The 26S proteasome is a large biomacromolecular complex which facilitates the targeted degradation
of intracellular proteins, and thus plays an essential role in keeping protein homeostasis. It
consists of a catalytic particle, made up of alpha rings and beta rings, controlled by regulatory particles,
which are made up of a number of proteins. The proteasome is an intricate molecular machine which requires complex
regulation to unfold and deubiquitilate the substrate, and push it through the catalytic machinery...
Necessarily, the proteasome undergoes large conformational changes
during its operation. However, due to its size, such changes are very
difficult to study. Recent work by Unverdorben et. al in
the field of cryo-electron microscopy has lead to the discovery of intermediate conformers during the
initial binding of ubiquitylated substrates. While the conformational
changes in the regulatory particle are easily distinguishable, the
changes in the catalytic particle are very subtle, due to the fact that
all studied conformers refer to the initial phase of substrate binding.
Using ACC, we calculated atomic charges in the 3 conformers of the 26S proteasome that are available
in the Protein Data Bank (PDB IDs 4CR2, 4CR3, 4CR4). The default ACC settings were used.
Specifically, the total molecular charge was considered neutral for all conformers.
The EEM parameter set used was EX-NPA_6-31Gd_gas, which covered the NPA charge definition at
the HF/6-31G* level of theory, and was developed for proteins. The computation method was EEM Cutoff Cover,
with a cutoff radius of 12Å. The computation took 130s.
The calculation produced 3 sets of atomic charges, one for each conformer. Since the proteasome is very large,
we analyzed the residue charges, which are also reported by ACC, and subsequently the charges for the various
subunits that make up the proteasome. Note that information about the subunits is given in the COMPND records
of the PDB entries used here. Residues with the chain identifier 1-7 correspond to subunits beta 1-7.
Residues with the chain identifier A-G correspond to subunits alpha 1-7. Finally, residues with the chain
identifier H-Z correspond to regulatory subunits. Therefore, the total charge for a specific subunit or
particle can be obtained as the sum of the charges for the residues with the corresponding chain identifiers.
We thus followed the change in total charge for the regulatory particle, the catalytic particle,
then separately for the alpha and beta rings.
The first observation was that, during the conformational changes from state 1, to state 2, and then to state 3,
a significant amount of electron density is transferred between the catalytic particle and the regulatory particle.
This suggests that, even though there is no significant movement observed for the catalytic particle, allosteric
information is exchanged with the catalytic particle, and this information can be tracked at the electrostatic level.
The next observation is that significant information is disseminated not only horizontally (within the alpha ring, or within the beta ring),
but also vertically. In the overall transition it appears that the alpha ring loses electron density to the regulatory particle. By checking the
intermediate state 2 it is possible to see that there is also transfer between the alpha ring and beta ring.
This vertical shuttling of electron density within the catalytic particle suggests that the
activity of alpha and beta subunits may cross-correlate. Such phenomena have indeed been reported.
For example, O'Hara et. al found that subunits alpha5 and beta1 may
translocate together, while Cron et. al found that knockdown of alpha1
leads to loss of chymotrypsin activity associated with beta5. Further analysis can even yield the residues involved
in the allosteric regulation, as those residues which exhibit a high variation in total charge (e.g., approximately 10 sites on the Rpn-13 regulatory subunit).
This case study shows how a brief calculation using only a crude structural approximation can give insight regarding allosteric
regulation in large biomolecular complexes.