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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

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          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.

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          Author and article information

          Contributors
          jysuh@snu.ac.kr
          Journal
          Protein Sci
          Protein Sci
          10.1002/(ISSN)1469-896X
          PRO
          Protein Science : A Publication of the Protein Society
          John Wiley and Sons Inc. (Hoboken )
          0961-8368
          1469-896X
          01 August 2016
          October 2016
          : 25
          : 10 ( doiID: 10.1002/pro.v25.10 )
          : 1803-1811
          Affiliations
          [ 1 ] Department of Agricultural Biotechnology Seoul National University 1 Gwanak‐Ro, Gwanak‐Gu Seoul 151‐742 Republic of Korea
          [ 2 ] Division of Magnetic Resonance Korea Basic Science Institute Ochang Chungbuk 363‐883 Republic of Korea
          [ 3 ] Department of Bioscience and Biotechnology Shinshu University, Ina Nagano 399‐4598 Japan
          [ 4 ] Institute for Biomedical Sciences, Interdisciplinary Cluster for Cutting Edge Research, Shinshu University Matsumoto Nagano 390‐8621 Japan
          Author notes
          [* ]Correspondence to: Jeong‐Yong Suh, Department of Agricultural Biotechnology, Seoul National University, 1 Gwanak‐ro, Gwanak‐gu, Seoul 151‐742, Korea. E‐mail: jysuh@ 123456snu.ac.kr
          Article
          PMC5029529 PMC5029529 5029529 PRO2988
          10.1002/pro.2988
          5029529
          27438678
          © 2016 The Protein Society
          Page count
          Figures: 4, Tables: 1, Pages: 9, Words: 4505
          Funding
          Funded by: National Research Foundation of Korea (NRF)
          Award ID: 2013R1A1A2010856
          Funded by: Cooperative Research Program for Agriculture Science & Technology Development, Rural Development Administration
          Award ID: PJ011112016
          Funded by: New & Renewable Energy Core Technology Program of the Korea Institute of Energy Technology Evaluation and Planning (KETEP) granted financial resource from the Ministry of Trade, Industry & Energy, Republic of Korea
          Award ID: 20143030090940
          Categories
          Article
          Articles
          Custom metadata
          2.0
          pro2988
          October 2016
          Converter:WILEY_ML3GV2_TO_NLMPMC version:4.9.4 mode:remove_FC converted:20.09.2016

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