Binding and hydrolysis of nucleotides in the chaperonin catalytic cycle: implications for the mechanism of assisted protein folding.
Cpn60 was labeled with pyrene maleimide in order to follow structural rearrangements in the protein triggered by the binding of nucleotides and cpn10. The conjugate binds ATP, AMP-PNP, and ADP(P(i)) with pyrene fluorescence enhancements of 60%, 60%, and 15%, respectively. In each case, binding is cooperative with half-saturation (K1/2) occurring at 10 microM, 290 microM, and 2500 microM and Hill constants (nH) of 4, 3, and 3, respectively. Inclusion of the co-protein, cpn10, tightens the binding of ATP, AMP-PNP, and ADP(P(i)) to give K1/2 values of 6 microM, 100 microM, and < 0.07 microM, respectively, and cooperativity is increased. Titration of the cpn60/ADP (14-mer) complex with cpn10 (7-mer) gives a stoichiometry of 14:7 with respect to subunits, confirming the molecular asymmetry shown by electron microscopy. Transient kinetics demonstrate that ATP initially forms a weak collision complex with cpn60 (Kd = 4 mM) which isomerizes to the strongly binding state at a rate of 180 s-1. We suggest that the slow structural rearrangement driven by ATP binding is the same event which lowers the affinity of the chaperonin for protein substrates; a suggestion reinforced by the loss of AMP-PNP binding affinity in the presence of an unstructured polypeptide. As such, this rearrangement of cpn60 is analogous to a force-generating step in energy transduction. Measurements of ATP hydrolysis (pH 7.5, 25 degrees C) show that it is slow (0.04 s-1) compared both with the structural rearrangement and with the dissociation of products. This defines the steady-state complex as cpn60/ATP, a form of the chaperonin which binds substrate proteins weakly. The rate of hydrolysis of ATP is stimulated 20-fold upon binding unfolded lactate dehydrogenase, and the yield of folded enzyme is increased even in the absence of cpn10. Addition of this co-protein inhibits hydrolysis on only half of the sites in cpn60 and leads to a faster release of folded LDH. A mechanism for the action of chaperonins is proposed which depends upon cpn60 being cycled between states which have, alternately, low and high affinity for unfolded proteins. This cycle is driven by the binding and hydrolysis of ATP.
Jackson
G S
GS
Molecular Recognition Centre, University of Bristol School of Medical Sciences, U.K.
Staniforth
R A
RA
Halsall
D J
DJ
Atkinson
T
T
Holbrook
J J
JJ
Clarke
A R
AR
Burston
S G
SG
D000227:Adenine Nucleotides / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D018834:Chaperonin 60; D018833:Chaperonins; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D008954:Models, Biological; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology; D011485:Protein Binding; D017510:Protein Folding; D011506:Proteins / Q000378:metabolism
D000227:Adenine Nucleotides; D001426:Bacterial Proteins; D018834:Chaperonin 60; D006360:Heat-Shock Proteins; D010455:Peptides; D011506:Proteins; D000255:Adenosine Triphosphate; D018833:Chaperonins