Particle Protein and Friends

To really appreciate the beauty of the SRP system, we should look more closely at the major players.  But first, let’s make things more manageable.  Lucky for us, the bacterium E. coli has a scaled-down version of the system that nevertheless functions much like the system seen in human cells [8].  The RNA is much smaller, being only 114 nucleotides in length and thus lacking the Alu domain [9].  Furthermore, instead of having six different proteins as part of the SRP, the E. coli version has only one, known as Ffh.  Since there is only one, we’ll just call Ffh the ‘particle protein.’  E. coli also has the receptor (FtsY) and the translocon (SecY).  Thus, the system is actually quite simple, being composed of a small RNA molecule (4.5S RNA) that is bound by the particle protein which in turn binds to the receptor and the translocon.

Let’s first put the particle protein under the microscope.

This protein is truly multifunctional.  It has the ability to bind to the ribosome, the signal sequence, the 4.5S RNA, GTP, and the receptor.  Each function is essential and to carry them all out, the 450 amino acids that make up the protein fold up to form three distinct domains: the N domain, followed by the G domain, followed by the M domain (see Figure 3).  The M domain has the ability to bind to the signal sequence and the RNA [9].  The G domain binds GTP and is an enzyme that also breaks it down.  Both the N and G domains form a structural and functional unit that binds to receptor [11].  The N domain also plays an important role in binding to the ribosome [12] and contributes to the binding of the signal sequence [13].

particle protein

Figure 3.  The particle protein (Ffh) bound to the 4.5S RNA.  The relative orientation of the domains and RNA is from ref. 10.  The functions associated with each domain and the RNA are also indicated.

The receptor is the other major player that interacts with the particle protein.  The receptor too has the N and G domains, but lacks the M domain.  Instead, another chunk of amino acids, known as the A domain, is typically present in front of the N and G domains.  The A domain is believed to help anchor the receptor to the membrane, but studies have shown it to be dispensable [14], meaning it is the N and G domain of the receptor that provide the core functions of binding to the membrane, the particle protein, and the translocon.

The remaining players include the RNA, which binds to the ribosome and may also regulate the interaction of the domains within the particle protein, and the translocon itself, that forms the membrane channel.  The translocon forms a ring-like tunnel with a central cavity that is sufficiently large to conduct the newly made protein across the membrane [9].

matrix2Now that we have all the players in place, let’s think about them in motion.  Even though we have a relatively few components to tend to, they play an essential role in targeting the membrane and secretory proteins to their proper destination, and as SRP researcher Peter Walter, from the Howard Hughes Medical Institute, notes, “like many other cellular processes, the targeting reaction involves a series of ordered steps that need to be closely coordinated” [16].  We should not underestimate the difficulty of this task.  To properly carry out the job, such precise coordination requires that the SRP and its receptor “switch between multiple functional states in response to cargo occupancy, spatial information, and time constraints” [16].

It all starts when the SRP, composed of particle protein bound to RNA, samples the ribosomes.  This sampling requires that that SRP bind weakly to ribosomes near the exit tunnel where the signal sequence from the newly made protein will emerge.  Keep in mind that the ribosome is much larger than the SRP.  While the SRP is composed of one protein and an RNA molecule 114 nucleotides in length, the ribosome is composed of over 50 proteins and three RNA molecules comprising 1000s of nucleotides.  Thus, the smaller SRP must find the particular patch on the larger ribosome and it does so by binding to a particular ribosomal protein known as L23, which happens to be adjacent to the exit tunnel [17].  Also, when anchored at this point, the RNA that is part of the SRP makes contact with RNA on the ribosome.  Yet we don’t want to SRP to bind too tightly yet, as it would be wasting its time latched on to a ribosome that was making a cytoplasmic protein.  So, the SRP binds weakly and briefly, allowing to check if it is needed, and if not, it dissociates to sample another ribosome.

If a signal sequence emerges from the ribosome, the M domain of the sampling SRP will bind it tightly.  The M domain contains a deep groove that can house the signal sequence and once they bind, the SRP itself is now tightly latched on to the ribosome.  What’s more, the interaction between the M domain and signal sequence communicates conformation changes throughout the particle protein, causing the N and G domain to swivel out such that the G domain can now bind GTP [13].  When the G domain binds GTP, it, along with the N domain, becomes more compact [18] and now is effectively primed for interaction with the receptor [13].  In the meantime, the receptor itself apparently undergoes priming in an analogous manner, where it binds GTP as a function of its association with the membrane or translocon.  These priming steps are important because they prevent the SRP and receptor from interacting until both are loaded with their appropriate cargo.  But once both are primed, they can finally bind to each other, bringing the exit channel of the ribosome into close proximity to the translocon.

The interaction between the SRP and its receptor is more complex than first suspected.  They interact pretty much along the entire interface of their respect N and G domains.  The interface is unusually large, being 3-6 times more extensive that the interface formed between antibodies and their antigens [12] and there are many sites along this interface that are crucial for SRP function.  After the particle protein and its receptor bind, a cascade of conformational changes ensues.  Both N and G domains re-adjust their shape to form a tighter binding pocket for their respective GTP molecules and further changes then form a composite active site, where the two GTP molecules are paired together within this interface.  This GTP twinning is essential to the mechanism of breaking down the GTP molecules, the receptor helps the particle protein break down its GTP molecule while the particle protein helps the receptor break down its GTP molecule. Once the GTP molecules are broken down into GDP and Pi (a process known as hydrolysis), this triggers more conformation changes that allow the receptor and particle protein to dissociate.  The breakdown of the GTP molecules acts as a timing devise.  Prior to such activity, the M domain on the particle protein transfers the signal sequence on the newly formed protein to the translocon where it is then threaded into or across the membrane .  The exact mechanism of this transfer remains obscure.  But once the handoff to the translocon is complete, the breakdown of the GTP molecules occurs and the particle protein is released from the receptor and ribosome for another round of reuse.

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