6MI6

STRUCTURE OF CHEA DOMAIN P4 IN COMPLEX WITH AN ADP ANALOG

Summary for 6MI6

• Entry DOI: 10.2210/pdb6mi6/pdb
• Descriptor: Chemotaxis protein CheA, SULFATE ION, 5'-O-[(S)-hydroxy{[(S)-hydroxy{[(1-hydroxy-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl]disulfanyl}phosphoryl]oxy}phosphoryl]adenosine, ... (4 entities in total)
• Functional Keywords: bacterial chemotaxis, histidine kinase, thermotoga maritima, signaling protein
• Biological source: Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
• Total number of polymer chains: 4
• Total formula weight: 86360.72

Authors

Crane, B.R.,Muok, A.R.,Chua, T.K.,Le, H. (deposition date: 2018-09-19, release date: 2018-12-05, Last modification date: 2024-12-25)

Primary citation

Muok, A.R.,Chua, T.K.,Le, H.,Crane, B.R.
Nucleotide Spin Labeling for ESR Spectroscopy of ATP-Binding Proteins.
Appl.Magn.Reson., 49:1385-1395, 2018

PubMed Abstract: Site-directed spin labeling of proteins by chemical modification of engineered cysteine residues with the molecule MTSSL (1-Oxyl-2,2,5,5-tetramethylpyrroline-3-methyl methanethiosulfonate) has been an invaluable tool for conducting double electron electron resonance (DEER) spectroscopy experiments. However, this method is generally limited to recombinant proteins with a limited number of reactive Cys residues that when modified will not impair protein function. Here we present a method that allows for spin-labeling of protein nucleotide binding sites by adenosine diphosphate (ADP) modified with a nitroxide moiety on the β-phosphate (ADP-β-S-SL). The synthesis of this ADP analog is straightforward and isolation of pure product is readily achieved on a standard reverse-phase high-performance liquid chromatography (HPLC) system. Furthermore, analyses of isolated ADP-β-S-SL by LC-mass spectrometry confirm that the molecule is extremely stable under ambient conditions. The crystal structure of ADP-β-S-SL bound to the ATP pocket of the histidine kinase CheA reveals specific targeting of the probe, whose nitroxide moiety is mobile on the protein surface. Continuous wave and pulsed ESR measurements demonstrate the capability of ADP-β-S-SL to report on active site environment and provide reliable DEER distance constraints.

PubMed: 30686862
DOI: 10.1007/s00723-018-1070-6

Experimental method

X-RAY DIFFRACTION (2.95 Å)