Comparative structural analyses and nucleotide-binding characterization of the four KH domains of FUBP1

Xiaomin Ni,Stefan Knapp,Apirat Chaikuad

doi:10.1038/s41598-020-69832-z

Xiaomin Ni, Stefan Knapp + Show 1 more

Open Access

https://doi.org/10.1038/s41598-020-69832-z

Copy DOI

Abstract

The FUBP1-FUSE complex is an essential component of a transcription molecular machinery that is necessary for tight regulation of expression of many key genes including c-Myc and p21. FUBP1 utilizes its four articulated KH modules, which function cooperatively, for FUSE nucleotide binding. To understand molecular mechanisms fundamental to the intermolecular interaction, we present a set of crystal structures, as well ssDNA-binding characterization of FUBP1 KH domains. All KH1-4 motifs were highly topologically conserved, and were able to interact with FUSE individually and independently. Nevertheless, differences in nucleotide binding properties among the four KH domains were evident, including higher nucleotide-binding potency for KH3 as well as diverse nucleotide sequence preferences. Variations in amino acid compositions at one side of the binding cleft responsible for nucleobase resulted in diverse shapes and electrostatic charge interaction, which might feasibly be a contributing factor for different nucleotide-binding propensities among KH1-4. Nonetheless, conservation of structure and nucleotide-binding property in all four KH motifs is essential for the cooperativity of multi KH modules present in FUBP1 towards nanomolar affinity for FUSE interaction. Comprehensive structural comparison and ssDNA binding characteristics of all four KH domains presented here provide molecular insights at a fundamental level that might be beneficial for elucidating the mechanisms of the FUBP1-FUSE interaction.

Highlights

Far upstream element (FUSE) binding protein 1 (FUBP1 or FBP1) is a multifunctional single-stranded DNA(ssDNA) and RNA-binding protein, which acts as a master regulator of diverse cellular processes including transcription, mRNA stability and translation as well as RNA splicing[1,2,3]
Comparison among four FUBP1 K homology (KH) domains revealed that the highly conserved residues from α1-α2 region harbouring the canonical GXXG motif within the connecting loop clustered within one side of their DNA binding pockets (Fig. 1E)
We aimed to investigate whether each KH domain might prefer specific interacting sites, and tested the ability of the 4 individual KH domains to bind to a series of randomly-truncated 10-mer c-Myc FUSE fragments that harboured an AA-double nucleotide on their 3′ ends in order to increase the migration of the proteins in native gels

Summary

Introduction

Far upstream element (FUSE) binding protein 1 (FUBP1 or FBP1) is a multifunctional single-stranded DNA(ssDNA) and RNA-binding protein, which acts as a master regulator of diverse cellular processes including transcription, mRNA stability and translation as well as RNA splicing[1,2,3]. It is best known for its role as a positive regulator of c-Myc oncoprotein mediated by the interaction with the supercoiled, A/T-rich non-coding strand of FUSE located upstream of the c-Myc promoter[4,5,6] which leads to the recruitment and activation of transcription factor TFIIH that enhances transcription of c-Myc gene[7]. These early studies have suggested consistently that all four KH domains of FUBP1 likely function as articulated modules and exploit hierarchical mechanisms to specify their binding sites on F USE6,31

Methods

Results

Conclusion