Inhibitor Binding Site—ATP pocket
The inhibitors are ATP competitive and bind to the ATP pocket made up of residues from the glycine loop (Leu49, Gly50, Thr51, Val57), b-sheet 3 (Ala70), hinge region (Met120, Glu121, Tyr122, Val123, Glu127), catalytic loop (Glu170, Asn171, Leu173), beginning of the activation loop (Thr183, Asp184), and C-terminal stretch (Phe327) (Fig. 3).
The buried surface of the inhibitors in their pocket in PKA correlates with their affinity: H-1152P (1.1 mM) with the highest affinity also has the largest buried surface of the three inhibitors (215.7 A2); Y-27632 (25 mM) has the smallest buried surface of the three (188 A2). A correlation of buried inhibitor surface and binding affinity has previously been observed for PKA inhibitors (Engh and Bossemeyer 2002). Rho kinase and PKA differ, however, in eight positions in the ATP binding pocket. Four of their sidechains are
in close (<4 A) contact with the inhibitors, corresponding to the following PKA^Rho substitutions: Leu49Ile, Val123Met, Thr183Ala and Glu127Asp. Because the protein kinase fold is so highly conserved, variations of amino acid residues that line the ATP subsite belong to the most important factors in defining inhibitor selectivity.
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