Ff gene 5 protein-d(pA)40-60 complex: proton NMR supports a localized base-binding model

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Abstract
The interaction between Ff gene 5 protein (G5P) and d(pA)40-60 serves as an improved model system for a 1H NMR examination of the G5P-ssDNA interface under cooperative binding conditions. Selective deuteriation of aromatic residues enables individual Tyr (3,5)H and (2,6)H resonances to be monitiored in spectra of high molecular weight nucleoprotein assemblies. Analysis of complexation-induced chemical shift changes and intermolecular NOEs indicates that Tyr 26 is the only tryosine to interact directly with ssDNA. Tyr 41, which is immobilized upon binding, is implicated in a dimer-dimer contact role. These and other NMR data are consistent with a previously outlined model of the protein-DNA interface in which Phe 73'', Leu 28, and Try 26 form components of a base-binding pocket or "dynamic clamp" fringed by a cluster of positively charged residues [King, G. C., and Coleman, J.E.(1987) Biochemistry 26, 2929-2937]. In the present version of this model, the Phe and Leu side chains are proposed to stack on either side of a single base, while there is the possibility that Tyr 26 may H-bound to the sugar-phosphate backbone in addition to or instead of stacking. Chemical-exchange effects underscore the dynamic nature of binding at the pocket. A comparison of d(pA)40-60 and oligo(dA)-induced chemical shift changes suggests that polyand oligonucleotide complexes have indistinguishable base-binding loci but appear to differ in their dimer-dimer interactions. Alternative polynucleotide binding stoichiometries are explicable in terms of a single base-binding model: chemical shift data are consistent with a proposal that the common n = 3 and n = 4 modes differ basically in the extent to which the sugar-phosphate backbone is stretched between binding pockets on adjacent G5P dimers.