Irmgard Sinning

Following the signal sequence from ribosomal tunnel exit to signal recognition particle

Membrane and secretory proteins can be co-translationally inserted into or translocated across the membrane. This process is dependent on signal sequence recognition on the ribosome by the signal recognition particle (SRP), which results in targeting of the ribosome–nascent-chain complex to the protein-conducting channel at the membrane. Here we present an ensemble of structures at subnanometre resolution, revealing the signal sequence both at the ribosomal tunnel exit and in the bacterial and eukaryotic ribosome–SRP complexes. Molecular details of signal sequence interaction in both prokaryotic and eukaryotic complexes were obtained by fitting high-resolution molecular models. The signal sequence is presented at the ribosomal tunnel exit in an exposed position ready for accommodation in the hydrophobic groove of the rearranged SRP54 M domain. Upon ribosome binding, the SRP54 NG domain also undergoes a conformational rearrangement, priming it for the subsequent docking reaction with the NG domain of the SRP receptor. These findings provide the structural basis for improving our understanding of the early steps of co-translational protein sorting.

Delivering proteins for export from the cytosol

Correct protein function depends on delivery to the appropriate cellular or subcellular compartment. Following the initiation of protein synthesis in the cytosol, many bacterial and eukaryotic proteins must be integrated into or transported across a membrane to reach their site of function. Whereas in the post-translational delivery pathway ATP-dependent factors bind to completed polypeptides and chaperone them until membrane translocation is initiated, a GTP-dependent co-translational pathway operates to couple ongoing protein synthesis to membrane transport. These distinct pathways provide different solutions for the maintenance of proteins in a state that is competent for membrane translocation and their delivery for export from the cytosol.

High-resolution X-ray and NMR Structures of the SMN Tudor Domain: Conformational Variation in the Binding Site for Symmetrically Dimethylated Arginine Residues

The SMN protein, which is linked to spinal muscular atrophy (SMA), plays an important role in the assembly of the spliceosomal small nuclear ribonucleoprotein complexes. This function requires binding of SMN to the arginine-glycine (RG) rich C-terminal tails of the Sm proteins, which contain symmetrically dimethylated arginine residues (sDMA) in vivo. Using NMR titrations, we show that the SMN Tudor domain recognizes these sDMAs in the methylated RG repeats. Upon complex formation a cluster of conserved aromatic residues in the SMN Tudor domain interacts with the sDMA methyl groups. We present two high resolution structures of the uncomplexed SMN Tudor domain, a 1.8A crystal structure and an NMR structure that has been refined against a large number of backbone and side-chain residual dipolar couplings. The backbone conformation of both structures is very similar, however, differences are observed for the cluster of conserved aromatic side-chains in the sDMA binding pocket. In order to validate these variations we introduce a novel application of residual dipolar couplings for aromatic rings. We show that structural information can be derived from aromatic ring residual dipolar couplings, even in the presence of internal motions such as ring flipping. These residual dipolar couplings and ring current shifts independently confirm that the SMN Tudor domain adopts two different conformations in the sDMA binding pocket. The observed structural variations may play a role for the recognition of sDMAs.

Anionic phospholipids are involved in membrane association of FtsY and stimulate its GTPase activity

FtsY, the Escherichia coli homologue of the eukaryotic signal recognition particle (SRP) receptor α‐subunit, is located in both the cytoplasm and inner membrane. It has been proposed that FtsY has a direct targeting function, but the mechanism of its association with the membrane is unclear. FtsY is composed of two hydrophilic domains: a highly charged N‐terminal domain (the A‐domain) and a C‐terminal GTP‐binding domain (the NG‐domain). FtsY does not contain any hydrophobic sequence that might explain its affinity for the inner membrane, and a membrane‐anchoring protein has not been detected. In this study, we provide evidence that FtsY interacts directly with E.coli phospholipids, with a preference for anionic phospholipids. The interaction involves at least two lipid‐binding sites, one of which is present in the NG‐domain. Lipid association induced a conformational change in FtsY and greatly enhanced its GTPase activity. We propose that lipid binding of FtsY is important for the regulation of SRP‐mediated protein targeting.

Protein targeting by the signal recognition particle

Abstract Protein targeting by the signal recognition particle (SRP) is universally conserved and starts with the recognition of a signal sequence in the context of a translating ribosome. SRP54 and FtsY, two multidomain proteins with guanosine triphosphatase (GTPase) activity, are the central elements of the SRP system. They have to coordinate the presence of a signal sequence with the presence of a vacant translocation channel in the membrane. For coordination the two GTPases form a unique, nearly symmetric heterodimeric complex in which the activation of GTP hydrolysis plays a key role for membrane insertion of substrate proteins. Recent results are integrated in an updated perception of the order of events in SRP-mediated protein targeting.

Membrane curvature induced by Arf1-GTP is essential for vesicle formation.

The GTPase Arf1 is considered as a molecular switch that regulates binding and release of coat proteins that polymerize on membranes to form transport vesicles. Here, we show that Arf1-GTP induces positive membrane curvature and find that the small GTPase can dimerize dependent on GTP. Investigating a possible link between Arf dimerization and curvature formation, we isolated an Arf1 mutant that cannot dimerize. Although it was capable of exerting the classical role of Arf1 as a coat receptor, it could not mediate the formation of COPI vesicles from Golgi-membranes and was lethal when expressed in yeast. Strikingly, this mutant was not able to deform membranes, suggesting that GTP-induced dimerization of Arf1 is a critical step inducing membrane curvature during the formation of coated vesicles.

Signal recognition particle mediates post‐translational targeting in eukaryotes

Signal recognition particle (SRP) plays a central role in the delivery of classical secretory and membrane proteins to the endoplasmic reticulum (ER). All nascent chains studied to date dissociate from SRP once released from the ribosome, thereby supporting a strictly cotranslational mode of action for eukaryotic SRP. We now report a novel post‐translational function for SRP in the targeting of tail‐anchored (TA) proteins to the ER. TA proteins possess a hydrophobic membrane insertion sequence at their C‐terminus such that it can only emerge from the ribosome after translation is terminated. We show that SRP can associate post‐translationally with this type of ER‐targeting signal, and deliver newly synthesised TA proteins to the ER membrane by a pathway dependent upon GTP and the SRP receptor. We find that dependency upon this SRP‐dependent route is precursor specific, and propose a unifying model to describe the biogenesis of TA proteins in vivo.

FlhA provides the adaptor for coordinated delivery of late flagella building blocks to the type III secretion system

Flagella are the bacterial organelles of motility and can play important roles in pathogenesis. Flagella biosynthesis requires the coordinated export of huge protein amounts from the cytosol to the nascent flagellar structure at the cell surface and employs a type III secretion system (T3SS). Here we show that the integral membrane protein FlhA from the gram-positive bacterium Bacillus subtilis acts as an adaptor for late export substrates at the T3SS. The major filament protein (flagellin) and the filament-cap protein (FliD) bind to the FlhA cytoplasmic domain (FlhA-C) only in complex with their cognate chaperones (FliS and FliT). To understand the molecular details of these interactions we determined the FlhA-C crystal structure at 2.3 Å resolution. FlhA-C consists of an N-terminal linker region, three subdomains with a novel fold, and a disordered region essential for the adaptor function. We show that the export protein FliJ associates with the linker region and modulates the binding properties of FlhA-C. While the interaction of FliD/FliT is enhanced, flagellin/FliS is not affected. FliJ also keeps FliT associated with FlhA-C and excess of FliT inhibits binding of FliD/FliT, suggesting that empty FliT chaperones stay associated with FliJ after export of FliD. Taken together, these results allow to propose a model that explains how the T3SS may switch from the stoichiometric export of FliD to the high-throughput secretion of flagellin.

Crystal structure of the complete core of archaeal signal recognition particle and implications for interdomain communication

Targeting of secretory and membrane proteins by the signal recognition particle (SRP) is evolutionarily conserved, and the multidomain protein SRP54 acts as the key player in SRP-mediated protein transport. Binding of a signal peptide to SRP54 at the ribosome is coordinated with GTP binding and subsequent complex formation with the SRP receptor. Because these functions are localized to distinct domains of SRP54, communication between them is essential. We report the crystal structures of SRP54 from the Archaeon Sulfolobus solfataricus with and without its cognate SRP RNA binding site (helix 8) at 4-Å resolution. The two structures show the flexibility of the SRP core and the position of SRP54 relative to the RNA. A long linker helix connects the GTPase (G domain) with the signal peptide binding (M) domain, and a hydrophobic contact between the N and M domains relates the signal peptide binding site to the G domain. Hinge regions are identified in the linker between the G and M domains (292-LGMGD) and in the N-terminal part of the M domain, which allow for structural rearrangements within SRP54 upon signal peptide binding at the ribosome.

Glutamate-binding affinity of Drosophila metabotropic glutamate receptor is modulated by association with lipid rafts

Metabotropic glutamate receptors (mGluRs) are responsible for the effects of glutamate in slow synaptic transmission, and are implicated in the regulation of many processes in the CNS. Recently, we have reported the expression and purification of a mGluR from Drosophila melanogaster (DmGluRA), a homologue of mammalian group II mGluRs. We have shown that ligand binding to reconstituted DmGluRA requires the presence of ergosterol in the liposomes [Eroglu, C., Cronet, P., Panneels, V., Beaufils, P. & Sinning, I. (2002) EMBO Rep. 3, 491-496]. Here we demonstrate that the receptor exists in different affinity states for glutamate, depending on the membrane composition. The receptor is in a high-affinity state when associated with sterol-rich lipid microdomains (rafts), and in a low-affinity state out of rafts. Enrichment of the membranes with cholesterol shifts the receptor into the high-affinity state, and induces its association with rafts. The receptor was crosslinked to photocholesterol. Our data suggest that sterol-rich lipid rafts act as positive allosteric regulators of DmGluRA.