title: Biophysical characterization of the domain association between cytosolic A and B domains of the mannitol transporter enzymes II(Mtl) in the presence and absence of a connecting linker.
abstract: The mannitol transporter enzyme II(Mtl) of the bacterial phosphotransferase system is a multi-domain protein that catalyzes mannitol uptake and phosphorylation. Here we investigated the domain association between cytosolic A and B domains of enzyme II(Mtl) , which are natively connected in Escherichia coli, but separated in Thermoanaerobacter tengcongensis. NMR backbone assignment and residual dipolar couplings indicated that backbone folds were well conserved between the homologous domains. The equilibrium binding of separately expressed domains, however, exhibited ∼28-fold higher affinity compared to the natively linked ones. Phosphorylation of the active site loop significantly contributed to the binding by reducing conformational dynamics at the binding interface, and a few key mutations at the interface were critical to further stabilize the complex by hydrogen bonding and hydrophobic interactions. The affinity increase implicated that domain associations in cell could be maintained at an optimal level regardless of the linker.
authors.affiliation: Department of Agricultural Biotechnology, Seoul National University, 1 Gwanak-Ro, Gwanak-Gu, Seoul, 151-742, Republic of Korea.
authors.forename: Ko On
authors.initials: KO
authors.lastname: Lee
authors.affiliation: Division of Magnetic Resonance, Korea Basic Science Institute, Ochang, Chungbuk, 363-883, Republic of Korea.
authors.forename: Eun-Hee
authors.initials: EH
authors.lastname: Kim
authors.affiliation: Department of Agricultural Biotechnology, Seoul National University, 1 Gwanak-Ro, Gwanak-Gu, Seoul, 151-742, Republic of Korea.
authors.forename: Gowoon
authors.initials: G
authors.lastname: Kim
authors.affiliation: Department of Agricultural Biotechnology, Seoul National University, 1 Gwanak-Ro, Gwanak-Gu, Seoul, 151-742, Republic of Korea.
authors.forename: Jea Yeon
authors.initials: JY
authors.lastname: Jung
authors.affiliation: Department of Bioscience and Biotechnology, Shinshu University, Ina, Nagano, 399-4598, Japan.|Institute for Biomedical Sciences, Interdisciplinary Cluster for Cutting Edge Research, Shinshu University, Matsumoto, Nagano, 390-8621, Japan.
authors.forename: Shigeru
authors.initials: S
authors.lastname: Katayama
authors.affiliation: Department of Bioscience and Biotechnology, Shinshu University, Ina, Nagano, 399-4598, Japan.|Institute for Biomedical Sciences, Interdisciplinary Cluster for Cutting Edge Research, Shinshu University, Matsumoto, Nagano, 390-8621, Japan.
authors.forename: Soichiro
authors.initials: S
authors.lastname: Nakamura
authors.affiliation: Department of Agricultural Biotechnology, Seoul National University, 1 Gwanak-Ro, Gwanak-Gu, Seoul, 151-742, Republic of Korea. jysuh@snu.ac.kr.|Institute for Biomedical Sciences, Interdisciplinary Cluster for Cutting Edge Research, Shinshu University, Matsumoto, Nagano, 390-8621, Japan. jysuh@snu.ac.kr.
authors.forename: Jeong-Yong
authors.initials: JY
authors.lastname: Suh
mesh_terms: D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003600:Cytosol / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000072417:Protein Domains; D046968:Thermoanaerobacter / Q000201:enzymology* / Q000235:genetics
chemical_list: D002352:Carrier Proteins; D029968:Escherichia coli Proteins
keywords: NMR; domain association; domain interface; linker; multi-domain protein