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New Crystal Structures Lift Fog around Protein Folding Print

Nature's proteins set a high bar for nanotechnology. Macromolecules forged from peptide chains of amino acids, these biomolecular nanomachines must first be folded into a dazzling variety of shapes and forms before they can perform the multitude of functions fundamental to life. However, the mechanisms behind the protein-folding process have remained a foggy mystery. Now the fog is lifting: a team of researchers from Berkeley Lab, Stanford University, and the Massachusetts Institute of Technology has deciphered the crystal structure of a critical control element within chaperonin, the protein complex responsible for the correct folding of other proteins.

Chaperonins promote the proper folding of newly translated proteins and proteins that have been stress-denatured—meaning they've lost their structure—by encapsulating them inside a protective chamber formed from two rings of molecular complexes stacked back-to-back. There are two classes of chaperonins, group I found in prokaryotes and group II found in eukaryotes and archaea (organisms with no cell membrane or internal membrane-bound organelles). Much of the basic architecture has been evolutionarily preserved (conserved) across these two classes but they do differ in how the protective chamber is opened to accept proteins and closed to fold them. Whereas group I chaperonins require a detachable ring-shaped molecular lid to open and close the chamber, group II chaperonins have a built-in lid.

A Molecular Origami Machine

The genetic code embodied by the nucleotide sequences in DNA and collected in the form of genes is well known. Biological macromolecules like proteins comprise strings of amino acids coded for by the genes, but the story doesn't end there. The amino-acid strings must then be folded into a dazzling multitude of shapes and forms designed specifically to carry out the various functions fundamental to life. It turns out that the cell entrusts a special protein called a chaperonin to supervise the all-important task of folding proteins correctly. In humans, for example, incorrect or "misfolding" of proteins has been linked to many diseases, including Alzheimer's, Parkinson's, and some forms of cancer. So far, however, a complete molecular-level picture of how these amazing molecular nanomachines actually promote the folding has been missing.

Pereira et al. have now filled in some of the blanks by studying the molecular structure of chaperonin in various stages of its protein-folding task. By identifying a key structural feature called a nucleotide-sensing loop and its controlling role, the researchers may have found a new way to modify protein-folding activities. For example, engineering the nucleotide-sensing loop so that it promotes the desired rate of protein folding in a human chaperonin could perhaps reduce the cellular accumulation of misfolded proteins that can cause disease and other problems.

Biologists had known that the binding of ATP and subsequent hydrolysis promotes the closure of the multi-subunit rings where protein folding occurs, but the mechanism by which local changes in the nucleotide-binding site are communicated between individual subunits was unknown. To attack this problem, the research team studied the group II chaperonin in the archeon Methanococcus maripaludis, using ALS Beamlines 8.2.1 and 8.2.2.

They determined crystal structures at sufficient resolution to allow examining, in detail, the effects that ATP binding and hydrolysis have in this group II chaperonin. From these structures, they identified a region called the nucleotide-sensing loop, which monitors ATP binding by the protein and communicates this information throughout the chaperonin. Functional analysis further suggested that the nucleotide-sensing loop region uses this information to control the rate of ATP binding and hydrolysis, which in turn controls the timing of the protein-folding reaction.

Group II chaperonin structure. The nucleotide-sensing loop synchronizes conformational changes in the three domains (apical, intermediate, equatorial) for the proper folding of proteins. The diagram indicates the rearrangement of the nucleotide-sensing loop between two states: Cpn–AMP–PNP (magenta) is chaperonin in complex with AMP–PNP (an ATP analog) and Cpn–ADP is chaperonin in complex with ADP.

The double-ring chaperonin complex features multiple subunits that are grouped into three domains—apical, intermediate, and equatorial. For group II chaperonins, the closing of the lid for protein-folding causes all three domains to rotate as a single rigid body, resulting in conformational changes to the chamber that enable the proteins within to be folded. The synchronized rotation of the chaperonin domains is dependent upon the communication to all the subunits that is provided by the nucleotide-sensing loop. In identifying the nucleotide-sensing loop and its controlling role in group II chaperonin protein-folding, the researchers believe they may have opened a new avenue by which modified protein-folding activities could be engineered.

Click on the image above to see a movie showing the local conformational changes as ATP is hydrolyzed to ADP. Residue Lys-161 interacts with the γ-phosphate of ATP but shows a different orientation in the presence of ADP. The loss of the ATP γ-phosphate interaction with Lys-161 in the ADP state promotes a significant rearrangement of a loop consisting of residues 160–169. It appears that Lys-161 functions as an ATP sensor and that residues 160–169 constitute a nucleotide-sensing loop that monitors the presence of the γ-phosphate.

The strong relationship between incorrectly folded proteins and pathological states is well documented. In humans, misfolding of proteins has been linked to many diseases, including Alzheimer's, Parkinson's, and some forms of cancer. Since ATP hydrolysis is required for protein folding, it could be possible to engineer a nucleotide-sensing loop that promotes slower or faster protein-folding activity in a given chaperonin. This could, for example, be used to increase the protein-folding activity of human chaperonin, or perhaps reduce the cellular accumulation of misfolded proteins that can cause disease and other problems.

In the belief that chaperonins have evolved to work on specific substrates and that the rates of protein folding may vary greatly between chaperonins in different organisms, the team will next apply what they have learned to study the human chaperonin TRiC.



Research conducted by: J.H. Pereira and C.Y. Ralston (Berkeley Lab); N.R. Douglas (Stanford University); R. Kumar, T. Lopez, and J. Frydman (Stanford University); R.P. McAndrew (Berkeley Lab); K.M. Knee and J.A. King (Massachusetts Institute of Technology); and P.D. Adams (Berkeley Lab and University of California, Berkeley).

Funding: National Institutes of Health and Howard Hughes Medical Institute. Operation of the ALS is supported by the U.S. Department of Energy, Office of Basic Energy Sciences.

Publication about this research: J.H. Pereira, C.Y. Ralston, N.R. Douglas, R. Kumar, T. Lopez, R.P. McAndrew, K.M. Knee, J.A. King, J. Frydman, and P.D. Adams, "Mechanism of nucleotide sensing in group II chaperonins," EMBO J. 31, 731 (2012).

ALS Science Highlight #253


ALSNews Vol. 333