The motional dynamics and solvent-exchange behavior of free and DNA-bound forms of the minimal zinc-finger DNA-binding domain of the yeast transcription factor ADR1 (ADR1-DBD) are investigated using NMR. The parameters measured include the 1H-15N heteronuclear NOE, 15N and 1H T1 relaxation rates, 15N T2 relaxation rates, and solvent-exchange rates. The spin relaxation parameters, spectral density maps, and solvent-exchange behavior show that, exclusive of the N and C termini, three distinct regions of free ADR1-DBD exhibit different motions on multiple timescales. The N-terminal proximal, or accessory, region appears to be unstructured and highly flexible: it exhibits large amplitude motions on a picosecond timescale, little or no protection from solvent exchange, and random-coil proton chemical shifts. The two zinc fingers tumble anisotropically as folded domains, with the tumbling of the individual fingers being only partly correlated to each other, and are modestly protected from solvent exchange except near the tips of the fingers and in the linker joining them. Free ADR1-DBD exhibits exchange broadening around P97 in the proximal region, at the tip of finger 1, and throughout finger 2. Upon binding, most of the proximal region and both zinc fingers tumble as a single domain and exhibit significantly reduced picosecond timescale motions. This region becomes more protected from solvent exchange. The bound portion of the proximal region is proposed to lie exposed on the surface of the DNA. Exchange broadening remains around P97 but also becomes evident for residues in direct contact with the DNA and in the linker. We conclude that the region of ADR1-DBD essential for high-affinity binding undergoes a disorder-to-order transition upon binding to its cognate DNA and, together with the zinc fingers, forms a cohesive molecular complex with the nucleic acid.

• PMID: 9642072
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Journal Article | Research Support, Non-U.S. Gov't | Research Support, U.S. Gov't, P.H.S.

Authors

Hyre DE, Klevit RE

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