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. 2017 Jun 20;112(12):2552–2566. doi: 10.1016/j.bpj.2017.04.045

Figure 7.

Figure 7

Channeling and turnover of cAMP through RIα in the PDE8-PKA-RIα complex as seen in the inversion of HDXMS kinetics at CNB:A and CNB:B. (A) Differences in average deuterons exchanged (Y axis) between PDE8AC-RIα-cAMP (the ternary complex) and PDE8AC-RIα complex for pepsin fragment peptides of RIα as listed from the N- to the C-terminus (X axis). Peptides spanning contiguous regions are grouped by brace brackets. Negative differences indicate decreased deuterium exchange, and positive differences denote increased deuterium exchange in the ternary complex. The two domains CNB:A and CNB:B and the PDE8-binding sites are highlighted by pink and blue boxes, respectively. The deuterium labeling times for the peptides are depicted and colored according to the key. Standard deviations are shaded gray. (B) Cyclic nucleotide binding domains CNB:A and CNB:B are indicated by stacked mass spectra of a representative peptide spanning residues 203–218 (i) and 329–338 (ii), respectively, including spectra from the ternary complex PDE8AC-RIα-cAMP (left) and the binary complex PDE8AC-RIα (right) at different labeling times. The shift in centroids with deuterium exchange is represented by double-headed red arrows, and the reference point for undeuterated RIα is shown as a vertical dashed line. (C) Surface representation of the crystal structure of RIα (PDB: 4MX3), with regions showing significant differences in deuterium exchange in the ternary complex, are mapped with color coding as in (A). Relative differences between the two states at three different labeling times—60 min (iii), 10 min (ii), and 1 min (i)—are displayed. Regions showing decreased exchange are colored in shades of blue and regions showing increased exchange in shades of red. The two RIα monomers are indicated as R (left) and R′ (right). cAMP molecules are shown as yellow spheres. No-coverage regions are in gray. A cartoon schematic is also shown of processive hydrolysis by the PDE8-PKAR complex by channeling of cAMP through PKAR resulting in release of 5′-AMP.