Leu309 Plays a Critical Role in the Encapsulation of Substrate Protein into the Internal Cavity of GroEL

Ayumi Koike-Takeshita,Tatsuro Shimamura,Ken Yokoyama,Masasuke Yoshida,Hideki Taguchi

doi:10.1074/jbc.m506298200

Abstract

In the crystal structure of the native GroEL.GroES.substrate protein complex from Thermus thermophilus, one GroEL subunit makes contact with two GroES subunits. One contact is through the H-I helices, and the other is through a novel GXXLE region. The side chain of Leu, in the GXXLE region, forms a hydrophobic cluster with residues of the H helix (Shimamura, T., Koike-Takeshita, A., Yokoyama, K., Masui, R., Murai, N., Yoshida, M., Taguchi, H., and Iwata, S. (2004) Structure (Camb.) 12, 1471-1480). Here, we investigated the functional role of Leu in the GXXLE region, using Escherichia coli GroEL. The results are as follows: (i) cross-linking between introduced cysteines confirmed that the GXXLE region in the E. coli GroEL.GroES complex is also in contact with GroES; (ii) when Leu was replaced by Lys (GroEL(L309K)) or other charged residues, chaperone activity was largely lost; (iii) the GroEL(L309K).substrate complex failed to bind GroES to produce a stable GroEL(L309K).GroES.substrate complex, whereas free GroEL(L309K) bound GroES normally; (iv) the GroEL(L309K).GroES.substrate complex was stabilized with BeF(x), but the substrate protein in the complex was readily digested by protease, indicating that it was not properly encapsulated into the internal cavity of the complex. Thus, conformational communication between the two GroES contact sites, the H helix and the GXXLE region (through Leu(309)), appears to play a critical role in encapsulation of the substrate.

Highlights

Sulation of the substrate protein into an enlarged cavity inside the cis-ring, which is capped by GroES
We investigated the GXXLE region in the E. coli GroEL1⁄7GroES complex to determine whether it is in contact with GroES and whether or not Leu309 in E. coli GroEL plays a role for the efficient encapsulation of substrate protein into the cis-cavity
Conservation of Novel Contacts in the E. coli GroEL1⁄7GroES Complex—To investigate whether or not the GXXLE region is in contact with GroES in the E. coli GroEL1⁄7GroES complex, we conducted a series of cross-linking experiments using mutants of E. coli GroEL and GroES

Summary

Introduction

Sulation of the substrate protein into an enlarged cavity (the ciscavity) inside the cis-ring, which is capped by GroES. The residues Gly305, Leu308, and Glu309 of T. thermophilus GroEL are well conserved across species, and hereafter, we refer this region as the GXXLE region This region has an intrasubunit interaction with the H helix; the side chain of Leu308 (Leu309 in E. coli GroEL) points at the N terminus of the H helix to form a hydrophobic cluster with other residues (Fig. 1, A, circle, and B). We investigated the GXXLE region in the E. coli GroEL1⁄7GroES complex to determine whether it is in contact with GroES and whether or not Leu309 in E. coli GroEL plays a role for the efficient encapsulation of substrate protein into the cis-cavity. The two GroES contact sites can communicate with each other through a hydrophobic cluster formed by Leu in the GXXLE region and residues at the entrance formed by the H helix (red arrows)

Objectives

Results

Conclusion