Structure and interaction of Corynebacterium pseudotuberculosis cold shock protein A with Y-box single-stranded DNA fragment.

Cold shock proteins (Csps) function to preserve cell viability at low tem-peratures by binding to nucleic acids and consequently control gene expres-sion. The mesophilic bacterium Corynebacterium pseudotuberculosis is thecausative agent of caseous lymphadenitis in animals, and infection in live-stock is a considerable economic burden worldwide. In this report, thestructure of cold shock protein A from Cp ( Cp-CspA) and biochemicalanalysis of its temperature-dependent interaction with a Y-box ssDNAmotif is presented. The Cp-CspA structure contains five b-strands makingup a b-barrel fold with 11 hydrophobic core residues and two salt bridgesthat confers it with a melting temperature of ~ 54 °C that is similar tomesophilic Bs-CspB. Chemical shift perturbations analysis revealed thatresidues in the nucleic acid-binding motifs (RNP 1 and 2) and loop 3 areinvolved in binding to the Y-box fragment either by direct interaction orby conformational rearrangements remote from the binding region. Fluo-rescence quenching experiments of Cp-CspA showed that the dissociationconstants for Y-box ssDNA binding is nanomolar and the binding affinitydecreased as the temperature increased, indicating that the interaction isenthalpically driven and the hydrogen bonds and van der Waals forces areimportant contributions for complex stabilization. The Y31 of Cp-CspA isa particular occurrence among Csps from mesophilic bacteria that providea possible explanation for the higher binding affinity to ssDNA than thatobserved for Bs-CspB. Anisotropy measurements indicated that the reduc-tion in molecular mobility of Cp-CspA upon Y-box binding is character-ized by a cooperative process