Topologies of a substrate protein bound to the chaperonin GroEL.
The chaperonin GroEL assists polypeptide folding through sequential steps of binding nonnative protein in the central cavity of an open ring, via hydrophobic surfaces of its apical domains, followed by encapsulation in a hydrophilic cavity. To examine the binding state, we have classified a large data set of GroEL binary complexes with nonnative malate dehydrogenase (MDH), imaged by cryo-electron microscopy, to sort them into homogeneous subsets. The resulting electron density maps show MDH associated in several characteristic binding topologies either deep inside the cavity or at its inlet, contacting three to four consecutive GroEL apical domains. Consistent with visualization of bound polypeptide distributed over many parts of the central cavity, disulfide crosslinking could be carried out between a cysteine in a bound substrate protein and cysteines substituted anywhere inside GroEL. Finally, substrate binding induced adjustments in GroEL itself, observed mainly as clustering together of apical domains around sites of substrate binding.
Elad
Nadav
N
Department of Crystallography, Birkbeck College London, Malet Street, London WC1E 7HX, UK.
Farr
George W
GW
Clare
Daniel K
DK
Orlova
Elena V
EV
Horwich
Arthur L
AL
Saibil
Helen R
HR
D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D018834:Chaperonin 60 / Q000737:chemistry / Q000648:ultrastructure*; D003198:Computer Simulation; D020285:Cryoelectron Microscopy / Q000379:methods; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli; D057927:Hydrophobic and Hydrophilic Interactions; D007091:Image Processing, Computer-Assisted; D008291:Malate Dehydrogenase / Q000737:chemistry / Q000648:ultrastructure; D008958:Models, Molecular; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013552:Swine
D018834:Chaperonin 60; D004220:Disulfides; D008291:Malate Dehydrogenase; D003545:Cysteine