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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) Overall structure of PDB entry 3vqh: a ternary complex of the catalytic subunit α of protein kinase A (PKA; white), the peptidic pseudosubstrate ‘protein kinase inhibitor’ (PKI; grey) and the ATP-competitive inhibitor H-89 (yellow). (b) B/temperature factors of the H-89 conformers in the structure 3vqh plotted as spheres on the respective atoms. The values are indicated for selected atoms. (c) Binding environment of the H-89 conformers in the ATP pocket of PKA in structure 3vqh. Residues Val123, Glu127 and Asn171 form hydrogen bonds to the ligands; the bromine moieties pack against hydrophobic atoms from the side chains of Phe54 and Lys174 and some main-chain atoms of the glycine-rich loop. The inner surface of the ATP pocket is represented in red (maximum distance of 2 Å from the inhibitor molecule).
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fig3: (a) Overall structure of PDB entry 3vqh: a ternary complex of the catalytic subunit α of protein kinase A (PKA; white), the peptidic pseudosubstrate ‘protein kinase inhibitor’ (PKI; grey) and the ATP-competitive inhibitor H-89 (yellow). (b) B/temperature factors of the H-89 conformers in the structure 3vqh plotted as spheres on the respective atoms. The values are indicated for selected atoms. (c) Binding environment of the H-89 conformers in the ATP pocket of PKA in structure 3vqh. Residues Val123, Glu127 and Asn171 form hydrogen bonds to the ligands; the bromine moieties pack against hydrophobic atoms from the side chains of Phe54 and Lys174 and some main-chain atoms of the glycine-rich loop. The inner surface of the ATP pocket is represented in red (maximum distance of 2 Å from the inhibitor molecule).

Mentions: In PDB entry 3vqh the catalytic subunit α of protein kinase A appears in its usual conformation (Fig. 3 ▶a), similar to that in reference structures such as 1atp (Zheng et al., 1993 ▶) and 1cdk (Bossemeyer et al., 1993 ▶) and nearly identical to an earlier structure of a PKA–H-89 complex (PDB entry 1ydt; Engh et al., 1996 ▶). The root-mean-square deviation (r.m.s.d.) between the structures 1ydt and 3vqh is 0.33 Å. In 3vqh the fragment 5–24 of the PKI (‘protein kinase inhibitor’) peptide, which is routinely used for structural work on PKA as it stabilizes the kinase domain and facilitates crystallization, occupies the peptide-substrate site, which is formed primarily by the surface of the α-helical C-terminal lobe of the protein. The N-terminal lobe, which is mainly comprised of a five-stranded antiparallel β-sheet, is linked covalently to the C-terminal lobe by a single peptide chain (the hinge). Their interface forms a deep cleft which constitutes the binding pocket for the nucleotide substrate ATP. In the structure described here, the ATP-competitive inhibitor H-89 occupies the ATP-binding site (Fig. 3 ▶a). As shown in Fig. 1 ▶, H-89 binds with respect to ATP such that the isoquinoline group of H-89 occupies the adenine-binding pocket, the sulfonamide mimics the ribose group of ATP and the bromobenzene moiety occupies the site of the phosphate groups beneath the glycine-rich loop (formed by β-strands 1 and 2 and the β-turn that links them).


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) Overall structure of PDB entry 3vqh: a ternary complex of the catalytic subunit α of protein kinase A (PKA; white), the peptidic pseudosubstrate ‘protein kinase inhibitor’ (PKI; grey) and the ATP-competitive inhibitor H-89 (yellow). (b) B/temperature factors of the H-89 conformers in the structure 3vqh plotted as spheres on the respective atoms. The values are indicated for selected atoms. (c) Binding environment of the H-89 conformers in the ATP pocket of PKA in structure 3vqh. Residues Val123, Glu127 and Asn171 form hydrogen bonds to the ligands; the bromine moieties pack against hydrophobic atoms from the side chains of Phe54 and Lys174 and some main-chain atoms of the glycine-rich loop. The inner surface of the ATP pocket is represented in red (maximum distance of 2 Å from the inhibitor molecule).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: (a) Overall structure of PDB entry 3vqh: a ternary complex of the catalytic subunit α of protein kinase A (PKA; white), the peptidic pseudosubstrate ‘protein kinase inhibitor’ (PKI; grey) and the ATP-competitive inhibitor H-89 (yellow). (b) B/temperature factors of the H-89 conformers in the structure 3vqh plotted as spheres on the respective atoms. The values are indicated for selected atoms. (c) Binding environment of the H-89 conformers in the ATP pocket of PKA in structure 3vqh. Residues Val123, Glu127 and Asn171 form hydrogen bonds to the ligands; the bromine moieties pack against hydrophobic atoms from the side chains of Phe54 and Lys174 and some main-chain atoms of the glycine-rich loop. The inner surface of the ATP pocket is represented in red (maximum distance of 2 Å from the inhibitor molecule).
Mentions: In PDB entry 3vqh the catalytic subunit α of protein kinase A appears in its usual conformation (Fig. 3 ▶a), similar to that in reference structures such as 1atp (Zheng et al., 1993 ▶) and 1cdk (Bossemeyer et al., 1993 ▶) and nearly identical to an earlier structure of a PKA–H-89 complex (PDB entry 1ydt; Engh et al., 1996 ▶). The root-mean-square deviation (r.m.s.d.) between the structures 1ydt and 3vqh is 0.33 Å. In 3vqh the fragment 5–24 of the PKI (‘protein kinase inhibitor’) peptide, which is routinely used for structural work on PKA as it stabilizes the kinase domain and facilitates crystallization, occupies the peptide-substrate site, which is formed primarily by the surface of the α-helical C-terminal lobe of the protein. The N-terminal lobe, which is mainly comprised of a five-stranded antiparallel β-sheet, is linked covalently to the C-terminal lobe by a single peptide chain (the hinge). Their interface forms a deep cleft which constitutes the binding pocket for the nucleotide substrate ATP. In the structure described here, the ATP-competitive inhibitor H-89 occupies the ATP-binding site (Fig. 3 ▶a). As shown in Fig. 1 ▶, H-89 binds with respect to ATP such that the isoquinoline group of H-89 occupies the adenine-binding pocket, the sulfonamide mimics the ribose group of ATP and the bromobenzene moiety occupies the site of the phosphate groups beneath the glycine-rich loop (formed by β-strands 1 and 2 and the β-turn that links them).

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