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One of the missense mutations identified in patients with osteoclast\u2010poor autosomal recessive osteopetrosis (ARO) is located in residue methionine 199 that is replaced with lysine (M199K) amid the TNF\u2010like core domain. However, the structure\u2013function relationship of this mutation is not clear. Sequence\u2010based alignment revealed that the fragment containing human M199 is highly conserved and equivalent to M200 in rat. Using site\u2010directed mutagenesis, we generated three recombinant RANKL mutants M200K\/A\/E (M200s) by replacing the methionine 200 with lysine (M200K), alanine (M200A), and glutamic acid (M200E), representative of distinct physical properties. TRAcP\u00a0staining and bone pit assay showed that M200s failed to support osteoclast formation and bone resorption, accompanied by impaired osteoclast\u2010related signal transduction. However, no antagonistic effect was found in M200s against wild\u2010type rat RANKL. Analysis of the crystal structure of RANKL predicted that this methionine residue is located within the hydrophobic core of the protein, thus, likely to be crucial for protein folding and stability. Consistently, differential scanning fluorimetry analysis suggested that M200s were less stable. Western blot analysis analyses further revealed impaired RANKL trimerization by M200s. Furthermore, receptor\u2013ligand binding assay displayed interrupted interaction of M200s to its intrinsic receptors. Collectively, our studies revealed the molecular basis of human M199\u2010induced ARO and elucidated the indispensable role of rodent residue M200 (equivalent to human M199) for the RANKL function.<\/jats:p>","DOI":"10.1002\/jcp.30045","type":"journal-article","created":{"date-parts":[[2020,9,23]],"date-time":"2020-09-23T09:45:35Z","timestamp":1600854335000},"page":"2800-2816","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":24,"title":["A missense mutation sheds light on a novel structure\u2013function relationship of RANKL"],"prefix":"10.1002","volume":"236","author":[{"given":"Heng","family":"Qiu","sequence":"first","affiliation":[{"name":"Division of Regenerative Biology, School of Biomedical Sciences University of Western Australia Perth Western Australia 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