Location of a folding protein and shape changes in GroEL–GroES complexes imaged by cryo-electron microscopy

Abstract

Protein folding mediated by the molecular chaperone GroEL occurs by its binding to non-native polypeptide substrates and is driven by ATP hydrolysis. Both of these processes are influenced by the reversible association of the co-protein, GroES (refs 2-4). GroEL and other chaperonin 60 molecules are large, cylindrical oligomers consisting of two stacked heptameric rings of subunits; each ring forms a cage-like structure thought to bind polypeptides in a central cavity. Chaperonins play a passive role in folding by binding or sequestering folding proteins to prevent their aggregation, but they may also actively unfold substrate proteins trapped in misfolded forms, enabling them to assume productive folding conformations. Biochemical studies show that GroES improves the efficiency of GroEL function, but the structural basis for this is unknown. Here we report the first direct visualization, by cryo-electron microscopy, of a non-native protein substrate (malate dehydrogenase) bound to the mobile, outer domains at one end of GroEL. Addition of GroES to GroEL in the presence of ATP causes a dramatic hinge opening of about 60 degrees. GroES binds to the equivalent surface of the GroEL outer domains, but on the opposite end of the GroEL oligomer to the protein substrate.

High resolution surface structure of E. coli GroES oligomer by atomic force microscopy

“…most EM images of GroES/GroEL complexes, the GroES appeared to be a domed cap at the end of GroEL, indicating that the surface of GroES facing the GroEL lumen is concave [8,17,19]. Therefore, the observed GroES surface with the AFM should be the surface facing away from the GroEL.…”

“…Each monomer of GroES has a nominal molecular weight of 10 kDa [2], and the GroES is normally found in an oligomeric configuration of seven subunits [2,13]. Although no specific functions have been identified with the GroES heptamer alone, it has been firmly established with both electron microscopy (EM) and biochemical methods that, in the presence of ATP or ADP, the GroES heptamer can form a stable complex with the GroEL tetradecamer [5,8,[18][19][20]. Upon the binding of GroES, the rate of ATP hydrolysis of GroEL is strongly affected [4,6,7,9,12,21], but the mechanism has not been well understood [14][15][16][17].…”

GroEL‐Mediated protein folding

“…The suggestion that the GroES mobile loops at least in part bind to and compete for the same surface of GroEL as polypeptide is supported both by observations in electron microscopy of the site of contact (Chen et al, 1994;Roseman et al, 1996) and by mutational findings that the same channel-facing residues critical for polypeptide binding (Fig. 1B) are also critical for binding GroES .…”

“…Electron microscopy studies have shown that non-native polypeptides are bound in these channel openings (Langer et al, 1992;Braig et al, 1993;Chen et al, 1994;Ishii et al, 1994). Because the amino acids forming the walls of the channel are hydrophobic in character (Fig.…”

“…2) that binds at one or both ends of the GroEL cylinder in the presence of nucleotide (Chandrasekhar et al, 1986;Saibil et al, 1991;Langer et al, 1992;Schmidt et al, 1994b), producing an upward and outward opening of the GroEL apical domains, which nearly doubles the volume of the central channel ( Fig. 3) (Chen et al, 1994). Crystal structure analyses of GroES reveal a dome-shaped structure about 70-80 8, in diameter and 30 8, high, with inside dimensions of -30 8, diameter and -20 8, high.…”