Ligand-induced Structural Changes of the CD44 Hyaluronan-binding Domain Revealed by NMR

Mitsuhiro Takeda,Hiroaki Terasawa,Haruko Hayasaka,Ichio Shimada,Kazuki N. Sugahara,Masayoshi Sakakura,Masahiro Kajiwara,Ryo Umemoto,Shinji Ogino,Masayuki Miyasaka

doi:10.1074/jbc.m608425200

Abstract

CD44, a major cell surface receptor for hyaluronan (HA), contains a functional domain responsible for HA binding at its N terminus (residues 21-178). Accumulating evidence indicates that proteolytic cleavage of CD44 in its extracellular region (residues 21-268) leads to enhanced tumor cell migration and invasion. Hence, understanding the mechanisms underlying the CD44 proteolytic cleavage is important for understanding the mechanism of CD44-mediated tumor progression. Here we present the NMR structure of the HA-binding domain of CD44 in its HA-bound state. The structure is composed of the Link module (residues 32-124) and an extended lobe (residues 21-31 and 125-152). Interestingly, a comparison of its unbound and HA-bound structures revealed that rearrangement of the beta-strands in the extended lobe (residues 143-148) and disorder of the structure in the following C-terminal region (residues 153-169) occurred upon HA binding, which is consistent with the results of trypsin proteolysis studies of the CD44 HA-binding domain. The order-to-disorder transition of the C-terminal region by HA binding may be involved in the CD44-mediated cell migration.

Highlights

HA, a major component of the extracellular matrix, is a very high molecular mass glycosaminoglycan, composed of a repeating disaccharide, D-glucuronic acid (GlcA) (␤1 3 3) N-acetylD-glucosamine (GlcNAc) (␤1 3 4) [4]
We found that the residues with large chemical shift changes induced by HA binding were mostly localized in the C-terminal extension and the first ␣-helix, and they generally differed from the contact residues revealed by the crosssaturation experiment
Our previous studies, based on cross-saturation and chemical shift perturbation experiments, showed that the structural elements consisting of the N- and C-terminal extensions of CD44 HABD contain part of the contact surface for HA and undergo significant conformational changes upon HA binding [14]

Summary

Introduction

HA, a major component of the extracellular matrix, is a very high molecular mass glycosaminoglycan, composed of a repeating disaccharide, D-glucuronic acid (GlcA) (␤1 3 3) N-acetylD-glucosamine (GlcNAc) (␤1 3 4) [4]. Comparisons of the structures and the backbone flexibility of CD44 HABD between its unbound and HA-bound states revealed that the C-terminal region of CD44 HABD became disordered upon HA binding Concomitant with this structural analysis, trypsin digestion experiments of CD44 HABD in the presence and absence of HA showed that the proteolytic susceptibility in the C-terminal region increased upon HA binding. This ligand-induced order-to-disorder transition of the C terminus of CD44 HABD provides a plausible explanation for the mechanism underlying the HA-induced CD44-mediated cell migration

Methods

Results

Conclusion