PROTEINS: Structure, Function, and Genetics zyxw 22:199-209 (1995) PREDICTION REPORT Prediction of the Structure of GroES zy and Its Interaction With GroEL Alfonso Valencia,' Tim J. Hubbard; Arturo Muga? Sonia Banuelos? Oscar Llorca,' Jose L. Carrascosa,' and Jose Maria Valpuesta' zyxwvu 'Centro Nacional de Biotecnologia, zyxwvutsr C.S.Z.C. Universidad Autonoma de Madrid, 28049 Madrid, Spain; 'Centre for Protein Engineering (CPE), MRC Centre, Cambridge, CB2 zyxwvut 2QH, United Kingdom; 3Departamento de Bioquimica zy y Biologia Molecular, Facultad de Ciencias, Universidad del Pais Vasco, 48080 Bilbao, Spain ABSTRACT The three-dimensional struc- ture of the GroES monomer and its interaction with GroEL has been predicted using a combi- nation of prediction tools and experimental data obtained by biophysical [electron micro- scope (EM),Fourier transform infrared (FTIR), and nuclear magnetic resonance (NMR)] and biochemical techniques. The GroES monomer, according to the prediction, is composed of eight @-strands forming a @barrel with loose ends. In the model, p-strands zyxwvu 5-8 run along the outer surface of GroES, forming an antiparallel p-sheet with zyxwvuts p4 loosely bound to one of the edges. p-strands 13 would then be parallel and placed in the interior of the molecule. Loops 13 would face the internal cavity of the GroEL- GroES complex, and together with conserved residues in loops 5 and 7, would form the active surface interacting with GroEL. 0 1995 Wdey-Liss, Inc. Key words: chaperonins, electron microscopy, FTIR, molecular modeling, struc- ture prediction, contact prediction, active site prediction INTRODUCTION Chaperonins are a family of proteins involved in the cellular response to stress and in the proper fold- ing of proteins, both in vivo and in ~ i t r o . l - ~ One of the best characterized chaperonin systems is the one formed by the bacterial GroEL (Hsp 60) and GroES (Hsp 10). They share extensive homology with cor- responding members of the family from bacterial, mitochondrial, chloroplast, and eukaryote cyto- plasm origin. Both proteins are needed, together with ATP, Mg2+ and K', to assist in the proper folding of some proteins, although the exact mecha- nism of their function is still ur~known.~*~ GroEL forms an oligomer of 14 identical subunits arranged as a double toroid of around 800 KDa,6,7 while GroES forms a ring of around 70 KDa, built up by 7 0 1995 WILEY-LISS. INC identical subunit^.^,^ Central to the study of the role of the chaperonins in protein folding is analysis of the structure of the complex formed by GroEL and GroES. The three-dimensional structure of the GroEL oligomer a t atomic resolution," together with the low-resolution three-dimensional maps of the complex derived from electron microscopy (EM) have shed some light on this subject; how- ever, there are a number of open questions related to the functional role of asymmetric and symmetric GroEGGroES complexes.'3-16 Also, the details of the interaction between GroEL and GroES oligo- mers is still unknown. Although it is clear that only X-ray crystallogra- phy can provide a high-resolution structure of the complex, this is a good system for trying to combine data from different approaches to reveal some as- pects of the structure. In particular, since the three- dimensional structure at atomic resolution of GroES is close to being it is interesting to combine the results from structural prediction tech- niques with data from spectroscopy and EM to ob- tain a predicted three-dimensional structure of GroES that can be compared later with the one ob- tained by X-ray diffraction techniques, to test the feasibility of this approach. It has recently been established that various new structural prediction methods can be quite useful in fold recognition and ab initio prediction of protein structure," namely, hidden Markov model^,'^ p-strand pairing prediction," and two-dimensional and "in/out" predictions by Neural Networks.21,22 Other tools have been tested on many known struc- tures (tree determinants or conserved residues) or across the entire database of known structures (cor- related mutations) but have never before been used Received January 27, 1995; accepted February 13, 1995. Address reprint requests to Jose Maria Valpuesta, Centro Nacional de Biotecnologia, C.S.I.C. Universidad Autonoma de Madrid, 28049 Madrid, Spain.