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NMR dynamics distinguish between hard and soft hydrophobic cores in the DNA-binding domain of PhoB and demonstrate different roles of the cores in binding to DNA.

Okamura H, Makino K, Nishimura Y

Graduate School of Supramolecular Biology, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan.

The transcription factor PhoB contains an N-terminal regulatory domain and a C-terminal DNA-binding/transactivation domain. The DNA-binding/transactivation domain alone can bind specifically to DNA and consequently activate transcription. It consists of an N-terminal four-stranded beta-sheet and a winged helix domain, containing a three-helix bundle and a C-terminal beta-hairpin. The second and third helices, together with the beta-hairpin, contact DNA and the loop between the second and third helices is responsible for the transactivation. Here, we have examined the backbone and side-chain dynamics of the DNA-binding domain in its DNA-free and bound forms by NMR. The side-chain dynamics identified two apparent hydrophobic cores: one, a soft hydrophobic core, shows inherently flexible dynamics on the pico-to nanosecond timescale and maintains the DNA-binding and transactivation surfaces; the other is a hard hydrophobic core formed between the N-terminal beta-sheet and the three-helix bundle, which maintains the other non-functional surface. Upon binding to DNA, the flexibility of the soft core decreases but remains more flexible than the hard core. The winged helix domain itself has inherent flexibility in the DNA-binding and transactivation functions. However, the back surface of both functional surfaces seems to be covered by the N-terminal beta-sheet in order to mask a possible function arising from the inherent flexibility of the winged helix domain.

Published 19 March 2007 in J Mol Biol, 367(4): 1093-117.
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