Abstract
We report on a study that combines advanced fluorescence methods with molecular dynamics (MD) simulations to cover timescales from nanoseconds to milliseconds for a large protein. This allows us to delineate how ATP hydrolysis in a protein causes allosteric changes at a distant protein binding site, using the chaperone Hsp90 as test system. The allosteric process occurs via hierarchical dynamics involving timescales from nano- to milliseconds and length scales from Ångstroms to several nanometers. We find that hydrolysis of one ATP is coupled to a conformational change of Arg380, which in turn passes structural information via the large M-domain α-helix to the whole protein. The resulting structural asymmetry in Hsp90 leads to the collapse of a central folding substrate binding site, causing the formation of a novel collapsed state (closed state B) that we characterise structurally. We presume that similar hierarchical mechanisms are fundamental for information transfer induced by ATP hydrolysis through many other proteins.
Highlights
Allosteric communication, i.e., coupling between distant regions of a protein, is an elementary mechanism for the regulation of protein function, signalling and energy transfer, as it enables alteration of protein structures at active sites by small changes in a binding pocket several nanometers away.[1,2] In spite of its importance, there is surprisingly little known about the underlying dynamical process and the timescales of allosteric communication.[3,4,5] Understanding the molecular mechanisms of allostery is crucial to, e.g., elucidate the effects of point mutations in cancer development[6] or to exploit it for the design of small molecule drugs.[7]In this work, we investigate allostery following ATP hydrolysis within the yeast molecular chaperone heat shock protein 90 (Hsp[90])
To monitor intra-protein changes associated with this allosteric communication, we focus on (i) the distance dArgP between the Arg[380] side chain guanidinium carbon atom and the Pa,b mass center, (ii) the Ca-RMSD of Mdomain position and (iii) the Ca-RMSD of the M-loop arrangement in reference to the cryo-EM structure 5FWK. dArgP highlights changes within the binding pocket directly a er hydrolysis at a distance of $0.4 nm from the triphosphate, the M-domain RMSD protein report on conformational changes at intermediate ($2.0 nm) distance, and the M-loop RMSD re ects conformational changes at the folding client binding site ($4.0 nm distance)
We are in a position to formulate a model of allosteric communication involving Arg[380] and the M-helix depicted as steps (1)–(5) in Fig. 5: starting with ATP (1), Arg[380] forms a salt bridge with Pg, and the resulting electrostatic interaction is transmitted via the M-domain to the M-loops, keeping the folding substrate binding site open
Summary
Allosteric communication, i.e., coupling between distant regions of a protein, is an elementary mechanism for the regulation of protein function, signalling and energy transfer, as it enables alteration of protein structures at active sites by small changes in a binding pocket several nanometers away.[1,2] In spite of its importance, there is surprisingly little known about the underlying dynamical process and the timescales of allosteric communication.[3,4,5] Understanding the molecular mechanisms of allostery is crucial to, e.g., elucidate the effects of point mutations in cancer development[6] or to exploit it for the design of small molecule drugs.[7]In this work, we investigate allostery following ATP hydrolysis within the yeast molecular chaperone heat shock protein 90 (Hsp[90]). A single hydrolysis event already seems to destabilise nucleotide binding, which is consistent with our experimental results of Hsp[90] heterodimers containing a non-hydrolizable E33A mutant on one monomer site (see Fig. S5† and below) and the observation that ATP hydrolysis is only required in one subunit of the Hsp[90] dimer for correct function.[50]
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