Structure of IDP91191

Crystal structure of the aminoglycoside phosphotransferase APH(3')-Ia, with substrate kanamycin and small molecule inhibitor pyrazolopyrimidine PP1

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CSGID target
IDP91191 
PDB Id
4FEV (NCBI MMDB) 
Authors
P.J.Stogios,E.Evdokimova,Z.Wawrzak,G.Minasov,O.Egorova,R.Di Leo,T.Shakya,P.Spanogiannopoulos,G.D.Wright,A.Savchenko,W.F.Anderson,Center For Structural Genomics Of Infectious Diseases (Csgid) 
Responsible person
Peter Stogios 
Responsible lab
University of Calgary 
Deposition Date
May 30, 2012 
Release Date
Jun 20, 2012 

Annotation

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Description
Activity of the aminoglycoside phosphotransferase APH(3')-Ia leads to resistance to aminoglycoside antibiotics in pathogenic Gram-negative bacteria, and contributes to the clinical obsolescence of this class of antibiotics. One strategy to rescuing compromised antibiotics such as aminoglycosides is targeting the enzymes conferring resistance with small molecules. Previously we demonstrated that eukaryotic protein kinase (ePK) inhibitors could inhibit APH enzymes, due to the structural similarity between these two enzyme families. However, limited structural information of enzyme-inhibitor complexes hindered interpretation of the results. As well, cross-reactivity of compounds between APHs and ePKs represents an obstacle to their use as aminoglycoside adjuvants to rescue aminoglycoside antibiotic activity. Here, we structurally and functionally characterize inhibition of APH(3')-Ia by three diverse chemical scaffolds - anthrapyrazolone, 4-anilinoquinazoline and pyrazolopyrimidine (PP) - and reveal distinctions in the binding mode of anthrapyrazolone and PP compounds to APH(3')-Ia versus ePKs. Using this observation, we identify PP-derivatives that select against ePKs, attenuate APH(3')-Ia activity and rescue aminoglycoside antibiotic activity against a resistant E. coli strain. The structures presented here and these inhibition studies provide an important opportunity for structure-based design of compounds to target aminoglycoside phosphotransferases for inhibition, potentially overcoming this form of antibiotic resistance.  
Functional assignment
antibiotic resistance 

Ligands

Ligand code Name Ligand type
NA crystallization
PP1 1-ter-butyl-3-p-tolyl-1h-pyrazolo[3,4-d]pyrimidin-4-yla mine crystallization
KAN biological
175 3,5-dihydro-5-methylidene-4h-imidazol-4-on

Structure information

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Unit cell parameters

Space Group
P 1  
Unit Cell

a=57.57Å, b=94.22Å, c=96.88Å
α=61.12, β=73.09, γ=87.44 
Solvent content
 
Matthews coefficient
 

Refinement

Data for the highest resolution shell is in parentheses.
Resolution range
19.97-1.89Å (1.96-1.89Å)  
Rall(%)
15.4 
Rwork(%)
15.4 (20.3) 
Rfree(%)
20.4 (27.1) 
Num. observed reflections
130159 (11509) 
Num. Rfree reflections
2004 (174) 
Completeness(%)
94.6 (85.0) 

Model parameters

Num Atoms
12750  
Num Waters
1404  
Num Hetatoms
1768  
Model mean isotropic B factor
25.160Å2  
RMSD bond length
0.007Å  
RMSD bond angle
1.131°  
RMSD improper torsion angle
12.469°
 
Filename uploaded
4FEV.pdb (uploaded on Jun 26, 2013 10:41 AM)  
Inserted
May 30, 2012