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oai:figshare.com:article/34027252 2026-09-29T18:16:49Z category_1 category_4 category_8 category_12 category_13 category_14 category_19 category_46 category_734 category_61 category_64 category_132 portal_63 item_type_3 month_year_09_2026

Phosphorylation by the PknB Serine/Threonine Kinase Stimulates Dimer Formation by WalR Marina Suppi (11944902) Ian R. Monk (11338402) Liam K. R. Sharkey (22120444) Nichollas E. Scott (159935) Dweipayan Goswami (10880407) Aakash Natarajan (25138516) Stephanie Tan (200424) Katharine Myler (25138519) Sheila Marie Pimentel-Elardo (8080409) Timothy P. Stinear (143097) Sacha J. Pidot (7865252) Justin R. Nodwell (114148) Biophysics Biochemistry Microbiology Cell Biology Genetics Molecular Biology Pharmacology Immunology Biological Sciences not elsewhere classified Developmental Biology Cancer Infectious Diseases threonine residue 101 greatly increased susceptibility methionine conferred low likely involves phosphorylation t101 phosphate group walr dimerization interface threonine kinase pknb walr dimer formation t101m </ sub second kinase pknb </ walr dimer pknb serves pknb interacts vivo </ vitro </ silico </ tunicamycin along staphylococcus aureus similar experiments response regulators regulates genes receiver domain primary activation positive bacteria phenotypic traits living cells level resistance gc gram demonstrate similarities data supports data suggest critical proteins component system clearly phosphorylation bacillus subtilis WalKR is a two-component system that regulates cell wall homeostasis and other processes in low-GC Gram-positive bacteria. It is essential for viability in Staphylococcus aureus and regulates genes that encode the autolysins and other critical proteins. The sensor kinase WalK phosphorylates WalR at the aspartic acid residue 53 (D53) in the receiver domain, stimulating promoter DNA binding. Unlike most response regulators, WalR is reported to have a second kinase, the serine/threonine kinase PknB, which phosphorylates the receiver domain at the threonine residue 101. We previously reported that a walR mutation that changed T101 to a methionine conferred low-level resistance to vancomycin and greatly increased susceptibility to tunicamycin along with several other phenotypic traits. In this work, we demonstrate similarities between pknB null and the WalRT101M mutants. A combination of in vitro and in vivo data supports the idea that PknB interacts with WalR in living cells (consistent with similar experiments in Bacillus subtilis) and that this likely involves phosphorylation of WalR at T101. Using in silico modeling, we identified a possible intermolecular hydrogen bond between the T101 phosphate group and E108. This would have the effect of stabilizing the WalR dimerization interface. Consistent with this, we find that T101 is important for WalR dimer formation in vivo. The primary activation of WalR is clearly phosphorylation at D53 by WalK. However, our data suggest that PknB serves as a second potentiating input stabilizing the WalR dimer. 2026-09-29T00:00:00Z Dataset Dataset 10.1021/acs.biochem.6c00334.s007 https://figshare.com/articles/dataset/Phosphorylation_by_the_PknB_Serine_Threonine_Kinase_Stimulates_Dimer_Formation_by_WalR/34027252 CC BY-NC 4.0