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Anomalous dispersion analysis of inhibitor flexibility: a case study of the kinase inhibitor H-89.

Pflug A, Johnson KA, Engh RA - Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. (2012)

Bottom Line: Here, an analysis of the binding of the kinase inhibitor H-89 to protein kinase A (PKA) is presented.H-89 contains a bromobenzene moiety that apparently binds with multiple conformations in the kinase ATP pocket.Using anomalous dispersion methods, it was possible to resolve these conformations into two distinct binding geometries.

View Article: PubMed Central - HTML - PubMed

Affiliation: Norwegian Structural Biology Centre, Department of Chemistry, University of Tromsø, N-9037 Tromsø, Norway.

ABSTRACT
With its ability to show the interactions between drug-target proteins and small-molecule ligands, X-ray crystallography is an essential tool in drug-discovery programmes. However, its usefulness can be limited by crystallization artifacts or by the data resolution, and in particular when assumptions of unimodal binding (and isotropic motion) do not apply. Discrepancies between the modelled crystal structure and the physiological range of structures generally prevent quantitative estimation of binding energies. Improved crystal structure resolution will often not aid energy estimation because the conditions which provide the highest rigidity and resolution are not likely to reflect physiological conditions. Instead, strategies must be employed to measure and model flexibility and multiple binding modes to supplement crystallographic information. One useful tool is the use of anomalous dispersion for small molecules that contain suitable atoms. Here, an analysis of the binding of the kinase inhibitor H-89 to protein kinase A (PKA) is presented. H-89 contains a bromobenzene moiety that apparently binds with multiple conformations in the kinase ATP pocket. Using anomalous dispersion methods, it was possible to resolve these conformations into two distinct binding geometries.

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(a) Anomalous difference Fourier maps (1.95 Å) generated to cover all atoms of the asymmetric unit in PDB entry 3vqh contoured at levels of 3σ, 4σ and 5σ. (b) 2.3 Å resolution electron-density map (grey) and difference density map (green) surrounding the compound H-89 in PDB entry 1ydt (cyan; Engh et al., 1996 ▶). (c) 1.95 Å resolution OMIT electron-density map (grey) and anomalous difference density map (blue) carved around the two conformations of compound H-89 in PDB entry 3vqh (yellow).
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fig2: (a) Anomalous difference Fourier maps (1.95 Å) generated to cover all atoms of the asymmetric unit in PDB entry 3vqh contoured at levels of 3σ, 4σ and 5σ. (b) 2.3 Å resolution electron-density map (grey) and difference density map (green) surrounding the compound H-89 in PDB entry 1ydt (cyan; Engh et al., 1996 ▶). (c) 1.95 Å resolution OMIT electron-density map (grey) and anomalous difference density map (blue) carved around the two conformations of compound H-89 in PDB entry 3vqh (yellow).

Mentions: REFMAC5 (Murshudov et al., 2011 ▶) was used to generate weighted electron-density and difference maps (2mFo − DFc and mFo − DFc, respectively) for the refined structures (Figs. 2 ▶b and 2 ▶c). Fo and Fc refer to the observed and model structure factors, m is the figure of merit and D is the model error parameter. The REFMAC5 calculation of the weighting factors D and m (Murshudov et al., 2011 ▶) matches that in SIGMAA (Read, 1986 ▶) except that only the free-R-­flagged reflections are used to estimate m and D. For missing reflections, the map coefficients are replaced with DFc in electron-density maps and zero in difference maps.


Anomalous dispersion analysis of inhibitor flexibility: a case study of the kinase inhibitor H-89.

Pflug A, Johnson KA, Engh RA - Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. (2012)

(a) Anomalous difference Fourier maps (1.95 Å) generated to cover all atoms of the asymmetric unit in PDB entry 3vqh contoured at levels of 3σ, 4σ and 5σ. (b) 2.3 Å resolution electron-density map (grey) and difference density map (green) surrounding the compound H-89 in PDB entry 1ydt (cyan; Engh et al., 1996 ▶). (c) 1.95 Å resolution OMIT electron-density map (grey) and anomalous difference density map (blue) carved around the two conformations of compound H-89 in PDB entry 3vqh (yellow).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3412763&req=5

fig2: (a) Anomalous difference Fourier maps (1.95 Å) generated to cover all atoms of the asymmetric unit in PDB entry 3vqh contoured at levels of 3σ, 4σ and 5σ. (b) 2.3 Å resolution electron-density map (grey) and difference density map (green) surrounding the compound H-89 in PDB entry 1ydt (cyan; Engh et al., 1996 ▶). (c) 1.95 Å resolution OMIT electron-density map (grey) and anomalous difference density map (blue) carved around the two conformations of compound H-89 in PDB entry 3vqh (yellow).
Mentions: REFMAC5 (Murshudov et al., 2011 ▶) was used to generate weighted electron-density and difference maps (2mFo − DFc and mFo − DFc, respectively) for the refined structures (Figs. 2 ▶b and 2 ▶c). Fo and Fc refer to the observed and model structure factors, m is the figure of merit and D is the model error parameter. The REFMAC5 calculation of the weighting factors D and m (Murshudov et al., 2011 ▶) matches that in SIGMAA (Read, 1986 ▶) except that only the free-R-­flagged reflections are used to estimate m and D. For missing reflections, the map coefficients are replaced with DFc in electron-density maps and zero in difference maps.

Bottom Line: Here, an analysis of the binding of the kinase inhibitor H-89 to protein kinase A (PKA) is presented.H-89 contains a bromobenzene moiety that apparently binds with multiple conformations in the kinase ATP pocket.Using anomalous dispersion methods, it was possible to resolve these conformations into two distinct binding geometries.

View Article: PubMed Central - HTML - PubMed

Affiliation: Norwegian Structural Biology Centre, Department of Chemistry, University of Tromsø, N-9037 Tromsø, Norway.

ABSTRACT
With its ability to show the interactions between drug-target proteins and small-molecule ligands, X-ray crystallography is an essential tool in drug-discovery programmes. However, its usefulness can be limited by crystallization artifacts or by the data resolution, and in particular when assumptions of unimodal binding (and isotropic motion) do not apply. Discrepancies between the modelled crystal structure and the physiological range of structures generally prevent quantitative estimation of binding energies. Improved crystal structure resolution will often not aid energy estimation because the conditions which provide the highest rigidity and resolution are not likely to reflect physiological conditions. Instead, strategies must be employed to measure and model flexibility and multiple binding modes to supplement crystallographic information. One useful tool is the use of anomalous dispersion for small molecules that contain suitable atoms. Here, an analysis of the binding of the kinase inhibitor H-89 to protein kinase A (PKA) is presented. H-89 contains a bromobenzene moiety that apparently binds with multiple conformations in the kinase ATP pocket. Using anomalous dispersion methods, it was possible to resolve these conformations into two distinct binding geometries.

Show MeSH
Related in: MedlinePlus