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Important Interactions Promoting Recognition of Peptide Ligands at the Binding Pocket of the PDZ Signaling Domain of AF6
Afadin (AF6) is a scaffolding protein involved in the formation of protein complexes at tight and adherens junctions. AF6 plays vital roles in many cellular processes, such as structural development, cellular organization, directional cell movement, and polarity. The PDZ domain participates in multiple signaling networks to initiate and maintain cellular polarity, to facilitate protein trafficking, and allow neuronal communications; thus, misregulated PDZ domain-dependent pathways can induce cells to become cancerous. AF6 interacts with many other proteins via its PDZ signaling domain and exhibits multimodal specificity.
The PDZ signaling domain of AF6 (AF6_PDZ) binds predominantly hydrophobic peptide ligands. A biological array binding study has shown that AF6_PDZ can also recognize peptide ligands that contain diverse features compared to those of cognate sequence. The plasticity of the AF6_PDZ to recognize chemically diverse ligands raises questions regarding how AF6_PDZ achieves specificity in peptide recognition, as well as which binding pocket interactions are important for peptide binding.
Structures of the AF6_PDZ with several biological peptide ligands highlight intermolecular interactions that stabilize these ligands at the binding pocket. We hypothesize that these interactions play a key role in recruiting the ligand to a “hot spot” on the AF6_PDZ surface and promote binding. This hypothesis predicts that changing the interactions by introducing mutations into the peptide sequence will alter the peptide binding affinity. The protein-peptide interactions in this project were studied predominantly with 1H-15N-heteronuclear single quantum coherence nuclear magnetic resonance (HSQC NMR) titration.
We selected two non-cognate peptides suspected of binding to AF6_PDZ but found that these peptides only weakly interact with AF6_PDZ. Based on this result, we suggest that AF6_PDZ cannot recognize peptide with a deviated residue at the extreme C-terminal position of the peptide (P0). In specific, an arginine or a proline amino acid is likely not preferable residue at this position of the peptide. Using knowledge-based approach, we introduced a valine at the P0 of the peptide, successfully restoring binding. This result highlights the crucial role of the valine in promote binding. The disassociation constants KD for binding interaction of AF6_PDZ with cognate peptide (Class I ligand) and designed peptide (Class II ligand) are 154 µM and 26.4 µM respectively, and these values are consistent with what others have observed previously. We also studied shifted motif ligands, in which we investigated whether AF6_PDZ can accommodate ligands with an additional C-terminal residue to the P0 valine. We conclude that the AF6_PDZ does not have the ability to recognize these peptides, and the shifted motif binding mode is likely not relevant for AF6_PDZ in biological environment. Comparison of available structures reveals the possibility that αA helix of AF6_PDZ may impose some structural constraint, and thereby inhibit the protein recognition of shifted motif peptides. We also suggest that the list of the top 100 binding sequences from the peptide screening experiment does not reflect all biological binders; thus, this list needs to be used with precaution in future experiments. Altogether, we confirm the important interactions that the valine at P0 forms at the binding pocket and these interactions help targeting the peptide to the binding pocket to promote binding. This finding will aid scientists in designing therapeutic drugs that targets the AF6_PDZ, in specific, and PDZ-domain containing proteins, in generally
Preemptive analgesic effect of intrathecal applications of neuroactive steroids in a rodent model of post-surgical pain: Evidence for the role of T-type calcium channels
Preemptive management of post-incisional pain remains challenging. Here, we examined the role of preemptive use of neuroactive steroids with activity on low-voltage activated T-type C
Preemptive Analgesic Effect of Intrathecal Applications of Neuroactive Steroids in a Rodent Model of Post-Surgical Pain: Evidence for the Role of T-Type Calcium Channels
Preemptive management of post-incisional pain remains challenging. Here, we examined the role of preemptive use of neuroactive steroids with activity on low-voltage activated T-type Ca2+ channels (T-channels) and γ-aminobutyric acid A (GABAA) receptors in the development and maintenance of post-incisional pain. We use neuroactive steroids with distinct effects on GABAA receptors and/or T-channels: Alphaxalone (combined GABAergic agent and T-channel inhibitor), ECN (T-channel inhibitor), CDNC24 (GABAergic agent), and compared them with an established analgesic, morphine (an opioid agonist without known effect on either T-channels or GABAA receptors). Adult female rats sustained the skin and muscle incision on the plantar surface of the right paw. We injected the agents of choice intrathecally either before or after the development of post-incisional pain. The pain development was monitored by studying mechanical hypersensitivity. Alphaxalone and ECN, but not morphine, are effective in alleviating mechanical hyperalgesia when administered preemptively whereas morphine provides dose-dependent pain relief only when administered once the pain had developed. CDNC24 on the other hand did not offer any analgesic benefit. Neuroactive steroids that inhibit T-currents—Alphaxalone and ECN—unlike morphine, are effective preemptive analgesics that may offer a promising therapeutic approach to the treatment of post-incisional pain, especially mechanical hypersensitivity