The Dynamics of Protein Translocation
Protein Localization in the Cell
The protein-conducting channel.
Proteins are the workhorses of the typical cell. Originally coded for by DNA and composed of a sequence of amino acids, proteins serve a variety of roles including binding other molecules and/or transporting them, serving as 'switches' for cellular activity, catalyzing chemical reactions, and even acting as structural building blocks. It should come as no surprise then that in order to accomplish all these goals, proteins are needed in a variety of locations both inside and outside of the cell. Therefore, a variety of mechanisms have evolved in order to correctly localize proteins, helping to guide them from their point of synthesis (at the ribosome) to their destination. One such set of mechanisms is the secretory pathway.
Also ubiquitous in biological systems are membranes, barriers between the cell and the outside world. Membranes also serve to divide compartments within some cells (those
of eukaryotes). In order for proteins to get to where they are needed inside these compartments or outside the cell, they need a way to cross the membrane without rupturing it at the same
time. The secretory pathway consists in part of a channel for just this purpose: the translocon (
![[Chembiochem 1:86]](paper-blue.jpg){kind=small}
).
A Dynamic Channel
A schematic of the translocation process. The ribosome docks with the translocon where the signal sequence (triangle) is recognized. This then opens the channel allowing the protein to
go through.
The translocon may be comprised of multiple proteins, but the core component is a heterotrimeric protein known as the Sec61 complex in eukaryotes, SecYEG in bacteria, and
SecYEβ in archaea. Despite their differences, all function in a similar way. The ribosome, the factory for all proteins, docks with the translocon where it begins feeding the new,
still linear, protein into the channel as it's being made, a process known as co-translational translocation. The channel appears to open up just a few angstroms wide, not enough for
a completely folded protein but enough for a perhaps a helix. To guarantee not just any molecule can cross the channel though, the translocon will only open for proteins displaying the right
'key', in this case, a short specific amino acid leader sequence. This key is recognized by SecY/Sec61 itself, although the manner in which this causes the channel to open is still a
mystery. Originally theorized over 30 years ago, it was the hypothesis of this key and channel combination that earned Günter Blobel the Nobel Prize in Physiology or Medicine in 1999
().
Once the channel has been opened, some may assume that it is a passive conduit for the nascent proteins being fed into it. However, this is actually where the channel
begins its most complex role, that of deciding whether to place the protein on the other side of the channel or to insert it into the membrane. While translocation of soluble proteins
across the membrane appears to be straightforward, there are different suggestions for how segments of protein destined for the membrane insert into it. Some ideas include modification of
SecY/Sec61 by the ribosome
()
or diffusion of the hydrophobic protein segments away from water and toward the hydrophobic membrane core while inside the channel
().
Regardless, it seems clear that large structural changes are required to open this channel both to membrane as well as to the opposite side.
SecYEβ, shown here both from the side and from above and also in its native membrane/water environment (click for a larger view).
Moving Proteins Across the Channel
A small polypeptide (protein segment) is crossing the channel and pushing open the plug of SecY.
The recent availability of a crystal structure of SecYEβ
()
has brought into focus some aspects of the protein translocation process. Two key structural elements were
identified that help keep the channel closed before and during translocation. First is a 'plug', a small
helix blocking the channel's periplasmic side (opposite of where proteins are fed into the channel).
Second is a 'pore ring', a constriction point at the center of the channel consisting of a few residues from
different helices. Molecular dynamics simulations using the program NAMD have allowed us to characterize
these elements and confirm their hypothesized function (described more thoroughly in a recent paper
).
During simulated translocation of short polypeptide helices, the plug was seen to leave the channel and
then, after translocation, return partially to its original location. Also, the pore ring blocked water and
ions before translocation; during translocation, it expanded enough to allow the polypeptide segment through
but did not allow any ions through and only a few water molecules.
Another question perhaps made more complicated by the atomic structure is that regarding
the oligomerization of SecYEβ. While some proteins function only as monomers (single copies), many
form larger structures such as dimers (two copies), tetramers (four copies), or, in general, polymers
(n copies). The SecY/Sec61 complex is known to form dimers and tetramers in nature but it is not
clear why. One original hypothesis was that the channel formed at the center of the four protein complexes.
However, the structure along with other experiments and our simulations seem to demonstrate that the monomer
is capable of forming the channel. The question then remains: what purpose does the tetramer serve? We
have attempted to begin answering this question by simulating a SecYEβ dimer in a back-to-back
conformation (although a front-to-front conformation has also recently been proposed
).
We discovered that during the simulation, the plug of each SecYEβ became destabilized, much more than in the simulation of a single SecYEβ. Thus, in this way, cooperative
interactions between the two copies of SecYEβ may aid translocation through a single monomer.
- Click the image above to see a movie made from a simulation of translocation through SecY. Shown from the top, the polypeptide is in blue, the plug in red, and the pore ring residues in yellow. Click here to see the same simulation from the side.
Lateral Gating of the Channel
Steered MD was used to force open the lateral gate (click for a larger view).
The other function of the translocon, inserting proteins into the membrane, is accomplished by means of a lateral gate, hypothesized based on the structure
().
Simulations of forced gate
opening and relaxation revealed how the structure responds. In particular, the two approximately symmetric halves of SecY were found to stay reasonably stable during opening, agreeing with
the "clamshell-like" behavior proposed previously. Additionally, SecE, the accessory protein surrounding SecY, did not affect the rate of gate closure; this was surprising as SecE had
been assumed to act as a clamp, holding the two halves of SecY together. Also, it was found that by opening the gate, interactions with the plug were broken, freeing it. This is potentially
how gating by a signal sequence occurs, where the inserted sequence pushes (or holds) open the lateral gate, thus freeing the plug and loosening the channel as a whole before translocation.
Click here for a movie of gate opening.
Also of interest was the behavior of lipids during gate opening. It has been suggested that if the lateral gate opens directly to the bilayer (as opposed to another
translocon next to it
),
it would become inactivated by lipids flooding the channel. To explore this suggestion in more detail, we simulated SecY with the lateral gate held open to the bilayer, both in an all-atom
representation and a reduced, coarse-grained representation. Surprisingly, we found after up to 1 microsecond, lipids did not invade the channel.
Instead, they clung to hydrophobic gating helices or remained firmly stuck in the bilayer. Given also that a transmembrane segment of an inserting protein probably occupies this region most
of the time, it is probably unlikely then that lipids pose any threat to the channel.
Maintaining the Permeability Barrier in the Channel
(left) The system used for simulating the channel. (middle) The channel shown with the native (green) and two mutant (red and blue) plugs. (right) A closeup of the pore ring in one of the mutants with two residues not shown in order to illustrate water permeating the channel (click for a larger version).
While previous simulations indicated that both the pore ring and plug played some role in closing the channel, it remained unclear what their independent roles are. Two
recently solved
structures of mutants further complicated the picture
).
Specifically, two mutants with either half or all of the native plug deleted formed new plugs from the remaining residues. These
mutants demonstrated that having a plug is important for the channel, but how well do the new plugs function? Various experiments indicated they do not function as well as the native plug,
but also do not kill the cell
) ).
Electrophysiology experiments demonstrated that the mutant channels permit intermittent permeation of water and ions whereas the native channel is water tight
).
However, the crystal structures of the two mutants still show the channel as closed by the pore ring at the channel's center.
The difference in the permeability is apparent when comparing the native (left) and one of the mutant (right) channels in simulation.
To reconcile the closed structures of the mutants with their experimental permeabilities, we performed multiple equilibrium simulations of the native channel and the two mutants. We measured the permeability of each channel to water, finding increasing diffusive and osmotic permeabilities corresponding to the amount of the plug deleted. The increase in permeability was also found to correspond to an increase in the motions of the new plugs and, in particular, the pore ring residues, suggesting that pore ring flexibility, controlled by the plug, is the primary determinant of channel openness. To confirm this suggestion, we performed a unique computational experiment in which the interactions between water and the plug in the native, closed channel were eliminated, making the plug "transparent" to water. By doing so, we separate the role of the plug as a purely steric barrier to the flow of water from its role as a lock, holding the channel closed. We found in simulations that this hybrid channel permitted almost no water permeation, significantly less than the mutants where even only a fraction of the plug was deleted. Thus it appears that the plug's role as a steric barrier alone is significantly less important than its role holding the pore ring closed.
Future Investigations
In our future work, we will explore further some of the current results put forth as well as look towards new ones. These include examining in more detail the gating mechanism of SecY, including the effects of channel partners.
Publications
Molecular dynamics studies of the archaeal translocon. James Gumbart and Klaus Schulten. Biophysical Journal, 90:2356-2367, 2006.
Structural determinants of lateral gate opening in the protein translocon. James Gumbart and Klaus Schulten. Biochemistry, 46:11147-11157, 2007.
The roles of pore ring and plug in the SecY protein-conducting channel. James Gumbart and Klaus Schulten. Journal of General Physiology, 132:709-719, 2008.
Investigators
Page created and maintained by JC Gumbart.