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Shu-ou Shan
Assistant Professor of Chemistry

The signal recognition particle (SRP) and its receptor (SR) comprise a universally conserved molecular machinery that couples protein biogenesis to its localization, thus allowing proteins to become selectively delivered to the endoplasmic reticulum (ER) in eukaryotic cells, or the plasma membrane in bacteria (Figure 1). Despite their universal conservation, little is understood about how SRP and SR work at the molecular level. As a sorting machine, SRP and SR must interact with different partners at the proper stages during protein targeting - with the signal sequences, the ribosomes, the membrane translocon, and with each other. Thus, their functional states need to be constantly switched. Hints about how this switch is built come from the finding that the targeting reaction involves two highly homologous GTPases, one in the SRP and one in the SR, that directly interact with each other. Our current goal is to decipher how these GTPases use the energy of GTP binding and hydrolysis to provide the driving force and promote fidelity of the protein targeting reaction.

Figure 1. Pathway for co-translational protein translocation by the signal recognition particle (SRP). SRP recognizes N-terminal signal sequences as the nascent polypeptide emerges from the ribosome. The ribosome and the cargo protein are then delivered to the ER or plasma membrane via the interaction of SRP with the SRP receptor (SR). There, SRP releases its ‘cargo’ to the translocation machinery, the ‘translocon’, where the nascent protein finishes its synthesis and is either integrated into the membrane or delivered into the secretory pathway.

Studies from our laboratory and others showed that the SRP and SR form a unique GTPase subfamily whose regulatory mechanism provides a notable exception to the 'GTPase switch' paradigm described for canonical signaling GTPases. 'Classical' GTPases convert slowly between an active, GTP-state and an inactive, GDP-bound state, and rely on external regulatory factors - such as guanine nucleotide exchange factors (GEFs) or GTPase activating proteins (GAPs) - for the inter-conversion. In contrast, SRP and SR appear to constitute a self-sufficient system, and no such external regulatory factors have been found to date. Instead, our kinetic analyses of the GTPase cycles of SRP and SR (Figure 2). showed that both proteins exchange nucleotides very quickly, so that they do not need GEFs. Further, once SRP and SR form a complex with each other, they reciprocally stimulate the GTPase activity of one another without the need for external regulatory factors. Clearly, these exceptional design features of the SRP and SR GTPases have been evolved to suit their biological function. Elucidation and comparison of the regulatory mechanism of the SRP-type GTPases to those of other members of the GTPase superfamily can provide new insights into the basic principles of molecular recognition and regulation in the cell, as well as shed light on the regulatory mechanism of other 'non-canonical' GTPases whose number is growing by the day.

Figure 2. A thermodynamic and kinetic framework for the GTPase cycles of SRP and SR. Each protein can bind and hydrolyze GTP without forming a higher-order complex (steps 1-3 for SRP and 1’-3’ for SR). SRP and SR, in their GTP-bound forms (T•SRP and SR•T, respectively), associate with one another to form a stable T•SRP•SR•T complex (step 4). The two GTPs are then hydrolyzed by both proteins to yield the D•SRP•SR•D complex (step 5). SRP and SR lose affinity for each other in their GDP-bound forms, leading to dissociation of the complex into individual protein components (step 6).

We use a combination of approaches - including biochemistry, biophysics, mechanistic enzymology and protein engineering - to decipher, at a biochemical and biophysical level, the intimate mechanisms by which this targeting machine works. For example, our kinetic analysis combined with the recent crystal structure of the SRPoSR complex (Figure 3; in collaboration with R. Stroud lab) revealed that SRP and SR use an unusual mechanism to achieve reciprocal activation: an intriguingly symmetric, composite active site is formed at the dimer interface, in which both enzymes reciprocally activate each other by forming direct hydrogen bonds between the two GTP molecules across the dimer interface in trans, and by bringing catalytic residues from each protein into the vicinity of the substrates through extensive conformational changes.

Figure 3. Crystal structure of the complex between the SRP and SR GTPases. Blue and yellow coloring indicate α-helices and β-strands in the SRP GTPase; the corresponding structures in SR are shown in green and pink. The two bound GMPPCP molecules (yellow sticks; GMPPCP is a non-hydrolyzable GTP analogue) are coordinated by two Mg2+ ions (cyan spheres) at the dimer interface.

We have also characterized an extensive array of mutant SRP and SRs that are defective in distinct stages during the SRP-SR interaction. The results show that during the SRP-SR interaction, both GTPases undergo a series of discrete conformational rearrangements that culminate in the activation of GTP hydrolysis from both proteins (Figure 4). A new working model emerges in which each of these conformational stages can provide a potential point at which regulation can be exerted by the interaction partners of SRP and SR - the ribosome, the nascent protein, and the translocon - thereby ensuring an ordered series of cargo loading, delivery and unloading events at the appropriate stages during the protein targeting reaction (Figure 5). Thus instead of using external regulatory factors, the intrinsic conformational flexibility of the SRP and SR GTPases may provide the alternative 'switch' that allows them to drive and regulate the protein targeting reaction.

Figure 4. Model for conformational changes during formation and activation of the SRP•SR complex. Step a is the open --> closed conformational change during complex formation. Step b is the coordinate docking of the conserved insertion box domain (IBD) loops into the active sites. Step c is the coordinate docking of the Arg191 residues in both active sites. Step d is the additional rearrangement of residues that completes one of the GTPase sites. Step e is the rearrangement that completes the other active site. GTP can be hydrolyzed from either the hemi-activated complexes (step f) or the activated complex (step g) to drive complex dissociation.

Figure 5. Conformational changes in the GTPase domains of SRP and SR provide potential regulatory points during the protein targeting reaction. Step 1, SR undergoes an open --> closed conformational change upon association with the membrane translocon. Step 2, SRP undergoes an open --> closed conformational change upon association with the ribosome and cargo protein. Step 3, complex formation between SRP and SR delivers the cargo to the membrane. Step 4, cargo release from SRP allows the SRP•SR complex to undergo additional rearrangements to activate GTP hydrolysis. Step 5, SRP dissociates from SR after GTP hydrolysis. Step 6, premature GTP hydrolysis leads to abortive targeting reactions.

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