTRACTdata extraction
MOSFLMdata reduction

Structure Validation

View Full Validation Report



Ligand Structure Quality Assessment 

Created with Raphaël 2.3.0Worse 01 BetterLigand structure goodness of fit to experimental dataBest fitted BZBClick on this verticalbar to view details

Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2010-02-16
    Type: Initial release
  • Version 1.1: 2011-07-13
    Changes: Version format compliance
  • Version 1.2: 2021-10-13
    Changes: Database references, Derived calculations
  • Version 1.3: 2024-02-21
    Changes: Data collection