The
gold standard for nanotechnology is nature’s own proteins. These
biomolecular nanomachines—macromolecules forged from peptide chains of
amino acids—are able to fold themselves into a dazzling multitude of
shapes and forms that enable them to carry out an equally dazzling
multitude of functions fundamental to life. As important as protein
folding is to virtually all biological systems, the mechanisms behind
this process have remained a mystery.
The fog, however, is being lifted.
A
team of researchers with the U.S. Department of Energy (DOE)’s Lawrence
Berkeley National Laboratory (Berkeley Lab), using the exceptionally
bright and powerful x-ray beams of the Advanced Light Source, have
determined the crystal structure of a critical control element within
chaperonin, the protein complex responsible for the correct folding of
other proteins. The incorrect or “misfolding” of proteins has been
linked to many diseases, including Alzheimer’s, Parkinson’s and some
forms of cancer.
The frequency at which droplets emerge is controlled by an acoustic trigger, which can be tuned so that each droplet containing a protein or virus meets an incoming pulse of x-rays. |
“We
identified, for the first time, a region within group II chaperonins we
call the nucleotide-sensing loop, which detects the presence of the ATP
molecules that fuel the chaperonin folding motion,” says Paul Adams, a
bioengineer with Berkeley Lab’s Physical Biosciences Division and
leading authority on X-ray crystallography who led this work. ““We knew
that ATP hydrolysis is important for promoting protein folding, but we
did not know how ATP activity was sensed and communicated.”
Adams is the corresponding author of a paper in The EMBO Journal that
describes this study, which was performed in collaboration with
colleagues at MIT and Stanford. The paper is titled “Mechanism of
nucleotide sensing in group II Chaperonins.” Co-authoring this paper
were Jose Pereira, Corie Ralston, Nicholai Douglas, Ramya Kumar, Tom
Lopez, Ryan McAndrew, Kelly Knee, Jonathan King and Judith Frydman.
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. Much of the basic architecture has been evolutionarily
preserved 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.
“We
obtained crystal structures at sufficient resolution to allow us to
examine, in detail, the effects that changes in nucleotides states have
on ATP binding and hydrolysis in group II chaperonins,” Adams says.
“From these structures we see that the nucleotide-sensing loop monitors
ATP binding sites for changes and communicates this information
throughout the chaperonin. Functional analysis further suggests 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.”
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, Adams
and his colleagues may have opened a new avenue by which modified
protein-folding activities could engineered.
“The
strong relationship between incorrectly folded proteins and
pathological states is well documented,” Adams says. “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.”
A
key factor that enabled Adams and his colleagues to solve the
three-dimensional crystal structure of the nucleotide sensing loop and
determine its pivotal role in the protein folding of group II
chaperonins was the unique protein crystallography capabilities of the
Berkeley Center for Structural Biology. The BCSB operates five protein
crystallography beamlines for Berkeley Lab’s Advanced Light Source
(ALS), a DOE Office of Science national user facility for synchrotron
radiation, and the first of the world’s third generation light sources.
For this particular study, Adams and his colleagues used ALS beamlines
8.2.1 and 8.2.2, which are powered by a superconducting bending magnetic
to yield higher energy x-rays that are optimized for the study of
single crystals of biological molecules.
In this study, Adams and his colleagues studied an archaeon chaperonin.
In their followup research, they will apply what they have learned to
study the chaperonin known as TRiC, which is the human chaperonin.
“We
believe that chaperonins have evolved to work on specific substrates
and that the rates of protein folding may vary greatly between
chaperonins in different organisms,” Adams says. “The structural and
biochemical identification of the changes related to ATP hydrolysis
provides important insights into the complex puzzle of protein folding
for each type of chaperonin.”
This
work was performed as part of the Center for Protein Folding Machinery,
an NIH Roadmap-supported Nanomedicine Development Center. The BCSB is
supported in part by the National Institutes of Health and National
Institute of General Medical Sciences, and the Howard Hughes Medical
Institute.
