Dissecting Orthosteric Contacts for a Reverse-Fragment-Based Ligand Design

Orthosteric sites on proteins are formed typically from noncontiguous interacting sites in three-dimensional space where the composite binding interaction of a biological ligand is mediated by multiple synergistic interactions of its constituent functional groups. Through these multiple interactions, ligands stabilize both the ligand binding site and the local secondary structure. However, relative energetic contributions of the individual contacts in these protein–ligand interactions are difficult to resolve. Deconvolution of the contributions of these various functional groups in natural inhibitors/ligand would greatly aid in iterative fragment-based drug discovery (FBDD). In this study, we describe an approach of progressive unfolding of a target protein using a gradient of denaturant urea to reveal the individual energetic contributions of various ligand-functional groups to the affinity of the entire ligand. Through calibrated unfolding of two protein–ligand systems: cAMP-bound regulatory subunit of Protein Kinase A (RIα) and IBMX-bound phosphodiesterase8 (PDE8), monitored by amide hydrogen–deuterium exchange mass spectrometry, we show progressive disruption of individual orthosteric contacts in the ligand binding sites, allowing us to rank the energetic contributions of these individual interactions. In the two cAMP-binding sites of RIα, exocyclic phosphate oxygens of cAMP were identified to mediate stronger interactions than ribose 2′-OH in both the RIα-cAMP binding interfaces. Further, we have also ranked the relative contributions of the different functional groups of IBMX based on their interactions with the orthosteric residues of PDE8. This strategy for deconstruction of individual binding sites and identification of the strongest functional group interaction in enzyme orthosteric sites offers a rational starting point for FBDD

Image1_Impaired cAMP processivity by phosphodiesterase-protein kinase A complexes in acrodysostosis.TIF

Acrodysostosis represents a group of rare genetic disorders characterized by defective skeletal development and is often accompanied by intellectual disabilities. Mutations in the 3′5′cyclic AMP (cAMP)-dependent protein kinase (PKA) type I regulatory subunit isoform α (RIα) and phosphodiesterase (PDE) PDE4D have both been implicated in impaired PKA regulation in acrodysostosis. How mutations on PDEs and RIα interfere with the regulation of cAMP-PKA signaling is not understood. cAMP-PKA signaling can be described in two phases. In the activation phase, cAMP binding to RIα dissociates the free C-subunit (Catalytic subunit). PDEs hydrolyze cAMP bound to RIα, priming the cAMP-free RIα for reassociation with the C-subunit, thereby completing one PKA activation cycle. Signal termination is thus critical for resetting PKA to its basal state and promoting adaptation to hormonal hyperstimulation. This proceeds through formation of a transient signal termination RIα: PDE complex that facilitates cAMP channeling from the cAMP-binding domain of RIα to the catalytic site of PDE. Signal termination of cAMP-PKA proceeds in three steps: Step 1) Channeling: translocation of cAMP from the CNB of RIα to the PDE catalytic site for hydrolysis. Step 2) Processivity: binding of free cAMP from the cytosol at both CNBs of RIα. Step 3) Product (5′AMP) release from the PDE hydrolysis site through competitive displacement by a new molecule of cAMP that triggers subsequent activation cycles of PKA. We have identified the molecular basis for two acrodysostosis mutants, PDE (PDE8 T690P) and RIα (T207A), that both allosterically impair cAMP-PKA signal termination. A combination of amide hydrogen/deuterium exchange mass spectrometry (HDXMS) and fluorescence polarization (FP) reveals that PDE8 T690P and RIα T207A both blocked processive hydrolysis of cAMP by interfering with competitive displacement of product 5′AMP release from the nucleotide channel at the end of each round of cAMP hydrolysis. While T690P blocked product 5′AMP release from the PDE, T207A greatly slowed the release of the substrate from RIα. These results highlight the role of processivity in cAMP hydrolysis by RIα: PDE termination complexes for adaptation to cAMP from GPCR hyperstimulation. Impairment of the signal termination process provides an alternate molecular basis for acrodysostosis.</p

DataSheet1_Impaired cAMP processivity by phosphodiesterase-protein kinase A complexes in acrodysostosis.PDF

Acrodysostosis represents a group of rare genetic disorders characterized by defective skeletal development and is often accompanied by intellectual disabilities. Mutations in the 3′5′cyclic AMP (cAMP)-dependent protein kinase (PKA) type I regulatory subunit isoform α (RIα) and phosphodiesterase (PDE) PDE4D have both been implicated in impaired PKA regulation in acrodysostosis. How mutations on PDEs and RIα interfere with the regulation of cAMP-PKA signaling is not understood. cAMP-PKA signaling can be described in two phases. In the activation phase, cAMP binding to RIα dissociates the free C-subunit (Catalytic subunit). PDEs hydrolyze cAMP bound to RIα, priming the cAMP-free RIα for reassociation with the C-subunit, thereby completing one PKA activation cycle. Signal termination is thus critical for resetting PKA to its basal state and promoting adaptation to hormonal hyperstimulation. This proceeds through formation of a transient signal termination RIα: PDE complex that facilitates cAMP channeling from the cAMP-binding domain of RIα to the catalytic site of PDE. Signal termination of cAMP-PKA proceeds in three steps: Step 1) Channeling: translocation of cAMP from the CNB of RIα to the PDE catalytic site for hydrolysis. Step 2) Processivity: binding of free cAMP from the cytosol at both CNBs of RIα. Step 3) Product (5′AMP) release from the PDE hydrolysis site through competitive displacement by a new molecule of cAMP that triggers subsequent activation cycles of PKA. We have identified the molecular basis for two acrodysostosis mutants, PDE (PDE8 T690P) and RIα (T207A), that both allosterically impair cAMP-PKA signal termination. A combination of amide hydrogen/deuterium exchange mass spectrometry (HDXMS) and fluorescence polarization (FP) reveals that PDE8 T690P and RIα T207A both blocked processive hydrolysis of cAMP by interfering with competitive displacement of product 5′AMP release from the nucleotide channel at the end of each round of cAMP hydrolysis. While T690P blocked product 5′AMP release from the PDE, T207A greatly slowed the release of the substrate from RIα. These results highlight the role of processivity in cAMP hydrolysis by RIα: PDE termination complexes for adaptation to cAMP from GPCR hyperstimulation. Impairment of the signal termination process provides an alternate molecular basis for acrodysostosis.</p

Table3_Impaired cAMP processivity by phosphodiesterase-protein kinase A complexes in acrodysostosis.XLSX

Acrodysostosis represents a group of rare genetic disorders characterized by defective skeletal development and is often accompanied by intellectual disabilities. Mutations in the 3′5′cyclic AMP (cAMP)-dependent protein kinase (PKA) type I regulatory subunit isoform α (RIα) and phosphodiesterase (PDE) PDE4D have both been implicated in impaired PKA regulation in acrodysostosis. How mutations on PDEs and RIα interfere with the regulation of cAMP-PKA signaling is not understood. cAMP-PKA signaling can be described in two phases. In the activation phase, cAMP binding to RIα dissociates the free C-subunit (Catalytic subunit). PDEs hydrolyze cAMP bound to RIα, priming the cAMP-free RIα for reassociation with the C-subunit, thereby completing one PKA activation cycle. Signal termination is thus critical for resetting PKA to its basal state and promoting adaptation to hormonal hyperstimulation. This proceeds through formation of a transient signal termination RIα: PDE complex that facilitates cAMP channeling from the cAMP-binding domain of RIα to the catalytic site of PDE. Signal termination of cAMP-PKA proceeds in three steps: Step 1) Channeling: translocation of cAMP from the CNB of RIα to the PDE catalytic site for hydrolysis. Step 2) Processivity: binding of free cAMP from the cytosol at both CNBs of RIα. Step 3) Product (5′AMP) release from the PDE hydrolysis site through competitive displacement by a new molecule of cAMP that triggers subsequent activation cycles of PKA. We have identified the molecular basis for two acrodysostosis mutants, PDE (PDE8 T690P) and RIα (T207A), that both allosterically impair cAMP-PKA signal termination. A combination of amide hydrogen/deuterium exchange mass spectrometry (HDXMS) and fluorescence polarization (FP) reveals that PDE8 T690P and RIα T207A both blocked processive hydrolysis of cAMP by interfering with competitive displacement of product 5′AMP release from the nucleotide channel at the end of each round of cAMP hydrolysis. While T690P blocked product 5′AMP release from the PDE, T207A greatly slowed the release of the substrate from RIα. These results highlight the role of processivity in cAMP hydrolysis by RIα: PDE termination complexes for adaptation to cAMP from GPCR hyperstimulation. Impairment of the signal termination process provides an alternate molecular basis for acrodysostosis.</p

Table4_Impaired cAMP processivity by phosphodiesterase-protein kinase A complexes in acrodysostosis.XLSX

Acrodysostosis represents a group of rare genetic disorders characterized by defective skeletal development and is often accompanied by intellectual disabilities. Mutations in the 3′5′cyclic AMP (cAMP)-dependent protein kinase (PKA) type I regulatory subunit isoform α (RIα) and phosphodiesterase (PDE) PDE4D have both been implicated in impaired PKA regulation in acrodysostosis. How mutations on PDEs and RIα interfere with the regulation of cAMP-PKA signaling is not understood. cAMP-PKA signaling can be described in two phases. In the activation phase, cAMP binding to RIα dissociates the free C-subunit (Catalytic subunit). PDEs hydrolyze cAMP bound to RIα, priming the cAMP-free RIα for reassociation with the C-subunit, thereby completing one PKA activation cycle. Signal termination is thus critical for resetting PKA to its basal state and promoting adaptation to hormonal hyperstimulation. This proceeds through formation of a transient signal termination RIα: PDE complex that facilitates cAMP channeling from the cAMP-binding domain of RIα to the catalytic site of PDE. Signal termination of cAMP-PKA proceeds in three steps: Step 1) Channeling: translocation of cAMP from the CNB of RIα to the PDE catalytic site for hydrolysis. Step 2) Processivity: binding of free cAMP from the cytosol at both CNBs of RIα. Step 3) Product (5′AMP) release from the PDE hydrolysis site through competitive displacement by a new molecule of cAMP that triggers subsequent activation cycles of PKA. We have identified the molecular basis for two acrodysostosis mutants, PDE (PDE8 T690P) and RIα (T207A), that both allosterically impair cAMP-PKA signal termination. A combination of amide hydrogen/deuterium exchange mass spectrometry (HDXMS) and fluorescence polarization (FP) reveals that PDE8 T690P and RIα T207A both blocked processive hydrolysis of cAMP by interfering with competitive displacement of product 5′AMP release from the nucleotide channel at the end of each round of cAMP hydrolysis. While T690P blocked product 5′AMP release from the PDE, T207A greatly slowed the release of the substrate from RIα. These results highlight the role of processivity in cAMP hydrolysis by RIα: PDE termination complexes for adaptation to cAMP from GPCR hyperstimulation. Impairment of the signal termination process provides an alternate molecular basis for acrodysostosis.</p

Table2_Impaired cAMP processivity by phosphodiesterase-protein kinase A complexes in acrodysostosis.XLSX

Acrodysostosis represents a group of rare genetic disorders characterized by defective skeletal development and is often accompanied by intellectual disabilities. Mutations in the 3′5′cyclic AMP (cAMP)-dependent protein kinase (PKA) type I regulatory subunit isoform α (RIα) and phosphodiesterase (PDE) PDE4D have both been implicated in impaired PKA regulation in acrodysostosis. How mutations on PDEs and RIα interfere with the regulation of cAMP-PKA signaling is not understood. cAMP-PKA signaling can be described in two phases. In the activation phase, cAMP binding to RIα dissociates the free C-subunit (Catalytic subunit). PDEs hydrolyze cAMP bound to RIα, priming the cAMP-free RIα for reassociation with the C-subunit, thereby completing one PKA activation cycle. Signal termination is thus critical for resetting PKA to its basal state and promoting adaptation to hormonal hyperstimulation. This proceeds through formation of a transient signal termination RIα: PDE complex that facilitates cAMP channeling from the cAMP-binding domain of RIα to the catalytic site of PDE. Signal termination of cAMP-PKA proceeds in three steps: Step 1) Channeling: translocation of cAMP from the CNB of RIα to the PDE catalytic site for hydrolysis. Step 2) Processivity: binding of free cAMP from the cytosol at both CNBs of RIα. Step 3) Product (5′AMP) release from the PDE hydrolysis site through competitive displacement by a new molecule of cAMP that triggers subsequent activation cycles of PKA. We have identified the molecular basis for two acrodysostosis mutants, PDE (PDE8 T690P) and RIα (T207A), that both allosterically impair cAMP-PKA signal termination. A combination of amide hydrogen/deuterium exchange mass spectrometry (HDXMS) and fluorescence polarization (FP) reveals that PDE8 T690P and RIα T207A both blocked processive hydrolysis of cAMP by interfering with competitive displacement of product 5′AMP release from the nucleotide channel at the end of each round of cAMP hydrolysis. While T690P blocked product 5′AMP release from the PDE, T207A greatly slowed the release of the substrate from RIα. These results highlight the role of processivity in cAMP hydrolysis by RIα: PDE termination complexes for adaptation to cAMP from GPCR hyperstimulation. Impairment of the signal termination process provides an alternate molecular basis for acrodysostosis.</p

Table1_Impaired cAMP processivity by phosphodiesterase-protein kinase A complexes in acrodysostosis.XLSX

Acrodysostosis represents a group of rare genetic disorders characterized by defective skeletal development and is often accompanied by intellectual disabilities. Mutations in the 3′5′cyclic AMP (cAMP)-dependent protein kinase (PKA) type I regulatory subunit isoform α (RIα) and phosphodiesterase (PDE) PDE4D have both been implicated in impaired PKA regulation in acrodysostosis. How mutations on PDEs and RIα interfere with the regulation of cAMP-PKA signaling is not understood. cAMP-PKA signaling can be described in two phases. In the activation phase, cAMP binding to RIα dissociates the free C-subunit (Catalytic subunit). PDEs hydrolyze cAMP bound to RIα, priming the cAMP-free RIα for reassociation with the C-subunit, thereby completing one PKA activation cycle. Signal termination is thus critical for resetting PKA to its basal state and promoting adaptation to hormonal hyperstimulation. This proceeds through formation of a transient signal termination RIα: PDE complex that facilitates cAMP channeling from the cAMP-binding domain of RIα to the catalytic site of PDE. Signal termination of cAMP-PKA proceeds in three steps: Step 1) Channeling: translocation of cAMP from the CNB of RIα to the PDE catalytic site for hydrolysis. Step 2) Processivity: binding of free cAMP from the cytosol at both CNBs of RIα. Step 3) Product (5′AMP) release from the PDE hydrolysis site through competitive displacement by a new molecule of cAMP that triggers subsequent activation cycles of PKA. We have identified the molecular basis for two acrodysostosis mutants, PDE (PDE8 T690P) and RIα (T207A), that both allosterically impair cAMP-PKA signal termination. A combination of amide hydrogen/deuterium exchange mass spectrometry (HDXMS) and fluorescence polarization (FP) reveals that PDE8 T690P and RIα T207A both blocked processive hydrolysis of cAMP by interfering with competitive displacement of product 5′AMP release from the nucleotide channel at the end of each round of cAMP hydrolysis. While T690P blocked product 5′AMP release from the PDE, T207A greatly slowed the release of the substrate from RIα. These results highlight the role of processivity in cAMP hydrolysis by RIα: PDE termination complexes for adaptation to cAMP from GPCR hyperstimulation. Impairment of the signal termination process provides an alternate molecular basis for acrodysostosis.</p

In situ dipteran larva abundances

In situ observations of the dipteran larvae inhabiting pitchers of Nepenthes gracilis at three different localities in Singapor

Digestion experiments

Digestion experiment

Fragments 1 and 2 differ in the nature of the allosteric effect in Hsp90.

<p><b>(A)</b> The absolute difference in numbers of deuterons (inferred from difference in mass in Daltons (Da) (y-axis) between the free and ligand bound state is plotted for each pepsin digest fragment listed from the N to C terminus (x-axis) of Hsp90 for each deuterium exchange time point (t = 0.5, 2, 5, 10 min) in a ‘difference plot’. Shifts in the positive scale represent decreases in deuterium exchange and shifts in the negative scale represent increases in deuterium exchange when compared to the apo-Hsp90. Regions showing significant differences above a threshold of 0.5 Da (red dashed line) are compared with orthosteric sites (blue boxes) to establish allosteric regions (red boxed). Fragment <b>2</b> does not show any changes in region A4, similar to 17-AAG, while fragment <b>1</b> shows differences, similar to Radicicol. In addition, fragment <b>1</b> shows an allosteric response at the regions A5 (residues 201–213 shown in orange box), which is not observed in the other three ligands. Time points are colored according to key. <b>(B,C)</b> The identified orthosteric (blue) and allosteric regions (red) for fragments are mapped on to the structure of Hsp90 in blue. <b>(C)</b> The allosteric site A5 in Hsp90, which is observed only fragment <b>2</b> is highlighted in orange. Radicicol bound at the ligand binding pocket is shown as sticks (PDB ID: 4EGK).</p