Aromatic ring currents at a protein surface: Use of H-1-NMR chemical shifts to refine the structure of a naked beta sheet

The naked beta sheet, a newly recognized motif of protein structure, exhibi ts ordered surfaces in the absence of a conventional hydrophobic core. A mo del is provided by an archaeal Zn ribbon homologous to eukaryotic RNA polym erase II subunit 9 (RPB9). This subunit, which regulates transcriptional st art-site selection and downstream pausing, contains Zn2+-binding motifs sim ilar to those of general transcription factors TFIIB and TFIIS. Interesting ly, distance-geometry yields two models of the archaeal Zn ribbon differing in the orientation of a conserved tyrosine side chain on the well-ordered surface of the naked beta -sheet. The models are equally consistent with co nventional restraints and otherwise contain indistinguishable structural fe atures, including a tetrahedral Cys(4) Zn2+-binding sites, four antiparalle l beta -strands, and disordered loop. Due to the change in tyrosine orienta tion and correlated changes in the configuration of neighboring side chains , the two models predict inequivalent patterns of aromatic ring-current shi fts. The observed secondary shifts of adjoining resonances are shown to be consistent with one model but not the other. In the consistent model the su rface of the beta -sheet contains successive aromatic edge-to-face contacts in accord with semi-classical and ab initio potentials. We speculate that the aromatic-rich surface of the hyperthermophilic RPB9 domain contributes its thermodynamic stability and provides a nucleic-acid-binding site in the eukaryotic and archaeal transcriptional machinery. The present study demon strates how the reduced dimensionality of a surface can lead to ambiguities in the interpretation of nuclear Overhauser enhancements. The results illu strate the utility of chemical shifts at such a surface in the cross-valida tion of a high-resolution solution structure.