Mutational analysis of the putative nucleic acid-binding surface of the cold-shock domain, CspB, revealed an essential role of aromatic and basic residues in binding of single-stranded DNA containing the Y-box motif

Mol Microbiol. 1995 May;16(4):699-708. doi: 10.1111/j.1365-2958.1995.tb02431.x.


The major cold-shock protein of Bacillus subtilis, CspB, is a member of a protein family widespread among prokaryotes and eukaryotes that share the highly conserved cold-shock domain (CSD). The CSD domain is involved in transcriptional and translational regulation and was shown to bind the Y-box motif, a cis-element that contains the core sequence ATTGG, with high affinity. The three-dimensional structure of CspB, a prototype of this protein family, revealed that this hydrophilic CSD domain creates a surface rich in aromatic and basic amino acids that may act as the nucleic acid-binding site. We have analysed the potential role of conserved aromatic and basic residues in nucleic acid binding by site-directed mutagenesis. In gel retardation and ultraviolet cross-linking experiments, the ability of CspB mutants to bind single-stranded oligonucleotides (ssDNA) that contain the Y-box motif was investigated. Single substitutions of three highly conserved phenylalanine residues (Phe-15, Phe-17, Phe-27) by alanine and substitution of one histidine (His-29) by glutamine, all located within the putative RNA-binding sites RNP-1 and RNP-2, abolished the nucleic acid-binding activity of CspB. Conservative substitutions of Phe-15 to tyrosine (F15Y) showed a small increase in binding affinity, whereas separate replacement of Phe-17 and Phe-27 by tyrosine caused a reduction in binding activity. These and other substitutions including the conserved basic residues Lys-7, Lys-13 and Arg-56 as well as the aromatic residues Trp-8 and Phe-30 strongly suggest that CspB uses the side-chains of these amino acids for specific interaction with nucleic acids. Ultraviolet cross-linking experiments for CspB mutants with ssDNA supported the idea of specific CspB/nucleic acid interaction and indicated an essential role for the aromatic and basic residues in this binding. In addition, two-dimensional nuclear magnetic resonance studies with F17A, K13Q, F15Y and F27Y revealed that the mutants have the same overall structure as the wild-type CspB protein.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacillus subtilis / genetics
  • Bacillus subtilis / metabolism
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism*
  • Base Sequence
  • Binding Sites
  • Conserved Sequence
  • DNA, Bacterial / genetics*
  • DNA, Bacterial / metabolism*
  • DNA, Single-Stranded / genetics*
  • DNA, Single-Stranded / metabolism*
  • Magnetic Resonance Spectroscopy
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Point Mutation
  • Protein Binding
  • RNA-Binding Proteins / chemistry
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism


  • Bacterial Proteins
  • DNA, Bacterial
  • DNA, Single-Stranded
  • RNA-Binding Proteins
  • cold-shock protein CspB, Bacteria