Functioning as key players in cellular regulation of membrane curvature, BAR-domain proteins bend bilayers and recruit interaction partners through poorly understood mechanisms. Using electron cryomicroscopy, we present reconstructions of full-length endophilin and its N-terminal N-BAR domain in their membrane-bound state. Endophilin lattices expose large areas of membrane surface, and are held together by promiscuous interactions between endophilin's amphipathic N-terminal helices. Coarse-grained molecular dynamics simulations reveal that endophilin lattices are highly dynamic, and that the N-terminal helices are required for formation of a stable and regular scaffold. Furthermore, endophilin accommodates different curvatures through a quantized addition or removal of endophilin dimers, which in some cases causes dimerization of endophilin's SH3 domains, suggesting that the spatial presentation of SH3-domains rather than affinity governs the recruitment of downstream interaction partners.

The signal recognition particle (SRP) GTPases Ffh and FtsY play a central role in co-translational targeting of proteins, assembling in a GTP-dependent manner to generate the SRP targeting complex at the membrane. A suite of residues in FtsY have been identified that are essential for the hydrolysis of GTP that accompanies disengagement. We have argued previously on structural grounds that this region mediates interactions that serve to activate the complex for disengagement and term it the activation region. We report here the structure of a complex of the SRP GTPases formed in the presence of GDP:AlF 4 . This complex accommodates the putative transition-state analog without undergoing significant change from the structure of the ground-state complex formed in the presence of the GTP analog GMPPCP. However, small shifts that do occur within the shared catalytic chamber may be functionally important. Remarkably, an external nucleotide interaction site was identified at the activation region, revealed by an unexpected contaminating GMP molecule bound adjacent to the catalytic chamber. This site exhibits conserved sequence and structural features that suggest a direct interaction with RNA plays a role in regulating the activity of the SRP targeting complex.

FtsY and Ffh are structurally similar prokaryotic Signal Recognition Particle GTPases that play an essential role in the Signal Recognition Particle (SRP)-mediated cotranslational targeting of proteins to the membrane. The two GTPases assemble in a GTP-dependent manner to form a heterodimeric SRP targeting complex. We report here the 2.1 Å X-ray structure of FtsY from T. aquaticus bound to GDP. The structure of the monomeric protein reveals, unexpectedly, canonical binding interactions for GDP. A comparison of the structures of the monomeric and complexed FtsY NG GTPase domain suggests that it undergoes a conformational change similar to that of Ffh NG during the assembly of the symmetric heterodimeric complex. However, in contrast to Ffh, in which the C-terminal helix shifts independently of the other subdomains, the C-terminal helix and N domain of T. aquaticus FtsY together behave as a rigid body during assembly, suggesting distinct mechanisms by which the interactions of the NG domain "module" are regulated in the context of the two SRP GTPases.

Ffh and FtsY are GTPase components of the signal recognition particle co-translational targeting complex that assemble during the SRP cycle to form a GTP-dependent and pseudo two-fold symmetric heterodimer. Previously the SRP GTPase heterodimer has been stabilized and purified for crystallographic studies using both the non-hydrolysable GTP analog GMPPCP and the pseudotransition state analog GDP:AlF 4 , revealing in both cases a buried nucleotide pair that bridges and forms a key element of the heterodimer interface. A complex of Ffh and FtsY from T. aquaticus formed in the presence of the analog GMPPNP could not be obtained, however. The origin of this failure was previously unclear, and it was thought to have arisen from either instability of the analog, or, alternatively, from differences in its interactions within the tightly conscribed composite active site chamber of the complex. Using insights gained from the previous structure determinations, we have now determined the structure of the SRP GTPase targeting heterodimer stabilized by the nonhydrolysable GTP analog GMPPNP. The structure demonstrates how the different GTP analogs are accommodated within the active site chamber despite slight differences in the geometry of the phosphate chain. It also reveals a K + coordination site at the highly conserved DARGG loop at the N/G interdomain interface.The Signal Recognition Particle (SRP) mediates signal peptide recognition and co-translational targeting of secreted and membrane proteins to the membrane translocon (Walter and Johnson, 1994;Keenan et al., 2001). SRP is a phylogenetically conserved ribonucleoprotein that comprises, in prokaryotes, Ffh, the SRP GTPase, and the 4.5S RNA (Luirink and Dobberstein, 1994). The primary structure of Ffh includes three domains, the N and G domains, and the M domain (Bernstein et al., 1989;Römisch et al., 1989). The M domain provides sites for signal sequence recognition and for interaction with the RNA (Zopf et al., 1990;Luirink et al., 1992;Lütcke et al., 1992). The N and G domains of the SRP GTPase, together the 'NG' domain, form a structural and functional unit (Freymann et al., 1997). The membrane associated receptor for SRP is also phylogenetically conserved, and its primary structure includes an NG GTPase as well (Montoya et al., 1997). The two SRP NG GTPases interact directly with each other forming a GTP-dependent heterodimeric targeting complex that plays a central role in co-translational protein targeting to the membrane (Powers and Walter, 1995;Powers and Walter, 1997;Rapiejko and Gilmore, 1997;Song et al., 2000;Mandon et al., 2003). * corresponding author: phone: 312/503-1877, fax: 312/503-5349, email: freymann@northwestern.edu. Author Contributions J.G.S. purified the GMPPNP-stabilized complex and setup the crystallization screen; D.M.F. carried out the crystallographic work and wrote the manuscript.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providi...

An insertion between iscA and hscB of the Xenorhabdus nematophila iscRSUA-hscBA-fdx locus, predicted to encode Fe-S assembly machinery, prevented colonization of Steinernema carpocapsae nematodes. The insertion disrupted cotranscription of iscA and hscB, but did not reduce hscBA expression, suggesting that X. nematophila requires coordinated expression of the isc-hsc-fdx locus for colonization.The intestines of Steinernema carpocapsae infective juvenilestage (IJ) nematodes are mutualistically colonized by Xenorhabdus nematophila bacteria (4). Germfree S. carpocapsae nematode eggs applied to lawns of X. nematophila will develop through juvenile and reproductive stages (32) until high nematode population density and low nutrient concentrations result in formation of progeny IJ nematodes colonized by X. nematophila (13,17). Our lab is investigating molecular mechanisms mediating X. nematophila-S. carpocapsae interactions by identifying X. nematophila genes required for IJ nematode colonization.Identification of a colonization-defective X. nematophila mutant. X. nematophila HGB081 (Table 1) was mutagenized with mini-Tn10, using plasmid pKV124 (31) transferred by conjugation from S17-1 (pir) (7). Exconjugants selected on rifampin (100 g/ml) and chloramphenicol (30 g/ml) were individually cultivated with S. carpocapsae (Strain All) nematodes. Progeny IJ nematodes were harvested from each coculture and microscopically examined for the presence or absence of X. nematophila colonizers (32). One of 692 bacterial mutants screened was deficient in colonization and was designated HGB166. This frequency (0.16%) is within the range found in an independent Tn5 screen (8) and suggests that colonization genes comprise a small mutagenesis target.In a quantitative colonization assay (8), HGB166 exhibited a severe colonization defect ( (Fig. 1 and Table 1). Plasmid isolation, sequencing, and sequence analysis were carried out as previously described (8).The HGB166 colonization defect is caused by Tn10 insertion in an isc-hsc-fdx locus. The transposon insertion of HGB166 is in a conserved locus with the gene order iscRSUA-hscBA-fdx, 3 nucleotides downstream of the predicted iscA stop codon and 56 nucleotides upstream of the putative hscB start codon (19,34) (Fig. 1). In Escherichia coli, this locus encodes iron-sulfur center assembly machinery (12,19,26,28,29). Iron-sulfur centers are components of many cellular proteins with redox, regulatory, or catalytic function (3), and the mechanism of their assembly by isc-hsc-fdx-encoded proteins has begun to be elucidated. IscS, a cysteine desulfurase, donates sulfur to a nascent cluster (6, 29, 34) forming on the scaffolding protein IscU (1). hscA and hscB encode Hsc66 and Hsc20, respectively (10,21,30), which interact with IscU, resulting in increased Hsc66 ATPase activity (9,23). IscA is proposed to be an alternative scaffold for cluster formation (12) or an iron donor for iron-sulfur assembly on Fdx, an electron-transferring ferredoxin (15).To determine if the HGB166 colonization defe...