Single-stranded DNA binding of the cold-shock protein CspB from Bacillus subtilis: NMR mapping and mutational characterization.
Cold-shock proteins (CSPs) bind to single-stranded nucleic acids, thereby acting as a "RNA chaperone." To gain deeper insights into the rather unspecific nature of ssDNA/RNA binding, we characterized the binding interface of CspB from Bacillus subtilis to a 25-mer of ssDNA (Y-Box25) using heteronuclear 2D NMR spectroscopy. Seventeen residues, including eight out of nine aromatic amino acids, are directly involved in the Y-Box25 interaction and were identified by extreme line broadening of their cross-peaks. Eight residues belong to the earlier proposed RNP binding motifs. A second set of seven backbone amides becomes evident by major chemical shift perturbations reporting remote conformational rearrangements upon binding. These residues are located in loop beta3-beta4 and loopbeta4-beta5, and include Ile18. The individual contributions of the so-identified residues were examined by fluorescence titration experiments of 15 CspB variants. Phenylalanine substitutions in- and outside the RNP motifs significantly reduce the binding affinity. Unrestricted possible backbone conformations of loop beta3-beta4 also markedly contribute to binding. Stopped-flow fluorescence kinetics revealed that the different binding affinities of CspB variants are determined by the dissociation rate, whereas the association rate remains unchanged. This might be of importance for the "RNA chaperone" activity of CspB.
Zeeb
Markus
M
Laboratorium für Biochemie, Universität Bayreuth, D-95440 Bayreuth, Germany.
Balbach
Jochen
J
D019943:Amino Acid Substitution; D001412:Bacillus subtilis / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D017124:Conserved Sequence; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010449:Peptide Mapping / Q000379:methods; D010649:Phenylalanine / Q000235:genetics; D011485:Protein Binding; D011487:Protein Conformation; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D016601:RNA-Binding Proteins; C077188:cold-shock protein CspB, Bacteria; D010649:Phenylalanine; D014364:Tryptophan