Conceived and designed the experiments: IC FF DF. Performed the experiments: IC ADS. Analyzed the data: IC ADS FF DF. Contributed reagents/materials/analysis tools: IC ADS FF DF. Wrote the paper: IC ADS FF DF.
The authors have declared that no competing interests exist.
The biological function of chaperone complexes is to assist the folding of non-native proteins. The widely studied GroEL chaperonin is a double-barreled complex that can trap non-native proteins in one of its two barrels. The ATP-driven binding of a GroES cap then results in a major structural change of the chamber where the substrate is trapped and initiates a refolding attempt. The two barrels operate anti-synchronously. The central region between the two barrels contains a high concentration of disordered protein chains, the role of which was thus far unclear. In this work we report a combination of atomistic and coarse-grained simulations that probe the structure and dynamics of the equatorial region of the GroEL/GroES chaperonin complex. Surprisingly, our simulations show that the equatorial region provides a translocation channel that will block the passage of folded proteins but allows the passage of secondary units with the diameter of an alpha-helix. We compute the free-energy barrier that has to be overcome during translocation and find that it can easily be crossed under the influence of thermal fluctuations. Hence, strongly non-native proteins can be squeezed like toothpaste from one barrel to the next where they will refold. Proteins that are already fairly close to the native state will not translocate but can refold in the chamber where they were trapped. Several experimental results are compatible with this scenario, and in the case of the experiments of Martin and Hartl, intra chaperonin translocation could explain why under physiological crowding conditions the chaperonin does not release the substrate protein.
Chaperonin complexes capture proteins that have not yet reached their functional (“native”) state. Non-native proteins cannot perform their function correctly and threaten the survival of the cell. The chaperonins help these proteins to reach their native state. The prokaryotic GroEL-GroES chaperonin is an ellipsoidal protein complex that is approximately 16 nm long. It consists of two chambers that are joined at the bottom. Interestingly, protein repair by this chaperonin is not a one-step process. Typically, several capture and release steps are needed before the target protein reaches its native state. It is commonly assumed that substrate proteins cannot translocate, i.e., move inside the complex from one chamber to the other. In the absence of translocation, proteins that have not yet reached their functional conformation have to be released into the cytosol before being recaptured by a chaperonin. We present multi-scale simulations that show that it is, in fact, surprisingly easy for substrate proteins to translocate between the two chambers via an axial pore that is filled with disordered protein filaments. This finding suggests that non-native proteins can be squeezed like toothpaste from one chamber to the other: the incorrect structure of the protein is broken up during translocation and the protein has an increased probability to find its native state when it reaches the other chamber. The possibility for intra-chaperonin translocation obviates the need for a potentially dangerous release of non-native proteins.
Proteins that have not yet folded to their native state may interfere with the machinery of the cell. For this reason, prokaryotic and eukaryotic cells have evolved special macro-molecular “chaperone” complexes that capture and refold partially folded proteins, thereby preventing them from indulging in cellular mischief
Colours represent the type of surface: all hydrophobic amino acids (A, V, L, I, M, F, P, Y) are in
The two pathways are shown in the schematic drawing above. In the initial configuration (1), the chaperonin barrel is open and exposes a hydrophobic rim for binding partially folded proteins. After a non-native protein is captured, the GroEL-GroES complex closes (i.e., the barrel gets capped) (2). After that, the protein can either refold in the original barrel (3A) or, if their structure is far from native
In the present paper we use atomistic and mesoscopic simulations to test whether such a translocation scenario is compatible with the available structural information on the GroEL complex. Our simulation studies focus on the equatorial regime of the GroEL complex that might be expected to act as a barrier against translocation. Crystallographic studies indicate that most protein units in the chaperonin complex have a fairly rigid structure both in the open and closed configurations
The presence of disordered protein chains in the pore that joins the two GroEL chambers will certainly affect the permeability of the equatorial plane, but they need not block translocation. There are, in fact, examples
We have performed fully atomistic and coarse-grained simulations that do reproduce the structural data of
The plot shows the experimental scattering intensity of solution of single ring GroEL obtained with SANS (o). Fitted over the data with the program Cryson
The data are obtained with the caterpillar model of the chains in the trans ring (circles), and with GROMACS full atomistic simulations for the trans (squares) and cis (diamonds) chains. The remarkable overlap between the distributions indicates that we do reproduce the static picture of a blocked hole. The density distribution generated by the coarse-grained model is more peaked than the one generated in the atomistic simulations. This indicates that, if anything, the coarse-grained model will overestimate the extent to which the disordered peptide chains hinder translocation. The coarse-grained model predicts that the chains in the cis region are far from the center of the hole and do not impede the passage of an α-helix.
To compute the free-energy barrier for protein translocation, the MD approach described above would have been prohibitively expensive. We therefore performed Monte Carlo simulations on a suitably coarse-grained model for the GroEL complex. We focused on the structural fluctuations within a spherical region (diameter 40 Å) around the trans side of the equatorial cavity (
We start by considering a very naive estimate that has the advantage that it is based on the fully atomistic simulations. From these simulations, we know the density profile of
Next, we compute the free energy barrier for the coarse-grained model system using the MC method described in the
First we considered the case of pure steric interactions between both the chains and the helix. In
For steric interactions (circles), the profile is rather symmetrical and presents a small barrier of 2
Of course, the interaction between a typical translocation protein segment and the ring chains is not purely steric. To consider the effect of both attractive and repulsive interactions, we consider the two cases separately. As the chains consist predominantly of
In fact, in the case of attractive interactions, there is virtually no barrier for translocation. This absence of a barrier may provide a rationale for the experimental observation that Krueger et al. observed in their SANS experiments
The translocation of encapsulated non-native proteins is most likely in cases where the initial structure is far native. The reason is two fold: first of all, for such conformation there should be a low free-energy cost associated with partial unfolding—a necessary first step in translocation. Secondly, non-native chains that are trapped in a hydrophilic cage tend to be compressed. They can lower their free energy by translocating out of the cage. The simulations of
In conclusion, our simulation results are not compatible with the assumption that the disordered protein chains in the cis or trans rings provide an effective barrier against translocation. The present findings may help explain a puzzling experimental finding concerning refolding experiments in the presence of crowding agents
The flexible nature of this region prevented accurate X-ray determination of the chains filling the interconnecting pore. To obtain a full-atomistic model, the program MODELLER
The Caterpillar model is a modification of the tube model of Maritan and co-workers
To sample the conformations of the protein chains anchored on the trans ring, we use two basic Monte-Carlo moves: branch rotation and an improved version of the biased Gaussian step
Root mean square displacement of the Cα atoms of the equatorial chains compared to the initial condition. The time scale starts from 7 ns and goes all the way to 11 ns. The plateau demonstrates that the dynamics reached equilibrium.
(0.15 MB EPS)
Schematic representation of the model used for the GROMACS full atomistic simulations. The part of the protein that was kept constrained in space is shown in grey. The chains that were free to fluctuate are shown in light blue. The water molecules that fully solvated the protein complex are not shown. The axes are drawn to indicate the coordinate system used in the calculation of the filament density profiles.
(0.71 MB EPS)
Real-space representation of the backbone of the caterpillar model. The large blue sphere represents the self-avoidance area of the Cα atoms in with a radius of 2.5 Å. The H and O atoms interact through a 10-12 Lennard-Jones potential tuned with a quadratic orientation term that selects for alignment of the C H O and N atoms involved in a bond. The backbone fluctuates only around the torsional angles
(1.11 MB EPS)
Real space snapshot of one configuration of the chains in the equatorial region equilibrated with the Monte Carlo simulation of the caterpillar model. The top and side view shows a fully blocked pore as seen in X-ray crystallography or Cryo-EM reconstruction.
(2.23 MB EPS)
Plot of translocation free energy
(0.21 MB EPS)
Plot of translocation free energy
(0.07 MB EPS)
IC thanks Michele Vendruscolo for many discussions.