Journal of Structural Biology 155 (2006) 482–492 www.elsevier.com/locate/yjsbi 1047-8477/$ - see front matter  2006 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2006.06.008 Crystal structure of the temperature-sensitive and allosteric-defective chaperonin GroEL E461K Aintzane Cabo-Bilbao a , Silvia Spinelli b , Begoña Sot a , Jon Agirre a , Ariel E. Mechaly a , Arturo Muga a , Diego M.A. Guérin a,c,¤ a Unidad de Biofísica (CSIC-UPV/EHU), P.O. Box 644, E-48080 Bilbao, Spain b AFMB-CNRS, UMR 6098. 163, Av. de Luminy, 13288 Marseille Cedex 09, France c Departamento de Física, Universidad Nacional del Sur, Bahía Blanca, Argentina Received 16 March 2006; received in revised form 16 June 2006; accepted 27 June 2006 Available online 8 July 2006 Abstract The chaperonin GroEL adopts a double-ring structure with various modes of allosteric communication. The simultaneous positive intra-ring and negative inter-ring co-operativities alternate the functionality of the folding cavities in both protein rings. Negative inter- ring co-operativity is maintained through diVerent inter-ring interactions, including a salt bridge involving Glu 461. Replacement of this residue by Lys modiWes the temperature sensitivity of the substrate-folding activity of this protein, most likely as a result of the loss of inter-ring co-operativity. The crystal structure of the mutant chaperonin GroEL E461K has been determined at 3.3 Å and compared with other structures: the wild-type GroEL, an allosteric defective GroEL double mutant and the GroEL–GroES-(ADP) 7 complex. The inter- ring region of the mutant exhibits the following characteristics: (i) no salt-bridge stabilizes the inter-ring interface; (ii) the mutated residue plays a central role in deWning the relative ring rotation (of about 22°) around the 7-fold axis; (iii) an increase in the inter-ring distance and solvent accessibility of the inter-ring interface; and (iv) a 2-fold reduction in the stabilization energy of the inter-ring interface, due to the modiWcation of inter-ring interactions. These characteristics explain how the thermal sensitivity of the protein’s fundamental proper- ties permits GroEL to distinguish physiological (37 °C) from stress (42 °C) temperatures.  2006 Elsevier Inc. All rights reserved. Keywords: GroEL; E461K; Co-operativity; Chaperonin 1. Introduction The GroEL–GroES chaperonin complex helps unfolded polypeptides to achieve the active conformation via a nucleotide-regulated cyclic reaction. GroEL is a double- heptameric 800 kDa toroid made of identical subunits that contains a central cavity. GroES is a dome-like 70 kDa homo-heptamer that binds to the same GroEL ring to which other ligands (non-native polypeptides and nucleo- tides) are already bound. In this way, the complex GroES– GroEL forms a hydrophobic cavity where the peptide searches for the productive structure in an isolated environ- ment, known as the AnWnsen cage, and is subsequently returned to the medium. Each GroEL subunit is composed of three domains: (1) an apical domain (186 residues) that interacts with non- folded substrate proteins and GroES; (2) an intermediate domain (89 residues) that forms the outer wall of the cylin- der and provides a Xexible covalent connection from the apical to the equatorial domain; and (3) the equatorial domain (243 residues), which in addition to securing the nucleotide binding site, yields most of the intra-ring and all of the inter-ring contacts (Bartolucci et al., 2005; Braig et al., 1994). GroES binding to the ATP-bound ring induces major conformational changes in the apical and intermediate * Corresponding author. Present address: Unidad de Biofísica (CSIC- UPV/EHU), Barrio Sarriena S/N, 48940 Leioa, Vizcaya, Spain. Fax: +34 946013360. E-mail address: diego.guerin@ehu.es (D.M.A. Guérin).