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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
Author(s) -
Lee Ko On,
Kim EunHee,
Kim Gowoon,
Jung Jea Yeon,
Katayama Shigeru,
Nakamura Soichiro,
Suh JeongYong
Publication year - 2016
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.2988
Subject(s) - pep group translocation , linker , biochemistry , chemistry , biophysics , allosteric regulation , enzyme , stereochemistry , escherichia coli , biology , gene , computer science , operating system
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.