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

Abstract

Intrinsically disordered proteins (IDPs) represent approximately 30% of the human genome and play key roles in cell proliferation and cellular signaling by modulating the function of target proteins via protein–protein interactions. In addition, IDPs are involved in various human disorders, such as cancer, neurodegenerative diseases, and amyloidosis. To understand the underlying molecular mechanism of IDPs, it is important to study their structural features during their interactions with target proteins. However, conventional biochemical and biophysical methods for analyzing proteins, such as X-ray crystallography, have difficulty in characterizing the features of IDPs because they lack an ordered three-dimensional structure. Here, we present biochemical and biophysical studies on nucleolar phosphoprotein 140 (Nopp140), which mostly consists of disordered regions, during its interaction with casein kinase 2 (CK2), which plays a central role in cell growth. Surface plasmon resonance and electron paramagnetic resonance studies were performed to characterize the interaction between Nopp140 and CK2. A single-molecule fluorescence resonance energy transfer study revealed conformational change in Nopp140 during its interaction with CK2. These studies on Nopp140 can provide a good model system for understanding the molecular function of IDPs.

Protein interactions of Magnaporthe oryzae protein kinase CK2 and secondary data analysis of a large number of transcriptomes suggest chaperone-like activity is integral to its function

“…63 overrepresentation of the motif among the pulldown proteins (P-value for the null hypothesis of same 123 frequency as in the whole proteome = 4 E-19, Fisher's Exact test) lending support for the proposed role 124 for this motif in these proteins as a target for CK2 phosphorylation and protein unfolding. The finding 125 of overrepresentation of this signal in the set of CK2 interacting proteins corroborates the previous 126 suggestion that CK2 is involved in the destabilization/binding of intrinsically disordered proteins 127 (Zetina, 2001; Tantos et al, 2013;Na et al, 2018) and is consistent with the strong accumulation of 128 both CK2 and intrinsically disordered proteins in the nucleolus (Frege and Uversky, 2015) and also at 129 pores between cell compartments (Lai et al, 2012;Zhang et al, 2019). 130 To further explore the general hypothesis that CK2 could interact with and possibly phosphorylate 131 intrinsically unfolded proteins we used FungiFun (https://elbe.hki-jena.de/fungifun/) to make a 132 functional classification of the pulldown proteins with a special interest for those containing the alpha 133 helix unfolding motif (Zetina, 2001).…”

“…1)further supporting the view 199 that CKa-dependent phosphorylation/dephosphorylation plays a major role in shaping protein200 interactions. Together with the high expression of CK2 in cells, this suggests an important function of 201 CK2 as a general temporary unfolder of intrinsically disordered proteins, that comprise roughly 30% of 202 eukaryotic proteins(Vucetic et al, 2003), in a similar way as it is known to interact with SRP40 and 203 influence its activity(Tantos et al, 2013;Na et al, 2018).204 CKa expression correlates with expression of genes associated with disaggregation and 205 autophagy 206…”

“…Ck2 is known to interact with the disordered nucleolar protein SRP40 that has a multitude of CK2 153 phosphorylation sites and in addition CK2 is known to change the protein binding activity towards 154 other proteins depending on the SRP40 phosphorylation status. When SRP40 becomes highly 155 phosphorylated it binds to and inhibits CK2 activity in a negative feedback loop ensuring that CK2 156 phosphorylation level will balance (Tantos et al, 2013;Na et al, 2018). We identified a putative 157 MoSRP40 (MGG_00613) and it is disordered in a similar way as other SRP40 proteins and it is well 158 conserved in filamentous fungi (Method S1).…”

Investigating the Characteristics and Evolution of Apical Membrane Antigen 1 (AMA1) of Plasmodium sp. using Phylogenetic Approach in Searching for Drug Candidate

“…The disordered region is the elusive part of the protein and it is well known for modulating the protein's function and enabling the protein to be involved in many cellular processes [30], [31]. The dynamic structure of this region is also contributing to its complexity [32] and even to the degree of oncogenicity [33]. The significance of the disordered region in protein is well understood which could serve as the target for drugs [30].…”

Conserved Eukaryotic Kinase CK2 Chaperone Intrinsically Disordered Protein Interactions

“…CK2 has been shown to phosphorylate a large number of proteins (1,2,5), and it seems to be involved in many cellular processes (5). Interestingly, the intrinsically highly disordered SRP40 protein in yeast (homologous to Nopp140; Insect, Drosophila, and Nolc1; Mammal, Human) has been shown to become phosphorylated to various degrees by CK2, with effects on SRP40 conformation, binding, and aggregation properties and further effects on diverse functions of the protein (6,7). Intrinsically disordered proteins are thus called because they contain long stretches of unstructured regions (more than 40 to 50 amino acids).…”