Structural basis of DNA binding to human YB-1 cold shock domain regulated by phosphorylation

Abstract

Human Y-box binding protein 1 (YB-1) is a multifunctional protein and overexpressed in many types of cancer. It specifically recognizes DNA/RNA through a cold shock domain (CSD) and regulates nucleic acid metabolism. The C-terminal extension of CSD and the phosphorylation of S102 are indispensable for YB-1 function. Until now, the roles of the C-terminal extension and phosphorylation in gene transcription and translation are still largely unknown. Here, we solved the structure of human YB-1 CSD with a C-terminal extension sequence (CSDex). The structure reveals that the extension interacts with several residues in the conventional CSD and adopts a rigid structure instead of being disordered. Either deletion of this extension or phosphorylation of S102 destabilizes the protein and results in partial unfolding. Structural characterization of CSDex in complex with a ssDNA heptamer shows that all the seven nucleotides are involved in DNA–protein interactions and the C-terminal extension provides a unique DNA binding site. Our DNA-binding study indicates that CSDex can recognize more DNA sequences than previously thought and the phosphorylation reduces its binding to ssDNA dramatically. Our results suggest that gene transcription and translation can be regulated by changing the affinity of CSDex binding to DNA and RNA through phosphorylation, respectively.

Autoantibody Formation and Mapping of Immunogenic Epitopes against Cold-Shock-Protein YB-1 in Cancer Patients and Healthy Controls

“…While untreated Flag-YB-1 decays over time in 2 additional N-terminal fragments at about 40 kDa and 35 kDa (4B, lane 3 >3 and >4) as well as corresponding C-terminal (4B, lane 5 >5 and >6) fragments, dephosphorylation leads to a delayed protein fragmentation ( Figure 4B, lane 2, 4 and 6). This finding stands in line with previously reported results showing a destabilization of protein conformation with YB-1 phosphorylation [22] Protein band >2 at about 50 kDa most likely reflects untagged eukaryotic YB-1, while it is not detected by Flag-targeting antibody. In Figure 4C treated and untreated Flag-YB-1 were probed with serum from tumor patients (lane 3-6) and healthy controls (lane 7-10).…”

“…This could be due to protein modifications like C-terminal amino acid extension which stabilize protein structure. Posttranslational modifications such as phosphorylation modify protein conformation [22]. It will be of interest to further analyze the identified linear antigenic epitopes for posttranslational modifications that may take place in YB-1 protein.…”

“…Zhang et al raised the possibility of an 11 amino acid extension of the C-terminal domain with eukaryotic YB-1, which stabilizes the protein and may prevent degradation. Those protein modifications are lacking in prokaryotic cold shock proteins [22,23]. Unexpectedly, bands corresponding to higher molecular weight than full-length YB-1 were detected as well.…”

Cold-Shock Domains—Abundance, Structure, Properties, and Nucleic-Acid Binding

“…Analyses by NMR and isothermal titration calorimetry (ITC) of DNA-heptamer binding to an extended YBX1 CSD reveals DNA-sequence preferences of YBX1, a binding mode resembling bacterial CSPs and the structural basis of the observed attenuated target DNA binding by S102 phosphorylation [ 9 ]. Conversely, dephosphorylation of S102 and other serine residues was proposed to unmask the nuclear localization signal of YBX1 and facilitate nuclear entry at specific stages during the cell cycle [ 141 ].…”

“…We focus on the common structural, biophysical and nucleic acid-binding properties of these evolutionarily related domains which share a conserved geometry of binding single-stranded DNA or RNA (ssDNA or ssRNA) and limited sequence or nucleic acid-type selectivity. Some of these common principles were revealed in very recent structural studies [ 8 , 9 , 10 , 11 , 12 , 13 , 14 ].…”