The Three-dimensional Structure of Two Mutants of the Signal Transduction Protein CheY Suggest its Molecular Activation Mechanism |
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| Institution: | 1. Departament de Biologia Molecular i Cel·lular, Centre d''Investigació i Desenvolupament-CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain;2. European Molecular Biology Laboratory, Meyerhofstrasse 1, 6900 Heidelberg, Germany;1. National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences (CEMPS), Institute of Plant Physiology and Ecology (SIPPE), Chinese Academy of Sciences (CAS), Shanghai 200032, China;2. School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China;3. Key Laboratory of Plant Carbon Capture, CAS, Shanghai 200032, China;4. New Cornerstone Science Laboratory, Shanghai 200032, China;1. National Key Laboratory of Plant Molecular Genetics (NKLPMG), CAS Center for Excellence in Molecular Plant Sciences (CEMPS), Institute of Plant Physiology and Ecology (SIPPE), Chinese Academy of Sciences (CAS), Shanghai 200032, China;2. School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China;3. University of Chinese Academy of Sciences, Shanghai 200032, China;4. Core Facility Center of CEMPS, SIPPE, CAS, Shanghai 200032, China;1. LBLGC, Université d’Orléans, INRAE, USC1328, 45067, Orléans, Cedex 2, France;2. Biomolécules et Biotechnologies Végétales (BBV), EA 2106, Université de Tours, 31 Avenue Monge, 37200, Tours, France;1. Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan;2. Chemical Biology and Molecular Biophysics, Taiwan International Graduate Program, Academia Sinica, Taipei 115, Taiwan;3. Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu 300, Taiwan;4. Genome and Systems Biology Degree Program, National Taiwan University and Academia Sinica, Taipei 106, Taiwan;5. Department of Agricultural Chemistry, National Taiwan University, Taipei 106, Taiwan |
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| Abstract: | The three-dimensional crystal structures of the single mutant M17G and the triple mutant F14G-S15G-M17G of the response regulator protein CheY have been determined to 2.3 and 1.9 Å, respectively. Both mutants bind the essential Mg2+cation as determined by the changes in stability, but binding does not cause the intrinsic fluorescence quenching of W58 observed in the wild-type protein. The loop β4-α4 appears to be very flexible in both mutants and helix α4, which starts at N94 in the native Mg2+-CheY and at K91 in the native apo-CheY, starts in both mutants at residue K92. The side-chain of K109 appears to be more mobile because of the space freed by the M17G mutation. In the triple mutant the main chain of K109 and adjacent residues (loop β5-α5) is displaced almost by 2Å affecting the main chain at residues T87 to E89 (C terminus of β4). The triple mutant structure has a Mg2+bound at the active site, but although the Mg2+coordination is similar to that of the native Mg2+-CheY, the structural consequences of the metal binding are quite different. It seems that the mutations have disrupted the mechanism of movement transmission observed in the native protein. We suggest that the side-chain of K109, packed between V86, A88 and M17 in the native protein, slides forwards and backwards upon activation and deactivation dragging the main chain at the loop β5-α5 and triggering larger movements at the functional surface of the protein. |
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