SOLUTION NMR STRUCTURE OF THE MAJOR COLD SHOCK PROTEIN (CSPA) FROM ESCHERICHIA-COLI - IDENTIFICATION OF A BINDING EPITOPE FOR DNA

Sequence-specific H-1 and N-15 resonance assignments have been determi ned for the major cold shock protein (CspA) from Escherichia coli with recently developed three-dimensional triple-resonance NMR experiments . By use of these assignments, five antiparallel beta-strands were ide ntified from analysis of NMR data. Strands 1-4 have a classical 3-2-1- 4 Creek key beta-sheet topology and there are two beta-bulges, at posi tions Lys(10)-Trp(11) and Gly(65)-Asn(66). Three-dimensional structure s of CspA were generated from NMR data by using simulated annealing wi th molecular dynamics. The overall chain fold of CspA is a beta-barrel structure, with a tightly packed hydrophobic core. Two-dimensional is otope-edited pulsed-field gradient N-15-H-1 heteronuclear single-quant um coherence spectroscopy was used to characterize the N-15-H-1 finger print spectrum with and without a 24-base oligodeoxyribonucleotide, 5' -AACGGTTTGACGTACAGACCATTA-3'. Protein-DNA complex formation perturbs a subset of the amide resonances that are located mostly on one face of the CspA molecule. This portion of the CspA molecular surface include s two putative RNA-binding sequence moths which contribute to an unusu al cluster of eight surface aromatic side chains: Trp(11), Phe(12), Ph e(18), Phe(20), Phe(31) His(33), Phe(34) and Tyr(42). These surface ar omatic groups, and also residues Lys(16) Ser(44), and Lys(60) located on this same face of CspA, are highly conserved in the family of CspA homologues. These isotope-edited pulsed-field gradient NMR data provid e a low-resolution mapping of a DNA-binding epitope on CspA.