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• W2984019444 abstract "Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract During infection chlamydial pathogens form an intracellular membrane-bound replicative niche termed the inclusion, which is enriched with bacterial transmembrane proteins called Incs. Incs bind and manipulate host cell proteins to promote inclusion expansion and provide camouflage against innate immune responses. Sorting nexin (SNX) proteins that normally function in endosomal membrane trafficking are a major class of inclusion-associated host proteins, and are recruited by IncE/CT116. Crystal structures of the SNX5 phox-homology (PX) domain in complex with IncE define the precise molecular basis for these interactions. The binding site is unique to SNX5 and related family members SNX6 and SNX32. Intriguingly the site is also conserved in SNX5 homologues throughout evolution, suggesting that IncE captures SNX5-related proteins by mimicking a native host protein interaction. These findings thus provide the first mechanistic insights both into how chlamydial Incs hijack host proteins, and how SNX5-related PX domains function as scaffolds in protein complex assembly. https://doi.org/10.7554/eLife.22311.001 eLife digest The bacterium Chlamydia trachomatis, commonly known as chlamydia, is a frequent cause of sexually transmitted infections, and a leading cause of blindness due to infection. The bacteria must directly enter the cells of its human host to grow and multiply. Inside a human cell, the bacteria form and then develop within specialized compartments called inclusions that are surrounded by membrane. The outside of the inclusion membrane becomes coated with dozens of unique bacterial proteins. The major role of these bacterial proteins is to hijack other proteins in the human cell to generate and maintain the membrane of the inclusion compartments. One bacterial protein in particular, called IncE, is able to bind to specific host proteins called sorting nexins. These host proteins normally control the formation of tube-like membrane structures, which transport fatty molecules and proteins throughout the cell. The IncE protein is thought to recruit sorting nexins to help shape the inclusion membrane and perhaps control which types of proteins and fatty molecules associate with it. However, until now it was unknown how IncE, or any similar protein for that matter, could specifically hijack a host cell protein. Now, Paul et al. have revealed the three-dimensional structure of a human sorting nexin protein, called SNX5, bound to a small fragment of the IncE protein from chlamydia. The structure shows that the part of SNX5 that associates with IncE is the part of the protein normally thought to interact with specific fatty molecules rather than proteins. Further experiments showed that SNX5 was still recruited to the inclusion compartment when the amount of these fatty molecules in human cells was reduced. However, this was not the case if SNX5 was prevented from interaction with the IncE protein. Paul et al. also observed that the site on SNX5 where IncE binds is almost identical in related proteins from many other species, including zebrafish and worms, most of which are not hosts for chlamydia. This lead them to suspect that IncE hijacks the sorting nexin proteins by mimicking an important host protein that is yet to be discovered. Proteins in the inclusion membrane play many important roles, and so this work on IncE only provides the first glimpse at how these proteins are able to manipulate the machinery of the host cell to their own ends. Further studies will therefore be needed to understand how these proteins exploit their host environment at the molecular level, and might be targeted in new antibacterial approaches. The findings also show how studying bacteria that live within host cells, like chlamydia, can provide insight into how other molecules are normally transported within cells: a process that is fundamental to all living cells. https://doi.org/10.7554/eLife.22311.002 Introduction To counter host defence mechanisms intracellular bacterial pathogens have evolved numerous strategies to evade immune detection, replicate and cause infection. Many pathogens manipulate endocytic pathways to gain entry into host cells and generate a membrane-enclosed replicative niche. This frequently involves hijacking or inhibiting the host cell trafficking machinery, first to generate the pathogen containing vacuole (PCV) and subsequently to prevent fusion with lysosomal degradative compartments. Concomitantly the pathogen often endeavors to decorate the PCV with host proteins and lipids that mimic other host cell organelles in order to circumvent innate immune detection, expand the replicative niche and acquire nutrients to support intracellular replication (Di Russo Case and Samuel, 2016; Personnic et al., 2016). This process is often orchestrated through the action of molecular syringe-like secretion systems that deliver bacterial effector proteins directly into the host cell cytoplasm. Chlamydia trachomatis is arguably one of the most successful human bacterial pathogens by virtue of its capacity to hijack host cell intracellular trafficking and lipid transport pathways to promote infection (Bastidas et al., 2013; Derré, 2015; Elwell et al., 2016; Moore and Ouellette, 2014). C. trachomatis causes nearly 100 million sexually transmitted infections annually worldwide, and if left unchecked leads to various human diseases including infection-induced blindness, pelvic inflammatory disease, infertility and ectopic pregnancy (Aral et al., 2006; Newman et al., 2015). Even though chlamydial infections can generally be treated with antibiotics, persistent infections remain a challenge (Kohlhoff and Hammerschlag, 2015; Mpiga and Ravaoarinoro, 2006). All Chlamydiae share a common dimorphic life cycle, where the bacteria alternates between the infectious but non-dividing elementary body (EB) form, and the non-infectious but replicative reticulate body (RB) form. Following internalization of EBs through a poorly defined endocytic process, the bacteria reside in a membrane-bound vacuole termed the inclusion, where they convert into RBs and replication occurs over 24–72 hr. RBs eventually redifferentiate back to EBs in an asynchronous manner, and are then released to infect neighboring cells (Di Russo Case and Samuel, 2016; Hybiske, 2015; Ward, 1983). The encapsulating inclusion membrane provides the primary interface between the bacteria and the host cell’s cytoplasm and organelles. From the initial stages of invasion until eventual bacterial egress the chlamydial inclusion is extensively modified by insertion of numerous Type-III secreted bacterial effector proteins called inclusion membrane proteins or ‘Incs’. The Incs modulate host trafficking and signaling pathways to promote bacterial survival at different stages, including cell invasion, inclusion membrane remodeling, avoidance of the host cell innate immune defense system, nutrient acquisition and interactions with other host cell organelles (Elwell et al., 2016; Moore and Ouellette, 2014; Rockey et al., 2002). Chlamydiae secrete more than fifty different Inc proteins. While Incs possess little sequence similarity, they share a common membrane topology with cytoplasmic N- and C-terminal domains, separated by two closely spaced transmembrane regions with a short intra-vacuolar loop (Dehoux et al., 2011; Kostriukova et al., 2008; Li et al., 2008; Lutter et al., 2012; Rockey et al., 2002) (Figure 1A). The cytoplasmic N- and C-terminal sequences of the Inc proteins act to bind and manipulate host cell proteins. Reported examples include the binding of the small GTPase Rab4A by CT229 (Rzomp et al., 2006), Rab11A by Cpn0585 (Cortes et al., 2007), SNARE proteins by IncA (Delevoye et al., 2008), centrosomal and cytoskeletal proteins by Inc850 and inclusion protein acting on microtubules (IPAM) (Dumoux et al., 2015; Mital et al., 2015, 2010), myosin phosphatase by CT228 (Lutter et al., 2013), 14-3-3 and Arf family proteins by IncG and InaC (Kokes et al., 2015; Scidmore and Hackstadt, 2001), and the lipid transfer protein CERT by IncD (Derré et al., 2011; Elwell et al., 2011). Despite these reports, there are no known structures of Inc family members either alone or in complex with host effectors. Figure 1 with 2 supplements see all Download asset Open asset SNX5, SNX6 and SNX32 tare recruited to C. trachomatis inclusions. (A) HeLa cells stably expressing the mCherry-Rab25 inclusion membrane marker (red) were infected with C. trachomatis serovar L2 (24 hr) and transfected with myc-tagged SNX expression constructs. The samples were fixed and immunolabeled with anti-myc (green) and anti-chlamydial HtrA antibodies (white) and counterstained with DAPI (blue). Similar experiments using GFP-tagged proteins are shown in Figure 1—figure supplement 1A. https://doi.org/10.7554/eLife.22311.003 Two recent studies have greatly expanded the repertoire of host cell proteins known to associate with chlamydial inclusions and Inc proteins (Aeberhard et al., 2015; Mirrashidi et al., 2015). Both reports confirmed that membrane trafficking proteins are major components of the inclusion proteome; and in particular members of the endosomal sorting nexin (SNX) family are highly enriched. Specifically it was shown that the C. trachomatis IncE/CT116 protein could recruit SNX proteins containing bin-amphiphysin-Rvs (BAR) domains SNX1, SNX2, SNX5 and SNX6 (Mirrashidi et al., 2015). SNX1 and SNX2 are highly homologous and form heterodimeric assemblies with either SNX5 or SNX6 to promote endosomal membrane tubulation and trafficking (van Weering et al., 2012). A fifth protein SNX32 is highly similar to SNX5 and SNX6 but is almost exclusively expressed in the brain and has not yet been characterized. SNX recruitment to the inclusion occurs via the C-terminal region of IncE interacting with the phox-homology (PX) domains of SNX5 or SNX6 (Mirrashidi et al., 2015) (Figure 1A). Interestingly, RNAi-mediated depletion of SNX5/SNX6 does not slow infection but rather increases the production of infectious C. trachomatis progeny suggesting that the SNX recruitment is not done to enable bacterial infection. Instead it was proposed that because SNX proteins regulate endocytic and lysosomal degradation, the manipulation by IncE could be an attempt to circumvent SNX-enhanced bacterial destruction (Aeberhard et al., 2015; Mirrashidi et al., 2015). Here we use X-ray crystallographic structure determination to define the molecular mechanism of SNX5-IncE interaction, and confirm this mechanism using mutagenesis both in vitro and in cells. When bound to SNX5, IncE adopts an elongated β-hairpin structure, with key hydrophobic residues docked into a complementary binding groove encompassing a helix-turn-helix structural extension that is unique to SNX5, SNX6, and the brain-specific homologue SNX32. A striking degree of evolutionary conservation in the IncE-binding groove suggests that IncE co-opts the SNX5-related molecules by displacing a host protein (as yet unidentified) that normally binds to this site. Our work thus provides both the first mechanistic insights into how protein hijacking is mediated by inclusion membrane proteins, and also sheds light on the functional role of the SNX5-related PX domains as scaffolds for protein complex assembly. Results IncE specifically binds and recruits SNX5, SNX6 and SNX32 to C. trachomatis inclusions It was previously shown that the sorting nexins SNX1, SNX2, SNX5 and SNX6 are recruited to the inclusion membrane in C. trachomatis infected cells (Aeberhard et al., 2015; Mirrashidi et al., 2015). We first confirmed this for myc-tagged SNX1, SNX2 and SNX5 in HeLa cells infected with C. trachomatis serovar L2 (MOI ~0.5) for 18 hr. All three proteins were recruited to the inclusion membrane as assessed by co-localisation with the inclusion marker mCherry-Rab25 (Figure 1B) (Teo et al., 2016), as were GFP-tagged SNX1 and SNX5 but not the more distantly homologous GFP-SNX8 (Figure 1—figure supplement 1A). We also observed localization of the SNX proteins to extensive inclusion-associated membrane tubules in a subset of infected cells as described previously (Figure 1—figure supplement 1B) (Aeberhard et al., 2015; Mirrashidi et al., 2015). Interestingly, when infected cells are treated with wortmannin, a pan-specific inhibitor of phosphoinositide-3-kinase (PI3K) activity, we see a loss of the SNX proteins from punctate endosomes, but not from the inclusion membrane (Figure 1—figure supplement 2; Video 1). A similar result is seen for specific inhibition of PtdIns3P production by Vps34 using Vps34-IN1 (Figure 1—figure supplement 2). This offers two possibilities; that either SNX recruitment to the inclusion occurs via protein-protein interactions, and does not depend on the presence of 3-phosphoinositide lipids that typically recruit SNX proteins to endosomal membranes, or alternatively that PI3Ks are not present at the inclusion and therefore wortmannin treatment has no effect at this particular compartment. Given our structural and mutagenesis studies below we favor the former explanation. Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Movie showing that wortmannin disrupts SNX5 recruitment to endosomes but not the chlamydial inclusion. HeLa cells stably expressing mCherry-Rab25 (red) were transfected transiently with GFP-SNX5 (green) and infected with Chlamydia trachomatis L2 for 24 hr. Time-lapse videomicroscopy was performed using an interval of 1 min on an inverted Nikon Ti-E deconvolution microscope with environmental control at 40 x magnification. 10 min into recording 100 nM wortmannin was added. https://doi.org/10.7554/eLife.22311.006 Mirrashidi et al. (2015), demonstrated an in vitro interaction between IncE and the SNX5 and SNX6 PX domains. To confirm their direct association we assessed the binding affinities using isothermal titration calorimetry (ITC) (Figure 2A; Table 1). Initial experiments with the human SNX5 and SNX6 PX domains showed robust interactions with the IncE C-terminal domain (residues 107–132). The affinities (Kd) for SNX5 and SNX6 were essentially indistinguishable (0.9 and 1.1 µM respectively), but we detected no interaction with the PX domain of SNX1 confirming the binding specificity. The PX domains of SNX5 and SNX6 possess a helix-turn-helix structural insert (Koharudin et al., 2009), which is not found in any other SNX family members except for SNX32 (Figure 2B), a homologue expressed primarily in neurons (BioGPS (Wu et al., 2009)). Confirming a common recruitment motif in the SNX5-related proteins, ITC showed a strong interaction between IncE and the SNX32 PX domain similar to SNX5 and SNX6 (Kd = 1.0 µM) (Figure 2A; Table 1), and SNX32 was robustly recruited to inclusion membranes in infected cells (Figure 1B; Figure 1—figure supplement 1A). Overall, our data indicates that a common structure within the SNX5, SNX6 and SNX32 PX domains is required for IncE interaction. Figure 2 Download asset Open asset IncE from C. trachomatis binds the PX domains of SNX5, SNX6 and SNX32. (A) Binding affinity between IncE peptide (residues 107–132) and SNX PX domains by ITC. Top panels show raw data and lower panels show normalised integrated data. See Table 1 for the calculated binding parameters. Truncation analyses of the IncE peptide by ITC are shown in Figure 3, Table 2. (B) Sequence alignment of human SNX1, SNX5, SNX6 and SNX32 PX domains. Conserved residues are indicated in red. Side-chains that directly contact IncE in the crystal structure are indicated with black circles. Mutations that block IncE binding are highlighted with red triangles, and mutations that do not affect binding indicated with green circles. Secondary structure elements derived from the SNX5 crystal structure are indicated above. (C) Sequence alignment of IncE from C. trachomatis and putative homologues from C. muridarum and C. suis. IncE side-chains that directly contact SNX5 in the crystal structure are indicated with black circles. Mutations that block SNX5 binding are highlighted with red triangles, and mutations that do not affect binding indicated with green circles. Predicted transmembrane regions are indicated and C-terminal IncE sequences that form β-strands in complex with SNX5 are shown. https://doi.org/10.7554/eLife.22311.007 Table 1 Thermodynamic parameters of IncE binding to SNX PX domains*. https://doi.org/10.7554/eLife.22311.008 Sample cellTitrantKd (µM)△H (kcal/mol)T△S (kcal/mol)△G (kcal/mol)NSNX5 PXIncE peptide†0.95 ± 0.07−6.9 ± 0.3−1.9 ± 0.05−8.2 ± 0.011.01 ± 0.01SNX6 PXIncE peptide1.13 ± 0.08−5.0 ± 0.9−3.0 ± 1−8.0 ± 0.071.01 ± 0.08SNX32 PXIncE peptide1.15 ± 0.07−6.9 ± 0.4−1.3 ± 0.8−8.2 ± 0.41.06 ± 0.005SNX1 PXIncE peptideNo binding *Values are the mean from three experiments ±SEM. b.†IncE synthetic peptide sequence PANGPAVQFFKGKNGSADQVILVTQ. Finally we tested a series of IncE truncation mutants for their binding to the SNX5 PX domain (Figure 3A, B and C; Table 2). Synthetic peptides were used with single amino acids removed sequentially from the N and C-terminus to determine the minimal sequence required for binding. These experiments showed that the shortest region of IncE able to support full binding to SNX5 encompasses residues 110–131 (GPAVQFFKGKNGSADQVILVT), while a shorter fragment containing residues 113–130 (VQFFKGKNGSADQVILV) can bind to SNX5 with a slightly reduced affinity. While variations are observed across the different C. trachomatis serovars (Harris et al., 2012) the SNX5-binding sequence appears to be preserved in all detected variants (Figure 3D). A comparison with other chlamydial species suggests that IncE is not very widely conserved in this Genus, being clearly identifiable only in the closely related mouse pathogen C. muridarum and swine pathogen C. suis (Figure 2C). Residues required for binding to SNX5 are preserved in these IncE homologues, but whether SNX proteins are also recruited during infection by these other chlamydial species remains to be determined. Figure 3 Download asset Open asset IncE residues 110–131 are sufficient for full recognition of the SNX5 PX domain. (A) Representative ITC experiments for truncated IncE peptides. These experiments were conducted using a single batch of SNX5 PX domain to minimize batch-to-batch protein variation. (B) Plots of the affinity constants for selected peptides to highlight the progressive loss of binding with N and C-terminal truncations. (C) Sequences of the truncated IncE peptides are given, with a qualitative indication of binding strength relative to the IncE_1 peptide containing residues 107–132. Full binding is indicated by ‘++’ reduced binding by ‘+’ and lack of binding by ‘−‘. All sequence information and their Kd values are given in Table 2. When compared to the reference ITC experiment the binding affinity of peptides was unaffected when the first three N-terminal residues were removed (IncE_2, IncE_3 and IncE_4) and gradually became weaker until IncE_7, after which binding was abolished. Results from IncE_6 are inconclusive due to the difficulty in successfully dissolving the peptides in buffer (n.d.). C-terminal truncations showed that IncE_14 and IncE_15 had similar high binding affinities to the reference, while the binding of IncE_16 and IncE_17 became progressively weaker and peptides shorter than IncE_17 showed no binding. This data indicates that the minimal IncE binding sequence retaining full SNX5 binding is GPAVQFFKGKNGSADQVILVT, and a shorter fragment VQFFKGKNGSADQVIL can bind to SNX5, albeit with a slightly reduced affinity. (D) Sequence alignment of IncE from different C. trachomatis serovars. https://doi.org/10.7554/eLife.22311.009 Table 2 ITC data for SNX5 PX domain binding to truncated and mutated IncE peptides*. https://doi.org/10.7554/eLife.22311.010 ProteinPeptideSequenceKd (µM)△H (kcal/mol)T△S (kcal/mol)△G (kcal/mol)NSNX5 PXIncE_1PANGPAVQFFKGKNGSADQVILVTQ0.95 ± 0.07−6.9 ± 0.3−1.9 ± 0.05−8.2 ± 0.011.01 ± 0.01IncE_2 ANGPAVQFFKGKNGSADQVILVTQ1−5.0−2.6−8.10.98IncE_3 NGPAVQFFKGKNGSADQVILVTQ0.93−6.7−1.4−8.11.03IncE_4 GPAVQFFKGKNGSADQVILVTQ0.87−6.8−1.2−8.21.03IncE_5 PAVQFFKGKNGSADQVILVTQ2−5.9−1.2−8.30.99IncE_6 AVQFFKGKNGSADQVILVTQ/////IncE_7 VQFFKGKNGSADQVILVTQ2.2−6.9−1.1−7.70.99IncE_8 QFFKGKNGSADQVILVTQNo binding////IncE_9 FFKGKNGSADQVILVTQNo binding////IncE_10 FKGKNGSADQVILVTQNo binding////IncE_11 KGKNGSADQVILVTQNo binding////IncE_12 GKNGSADQVILVTQNo binding////IncE_13 KNGSADQVILVTQNo binding////IncE_14PANGPAVQFFKGKNGSADQVILVT0.72−5.1−1.6−8.41IncE_15PANGPAVQFFKGKNGSADQVILV0.97−6.5−1.3−8.20.98IncE_16PANGPAVQFFKGKNGSADQVIL1.1−5.6−1.4−8.120.99IncE_17PANGPAVQFFKGKNGSADQVI8.7−2.7−2.5−6.90.99IncE_18PANGPAVQFFKGKNGSADQVNo binding////IncE_19PANGPAVQFFKGKNGSADQNo binding////IncE_20PANGPAVQFFKGKNGSADNo binding////IncE_21PANGPAVQFFKGKNGSANo binding////IncE_22PANGPAVQFFKGKNGSNo binding////IncE_23PANGPAVQFFKGKNGNo binding////IncE_24PANGPAVQFFKGKNNo binding////IncE Q115APANGPAVAFFKGKNGSADQVILVTQ6.3−5.3−1.6−6.90.90IncE F116DPANGPAVQAFKGKNGSADQVILVTQNo bindingIncE K118APANGPAVQFFAGKNGSADQVILVTQ2.8−6.0−1.5−7.50.91IncE V127DPANGPAVQFFKGKNGSADQDILVTQNo bindingSNX5 PX L133DIncE_1PANGPAVQFFKGKNGSADQVILVTQNo bindingSNX5 PX F136AIncE_1PANGPAVQFFKGKNGSADQVILVTQNo bindingSNX5 PX E144AIncE_1PANGPAVQFFKGKNGSADQVILVTQ15−9.9−3.1−130.99 *Except for IncE_1 all other peptide-binding experiments were performed only once. The crystal structure of IncE in complex with the SNX5 PX domain The canonical PX domain structure is composed of a three-stranded β-sheet (β1, β2 and β3) followed by three close-packed α-helices. The first and second α-helices are connected by an extended proline-rich sequence. Typically PX domains have been found to bind to the endosome-enriched lipid phosphatidylinositol-3-phosphate (PtdIns3P) via a basic pocket formed at the junction between the β3 strand, α1 helix and Pro-rich loop. In contrast SNX5, SNX6 and SNX32 possess major alterations in the PtdIns3P-binding pocket that preclude canonical lipid head-group docking (see below). In addition they possess a unique extended helix-turn-helix insert between the Pro-rich loop and α2 helix of unknown function (Figure 2B) (Koharudin et al., 2009). To determine the structure of the SNX5-IncE complex we generated a fusion protein encoding the human SNX5 PX domain (residues 22–170) and C. trachomatis IncE C-terminal sequence (residues 108–132) attached at the PX domain C-terminus Figure 4—figure supplement 1A). This construct readily crystallised in several crystal forms, and we were able to determine the structure of the complex in three different spacegroups (Figure 4; Table 3; Figure 4—figure supplement 1B). Confirming that the fusion does not alter complex formation, the short linker region is disordered, and the mode of IncE-binding to SNX5 is identical in all three structures (Figure 4—figure supplement 1C and D). Because of the higher resolution, we focus our discussions on the structure of the SNX5 PX-IncE complex observed in the P212121 crystal form. The first three IncE N-terminal residues (Pro107, Ala108, Asn109) and the last three IncE C-terminal residues (Val130, Thr131, Gln132) were not modeled due to lack of electron density, suggesting disorder and matching precisely with our mapping experiments showing these residues are not necessary for SNX5 association. Figure 4 with 1 supplement see all Download asset Open asset Structure of the SNX5 PX domain in complex with the IncE C-terminal domain. (A) Crystal structure of the SNX5 PX domain (yellow) in complex with IncE residues 107–132 (magenta) shown in cartoon representation. (B) Backbone atoms of the SNX5 and IncE proteins are shown to highlight the prominent β-sheet augmentation mediating the association between the two molecules. (C) Close up view of the SNX5-IncE interface highlighting specific contact areas at the N-terminus of the IncE peptide. (D) Close up of the SNX5-IncE interface highlighting specific contact areas at the hairpin loop of the IncE peptide shown at 90° to Figure 4C. (E). Close up of the SNX5-IncE interface highlighting contact areas at the C-terminus of the IncE peptide in approximately the same orientation as Figure 4C. Residues in SNX5 (Phe136) and IncE (Phe116) that are critical for binding based on mutagenesis are boxed. https://doi.org/10.7554/eLife.22311.011 Table 3 Summary of crystallographic structure determination statistics*. https://doi.org/10.7554/eLife.22311.013 CrystalSNX5 PX-IncE Form 1SNX5 PX-IncE Form 2SNX5 PX-IncE Form 3PDB ID5TGI5TGJ5TGHData collectionWavelength (Å)0.953700.953700.95370Space groupP212121I2P32Cell dimensions a, b, c (Å)60.7, 67.5, 88.258.4, 80.3, 94.6100.6, 100.6, 71.7 α, β, γ (°)90, 90, 9090, 97.2, 9090, 90, 120Resolution (Å)60.7–1.98 (2.03–1.98)31.9–2.6 (2.72–2.60)50.3–2.80 (2.95–2.80)Rmerge0.104 (0.525)0.153 (0.659)0.101 (0.713)Rmeas0.112 (0.572)0.18 (0.777)0.124 (0.873)Rpim0.042 (0.225)0.096 (0.408)0.051 (0.363)<I> / σI12.4 (3.4)39.6 (3.2)11.7 (2.3)Total number reflections178868 (11000)46691 (5757)115149 (16861)Total unique reflections26075 (1805)13432 (1632)20001 (2923)Completeness (%)100 (100)99.9 (100.0)100 (100)Multiplicity6.9 (6.1)3.5 (3.5)5.8 (5.8)Half-set correlation (CC(1/2))0.997 (0.868)0.986 (0.55)0.997 (0.683)RefinementResolution (Å)45.1–1.98 (2.02–1.98)31.9–2.6 (2.69–2.60)41.2–2.8 (2.87–2.80)No. reflections/No. Rfree26021/200013421/1342 (1208/134)19975/1972 (1301/144)Rwork/Rfree0.192/0.214 (0.221/0.246)0.199/0.242 (0.276/0.332)0.236/0.254 (0.329/0.372)No. atoms Protein257926195189 Solvent281690Average B-factor (Å2)31.842.556.0R.m.s deviations Bond lengths (Å)0.0120.0110.015 Bond angles (°)1.271.151.27 *Highest resolution shell is shown in parentheses. The IncE sequence forms a long β-hairpin structure that binds within a complementary groove at the base of the extended α-helical insertion of the SNX5 PX domain and adjacent to the β-sheet sub-domain (Figure 4A; Video 2). The β-hairpin structure of IncE (N-terminal βA and C-terminal βB strands) is directly incorporated as a β-sheet augmentation of the β1, β2 and β3 strands of SNX5 (Figure 4B). The N-terminal βA strand of the IncE sequence (Gly111-Lys118) forms the primary interface with SNX5, making main-chain hydrogen bonds with the β1 strand of the SNX5 PX domain for the stable positioning of the IncE structure. The two anti-parallel β-strands of IncE are connected by a short loop (Gly119-Ala124) that makes no direct contact with the SNX5 protein, and the C-terminal IncE βB strand (Asp125-Val130) forms an interface with the extended α-helical region of the SNX5 PX domain. Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Animation highlighting the mechanism of interaction between SNX5 and IncE. The SNX5 PX domain is shown in yellow ribbons and the IncE peptide is shown in magenta. https://doi.org/10.7554/eLife.22311.014 Detailed views of the SNX5-IncE interface are shown in Figure 4C, D and E. Aside from main-chain hydrogen bonding to form the extended β-sheet, IncE engages in several critical side-chain interactions with the relatively hydrophobic SNX5 binding groove. At the N-terminus of the βA strand Val114 of IncE inserts into a pocket formed primarily by Tyr132 and Phe136 on the SNX5 α’’ helix (Figure 4C). A major contribution comes from IncE Phe116, with π-stacking occurring with the Phe136 side-chain and hydrophobic docking with Val140 of SNX5 (Figure 4D). Adjacent to IncE Phe116 at the end of the βA strand Lys118 makes an electrostatic contact with SNX5 Glu144. Finally, at the C-terminal end of the IncE βB strand Val127 and Leu129 contact an extended SNX5 surface composed of Leu133, Tyr132 and Met106 (Figure 4E). Mutations in the SNX5-IncE interface disrupt complex formation in vitro and in cells To verify the crystal structure we mutated residues from both SNX5 and IncE and measured their affinities using ITC (Figure 5A and B; Table 2). At the interface between SNX5 and IncE several side chains make key contributions to peptide recognition. Because Leu133 and Phe136 residues in SNX5 are located at the core of the IncE-binding interface, and also due to the structural rearrangements these residues make on binding (see below), we reasoned that L133D and F136A mutations would inhibit the interaction. Indeed these mutants abolished association with the IncE peptide (Figure 5A). The reciprocal mutations in IncE residues F116A and V127D also abolished binding to the SNX5 PX domain (Figure 5B), and the SNX6 and SNX32 PX domains (Figure 5—figure supplement 1), demo" @default.
• W2984019444 created "2019-11-22" @default.
• W2984019444 creator A5015580385 @default.
• W2984019444 creator A5019522016 @default.
• W2984019444 creator A5035473732 @default.
• W2984019444 creator A5056823136 @default.
• W2984019444 creator A5071429877 @default.
• W2984019444 creator A5090169720 @default.
• W2984019444 date "2016-12-09" @default.
• W2984019444 modified "2023-09-23" @default.
• W2984019444 title "Author response: Structural basis for the hijacking of endosomal sorting nexin proteins by Chlamydia trachomatis" @default.
• W2984019444 doi "https://doi.org/10.7554/elife.22311.029" @default.
• W2984019444 hasPublicationYear "2016" @default.
• W2984019444 type Work @default.
• W2984019444 sameAs 2984019444 @default.
• W2984019444 citedByCount "0" @default.
• W2984019444 crossrefType "peer-review" @default.
• W2984019444 hasAuthorship W2984019444A5015580385 @default.
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