A Computational Investigation of Histone‐Deacetylase‐6’s role in Osteoclast Dislodgement from Bone

Human Bone constantly undergoes reconstruction via the actions of osteoclasts, which degrade old bone using powerful enzymes such as Tartrate-Resistant Acid Phosphatase. When osteoclasts adhere to the bone surface, they form sealing zones that maintain cellular attachment, thereby allowing for continuous digestion. After the process has been completed, osteoclasts dislodge before migrating to a new location. This study focuses on the molecular mechanisms underlying the physiology of osteoclast disengagement via the action of histone deacetylase 6 (HDAC6), a large protein involved in alpha-tubulin de-acetylation, podosome degradation, and enhanced cellular motility. Unlike other HDAC proteins, HDAC6 contains a second catalytic domain with deacetylase activity. Ras Homolog Family member A (RhoA) and Diaphanous-related formin-2 (mDia2) form a dimer (RhoA-mDia2) that allosterically activates HDAC6. Upon stimulation by RhoA-mDia2, HDAC6 targets alpha-tubulin, and by removing the acetyl-moiety on Lys-40, it enhances its susceptibility to ubiquitination. As microtubule integrity diminishes, the adherence of the osteoclast is weakened. While the details of the molecular mechanism of HDAC6 deacetylation have been elucidated, the molecular mechanism of its allosteric activation remains unexplored. This study focuses on RhoA-mDia2 stimulation of HDAC6, and the determination of which catalytic domain might be the major player in the process. Using computational tools, we have investigated the differential response of the first deacetylase domain (DD1) and the second deacetylase domain (DD2) to RhoA-mDia2 stimulation. Using the known structures of RhoA, mDia2, HDAC6, and Alpha-Tubulin, we performed protein-protein docking to explore the structure-function relationship of the protein players participating in the deacetylation process. Here, we present our investigations regarding the structural details and catalytic implications of the allosteric stimulation of HDAC6 by the RhoA-mDia2 dimer based on our docking results. We detail a functional comparison between the first and second deacetylase domains of HDAC6 and show that HDAC6 DD1 predominates the binding interface with alpha-tubulin before RhoA-mDia2 allosteric activation, and the pattern reverses once RhoA-mDia2 binds HDAC6. We also explored the pathways of HDAC6 inhibition by Cdc42 and Rac1, both of which promote podosome stability. Lastly, we compared DD1 and DD2 of HDAC6 with the HDAC10 deacetylase domain, the closest homologue that disfavors acetyl lysine hydrolysis. A comprehensive sequence analysis combined with analysis of binding interfaces, and in silico mutagenesis experiments all indicate greater similarity between HDAC10's deacetylase domain and HDAC6 DD1 than with HDAC6 DD2. We therefore speculate that DD1 may not be conducive to alpha-tubulin lysine-40 deacetylation in a similar manner as HDAC10. Combining all our findings, we can reasonably conclude that HDAC6 DD2 likely preferentially attracts alpha-tubulin, whereas DD1 plays a regulatory function. This work is pivotal not only for understanding bone physiology, but the research could also provide valuable information for developing treatments of chronic pathologies such as osteoporosis.