Biocomputational Analysis of ARAP1, a novel GTPase‐Activating‐Protein Involved in Osteoclast‐Mediated Bone Digestion

Abstract

RhoGTPases are important regulators of many cellular activities including cell proliferation, trafficking of proteins, DNA transcription, and apoptosis. RhoGTPases are also involved in bone remodeling, the continuous process of bone digestion, and formation that occurs during one's lifespan. Any abnormalities in these Rho Proteins can lead to physiological effects such as osteoporosis, characterized by porous bone, or osteopetrosis, characterized by highly-dense bone tissue. RhoA-GTP is responsible for disrupting sealing zone dynamics and promoting cell motility in osteoclasts. On the other hand, Cdc42-GTP and Rac1-GTP maintain osteoclast attachment to the bone surface, thereby allowing it to continue enzymatic degradation of bone. To employ RhoGTPases as potential drug targets, we need to further understand the complex physiology and control mechanisms for these G-proteins. ARAP1 has been shown to localize to the osteoclast sealing zones upon the release of phosphoinositide (3,4,5) trisphosphate, which binds to its first and third PH domains. ARAP1 belongs to the RhoGAP family of proteins and has a RhoGAP domain responsible for catalyzing the intrinsic GTPase activity of G proteins. However, it is still unknown which GTPase (i.e. RhoA, Cdc42, Rac1) preferentially binds to the ARAP1 catalytic domain. Delineating the substrate specificity for the RhoGAP domain is crucial to its characterization as a pro-migration or pro-digestion protein, allowing us to specifically target its function in managing osteoporosis or osteopetrosis-related illnesses. In this computational study, we have modeled the ARAP1 RhoGAP domain using homology modeling and ab initio techniques, characterized its biophysical features, and used docking analysis to scrutinize its interactions with potential substrates. The docking results suggest that ARAP1 shows a preferential substrate specificity for RhoA. This indicates that ARAP1-RhoGAP may behave in a similar manner as ARAP3-RhoGAP, which has been shown to prefer RhoA-targeting in an experimental setup. Lastly, we report an in silico mutagenesis analysis that induces RhoA-like mutations in Cdc42/Rac1 and provides structural data that supports our hypothesis of an ARAP1-RhoA preferential attraction. Overall, this study reveals new insights into the structure-function relations of the RhoGAP domain, specifically pertaining to its role in osteoclast-mediated bone digestion, using an integrated and comprehensive strategy of sequence and structure analyses. The novel findings from our study regarding the molecular mechanism of GTPases in the osteoclast lays the groundwork towards developing therapies that improve the quality of life for those suffering from chronic bone pathologies.