title: Leu309 plays a critical role in the encapsulation of substrate protein into the internal cavity of GroEL.
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.
authors.affiliation: Chemical Resources Laboratory, Tokyo Institute of Technology, Midori-ku, Yokohama, Japan.
authors.forename: Ayumi
authors.initials: A
authors.lastname: Koike-Takeshita
authors.forename: Tatsuro
authors.initials: T
authors.lastname: Shimamura
authors.forename: Ken
authors.initials: K
authors.lastname: Yokoyama
authors.forename: Masasuke
authors.initials: M
authors.lastname: Yoshida
authors.forename: Hideki
authors.initials: H
authors.lastname: Taguchi
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry; D001665:Binding Sites; D018835:Chaperonin 10 / Q000737:chemistry; D018834:Chaperonin 60 / Q000737:chemistry*; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D007930:Leucine / Q000737:chemistry*; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry; D008968:Molecular Conformation; D009154:Mutation; D010447:Peptide Hydrolases / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D016963:Thermus thermophilus / Q000378:metabolism; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D018835:Chaperonin 10; D018834:Chaperonin 60; D003432:Cross-Linking Reagents; D018832:Molecular Chaperones; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D010447:Peptide Hydrolases; D007930:Leucine; D003545:Cysteine