Biophysical characterization of the structural change of Nopp140, an intrinsically disordered protein, in the interaction with CK2α

Abstract

Nucleolar phosphoprotein 140 (Nopp140) is a nucleolar protein, more than 80% of which is disordered. Previous studies have shown that the C-terminal region of Nopp140 (residues 568–596) interacts with protein kinase CK2α, and inhibits the catalytic activity of CK2. Although the region of Nopp140 responsible for the interaction with CK2α was identified, the structural features and the effect of this interaction on the structure of Nopp140 have not been defined due to the difficulty of structural characterization of disordered protein. In this study, the disordered feature of Nopp140 and the effect of CK2α on the structure of Nopp140 were examined using single-molecule fluorescence resonance energy transfer (smFRET) and electron paramagnetic resonance (EPR). The interaction with CK2α was increased conformational rigidity of the CK2α-interacting region of Nopp140 (Nopp140C), suggesting that the disordered and flexible conformation of Nopp140C became more rigid conformation as it binds to CK2α. In addition, site specific spin labeling and EPR analysis confirmed that the residues 574–589 of Nopp140 are critical for binding to CK2α. Similar technical approaches can be applied to analyze the conformational changes in other IDPs during their interactions with binding partners.

How Do We Study the Dynamic Structure of Unstructured Proteins: A Case Study on Nopp140 as an Example of a Large, Intrinsically Disordered Protein

“…Although MTSSL is small and has a minor impact on the protein structure [ 26 ], it is important to confirm differences in the structure and function of the spin-labeled protein compared with that of the wild-type. Several IDRs of IDPs were studied using the SDSL EPR technique [ 25 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 ]. The EPR spectra of IDRs showed sharp peaks due to the fast motion of the EPR probe, which results from the IDRs’ inherent flexibility.…”

“…Single-molecule fluorescence techniques are good alternatives to overcome the limitations of the former methods. smFRET experiments, one of the single-molecule fluorescence methods, is useful for studying structural features of IDPs [ 27 , 41 , 42 , 43 , 44 , 45 , 46 , 47 ]. smFRET is based on the transfer of excited-state energy from the donor to acceptor fluorescence dyes.…”

“…It interacts with proteins essential for cellular function, such as RNA polymerase I [ 52 ], NAP57 [ 53 ], snRNPs [ 52 ], p80 coilin [ 50 ], and casein kinase 2 (CK2) [ 54 ]. Nopp140 contains 710 amino acids, and >80% of its structure is deemed to be disordered based on several disordered region prediction algorithms [ 27 , 55 ]. This prediction result together with CD spectra ( Figure 2 ) and high protease sensitivity of Nopp140 [ 55 ] shows that Nopp140 is IDP.…”

“…The interaction between the 568–596 region and CK2α is interrupted by D- myo -inositol 1,2,3,4,5,6-hexakisphosphate (IP 6 ) [ 64 ], an important regulator of many biological functions. These results together with the complex model structure of the 568–596 region and the CK2α [ 27 ] suggests that the phosphorylated Ser574 and IP 6 share the same binding site for CK2 [ 64 ], which is the key site for regulation of CK2 activity by Nopp140.…”

“…EPR spectra created using the spin-labeled (MTSSL) Nopp140 fragments showed sharp peaks because of the fast motion of the nitroxide group in MTSSL ( Figure 4 ) [ 27 ]. Among the MTSSL-labeled Nopp140s, only the EPR spectrum of the 589 fragments (those spin-labeled at residue 589) was changed to a broader line shape in the presence of CK2α ( Figure 4 ) [ 27 ]. These results imply that MTSSL-labeled Nopp140s were labeled in disordered regions and the region near amino acid 589 might be crucial for binding to CK2.…”