5LPA

AadA E87Q in complex with ATP, calcium and dihydrostreptomycin


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.40 Å
  • R-Value Free: 0.178 
  • R-Value Work: 0.146 
  • R-Value Observed: 0.148 

wwPDB Validation   3D Report Full Report


Ligand Structure Quality Assessment 


This is version 1.2 of the entry. See complete history


Literature

Structural mechanism of AadA, a dual-specificity aminoglycoside adenylyltransferase fromSalmonella enterica.

Stern, A.L.Van der Verren, S.E.Kanchugal P, S.Nasvall, J.Gutierrez-de-Teran, H.Selmer, M.

(2018) J Biol Chem 293: 11481-11490

  • DOI: https://doi.org/10.1074/jbc.RA118.003989
  • Primary Citation of Related Structures:  
    5G4A, 5LPA, 5LUH, 6FZB

  • PubMed Abstract: 

    Streptomycin and spectinomycin are antibiotics that bind to the bacterial ribosome and perturb protein synthesis. The clinically most prevalent bacterial resistance mechanism is their chemical modification by aminoglycoside-modifying enzymes such as aminoglycoside nucleotidyltransferases (ANTs). AadA from Salmonella enterica is an aminoglycoside (3″)(9) adenylyltransferase that O- adenylates position 3″ of streptomycin and position 9 of spectinomycin. We previously reported the apo-AadA structure with a closed active site. To clarify how AadA binds ATP and its two chemically distinct drug substrates, we here report crystal structures of WT AadA complexed with ATP, magnesium, and streptomycin and of an active-site mutant, E87Q, complexed with ATP and streptomycin or the closely related dihydrostreptomycin. These structures revealed that ATP binding induces a conformational change that positions the two domains for drug binding at the interdomain cleft and disclosed the interactions between both domains and the three rings of streptomycin. Spectinomycin docking followed by molecular dynamics simulations suggested that, despite the limited structural similarities with streptomycin, spectinomycin makes similar interactions around the modification site and, in agreement with mutational data, forms critical interactions with fewer residues. Using structure-guided sequence analyses of ANT(3″)(9) enzymes acting on both substrates and ANT(9) enzymes active only on spectinomycin, we identified sequence determinants for activity on each substrate. We experimentally confirmed that Trp-173 and Asp-178 are essential only for streptomycin resistance. Activity assays indicated that Glu-87 is the catalytic base in AadA and that the nonadenylating E87Q mutant can hydrolyze ATP in the presence of streptomycin.


  • Organizational Affiliation

    Department of Cell and Molecular Biology, Uppsala University, BMC, Box 596, SE-751 24 Uppsala, Sweden.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Streptomycin 3''-adenylyltransferase
A, B
270Salmonella enterica subsp. enterica serovar TyphimuriumMutation(s): 1 
Gene Names: aadASTM1264
EC: 2.7.7.47
UniProt
Find proteins for Q8ZPX9 (Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720))
Explore Q8ZPX9 
Go to UniProtKB:  Q8ZPX9
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ8ZPX9
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Ligands 5 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
71R
Query on 71R

Download Ideal Coordinates CCD File 
D [auth A],
J [auth B]
dihydrostreptomycin
C21 H41 N7 O12
ASXBYYWOLISCLQ-HZYVHMACSA-N
ATP
Query on ATP

Download Ideal Coordinates CCD File 
C [auth A],
I [auth B]
ADENOSINE-5'-TRIPHOSPHATE
C10 H16 N5 O13 P3
ZKHQWZAMYRWXGA-KQYNXXCUSA-N
EDO
Query on EDO

Download Ideal Coordinates CCD File 
H [auth A],
M [auth B],
O [auth B]
1,2-ETHANEDIOL
C2 H6 O2
LYCAIKOWRPUZTN-UHFFFAOYSA-N
CA
Query on CA

Download Ideal Coordinates CCD File 
E [auth A],
G [auth A],
K [auth B],
N [auth B]
CALCIUM ION
Ca
BHPQYMZQTOCNFJ-UHFFFAOYSA-N
CL
Query on CL

Download Ideal Coordinates CCD File 
F [auth A],
L [auth B]
CHLORIDE ION
Cl
VEXZGXHMUGYJMC-UHFFFAOYSA-M
Modified Residues  1 Unique
IDChains TypeFormula2D DiagramParent
CME
Query on CME
A, B
L-PEPTIDE LINKINGC5 H11 N O3 S2CYS
Binding Affinity Annotations 
IDSourceBinding Affinity
ATP Binding MOAD:  5LPA Kd: 5.00e+4 (nM) from 1 assay(s)
71R Binding MOAD:  5LPA Kd: 1.10e+4 (nM) from 1 assay(s)
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.40 Å
  • R-Value Free: 0.178 
  • R-Value Work: 0.146 
  • R-Value Observed: 0.148 
  • Space Group: P 32
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 82.885α = 90
b = 82.885β = 90
c = 79.782γ = 120
Software Package:
Software NamePurpose
PHENIXrefinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing

Structure Validation

View Full Validation Report



Ligand Structure Quality Assessment 


Entry History & Funding Information

Deposition Data


Funding OrganizationLocationGrant Number
Swedish Research CouncilSweden--
Knut and Alice Wallenberg FoundationSwedenRiboCORE

Revision History  (Full details and data files)

  • Version 1.0: 2018-01-17
    Type: Initial release
  • Version 1.1: 2019-06-12
    Changes: Data collection, Database references
  • Version 1.2: 2024-01-10
    Changes: Data collection, Database references, Derived calculations, Refinement description