Crystal and solution structures of human oncoprotein Musashi-2 N-terminal RNA recognition motif 1.

Lan Lan,Maithri Kashipathy,Robert Hanzlik,Minli Xing,Scott Lovell,Justin Douglas,Kevin Battaile,Liang Xu,Philip Gao

doi:10.1002/prot.25836

Abstract

Musashi‐2 (MSI2) belongs to Musashi family of RNA binding proteins (RBP). Like Musashi‐1 (MSI1), it is overexpressed in a variety of cancers and is a promising therapeutic target. Both MSI proteins contain two N‐terminal RNA recognition motifs and play roles in posttranscriptional regulation of target mRNAs. Previously, we have identified several inhibitors of MSI1, all of which bind to MSI2 as well. In order to design MSI2‐specific inhibitors and compare the differences of binding mode of the inhibitors, we set out to solve the structure of MSI2‐RRM1, the key motif that is responsible for the binding. Here, we report the crystal structure and the first NMR solution structure of MSI2‐RRM1, and compare these to the structures of MSI1‐RBD1 and other RBPs. A high degree of structural similarity was observed between the crystal and solution NMR structures. MSI2‐RRM1 shows a highly similar overall folding topology to MSI1‐RBD1 and other RBPs. The structural information of MSI2‐RRM1 will be helpful for understanding MSI2‐RNA interaction and for guiding rational drug design of MSI2‐specific inhibitors.

Highlights

| INTRODUCTIONThe RNA-binding protein (RBP) Musashi-2 (MSI2) is overexpressed in many cancers, including colorectal adenocarcinomas,[1,2,3] breast,[4,5] hematologic malignancies,[6,7,8,9,10,11,12] lung,[13] glioblastoma,[14] and pancreatic cancers.[15,16,17] As such, it mediates mRNA stability and translation of proteins involved in oncogenic pathways.[18,19,20] Overexpression and knockdown studies indicate that MSI2 is a promising therapeutic target for cancer.[3,13,16,21,22]
The highest degree of similarity was observed for the N-terminal RNA recognition motifs (RRMs) of human MSI2 which are 87% and 86% identical to the RNA-binding domains (RBDs) of human MSI1 and mouse MSI1, respectively
15 nt control RNA (Control15) containing a scrambled sequence displayed nonspecific binding at high protein concentration (Figure 2). These results indicate that the MSI2-RRM1 construct adopts a functional RNA-binding conformation in solution

Summary

| INTRODUCTION

The RNA-binding protein (RBP) Musashi-2 (MSI2) is overexpressed in many cancers, including colorectal adenocarcinomas,[1,2,3] breast,[4,5] hematologic malignancies,[6,7,8,9,10,11,12] lung,[13] glioblastoma,[14] and pancreatic cancers.[15,16,17] As such, it mediates mRNA stability and translation of proteins involved in oncogenic pathways.[18,19,20] Overexpression and knockdown studies indicate that MSI2 is a promising therapeutic target for cancer.[3,13,16,21,22]. The highest degree of similarity was observed for the N-terminal RRMs of human MSI2 (residues 21-187) which are 87% and 86% identical to the RNA-binding domains (RBDs) (residues 20-186) of human MSI1 and mouse MSI1, respectively. The C-terminal region of MSI2 has no known motifs or specific function, while the N-terminal RRMs mediate the binding to mRNAs,[25] including those involved in the proliferation of certain cancers. As such, targeting these interactions using structure-based drug design methods may provide a route for inhibitor development. A high degree of structural similarity was observed between the crystal and solution NMR structures, suggesting that an orthogonal approach using both crystallographic and solution NMR methods could potentially be utilized for subsequent ligand binding studies

| MATERIALS AND METHODS

| RESULTS AND DISCUSSION

| CONCLUSION