Lipid binding by the N-terminal motif mediates plasma membrane localization of Bordetella effector protein BteA
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100607
ISSN1083-351X
AutoresIvana Malcová, Ladislav Bumba, Filip Uljanic, Darya Kuzmenko, Jana Nedomova, Jana Kamanová,
Tópico(s)Escherichia coli research studies
ResumoThe respiratory pathogens Bordetella pertussis and Bordetella bronchiseptica employ a type III secretion system (T3SS) to inject a 69-kDa BteA effector protein into host cells. This effector is known to contain two functional domains, including an N-terminal lipid raft targeting (LRT) domain and a cytotoxic C-terminal domain that induces nonapoptotic and caspase-1–independent host cell death. However, the exact molecular mechanisms underlying the interaction of BteA with plasma membrane (PM) as well as its cytotoxic activity in the course of Bordetella infections remain poorly understood. Using a protein–lipid overlay assay and surface plasmon resonance, we show here that the recombinant LRT domain binds negatively charged membrane phospholipids. Specifically, we determined that the dissociation constants of the LRT domain–binding liposomes containing phosphatidylinositol 4,5-bisphosphate, phosphatidic acid, and phosphatidylserine were ∼450 nM, ∼490 nM, and ∼1.2 μM, respectively. Both phosphatidylserine and phosphatidylinositol 4,5-bisphosphate were required to target the LRT domain and/or full-length BteA to the PM of yeast cells. The membrane association further involved electrostatic and hydrophobic interactions of LRT and depended on a leucine residue in the L1 loop between the first two helices of the four-helix bundle. Importantly, charge-reversal substitutions within the L1 region disrupted PM localization of the BteA effector without hampering its cytotoxic activity during B. bronchiseptica infection of HeLa cells. The LRT-mediated targeting of BteA to the cytosolic leaflet of the PM of host cells is, therefore, dispensable for effector cytotoxicity. The respiratory pathogens Bordetella pertussis and Bordetella bronchiseptica employ a type III secretion system (T3SS) to inject a 69-kDa BteA effector protein into host cells. This effector is known to contain two functional domains, including an N-terminal lipid raft targeting (LRT) domain and a cytotoxic C-terminal domain that induces nonapoptotic and caspase-1–independent host cell death. However, the exact molecular mechanisms underlying the interaction of BteA with plasma membrane (PM) as well as its cytotoxic activity in the course of Bordetella infections remain poorly understood. Using a protein–lipid overlay assay and surface plasmon resonance, we show here that the recombinant LRT domain binds negatively charged membrane phospholipids. Specifically, we determined that the dissociation constants of the LRT domain–binding liposomes containing phosphatidylinositol 4,5-bisphosphate, phosphatidic acid, and phosphatidylserine were ∼450 nM, ∼490 nM, and ∼1.2 μM, respectively. Both phosphatidylserine and phosphatidylinositol 4,5-bisphosphate were required to target the LRT domain and/or full-length BteA to the PM of yeast cells. The membrane association further involved electrostatic and hydrophobic interactions of LRT and depended on a leucine residue in the L1 loop between the first two helices of the four-helix bundle. Importantly, charge-reversal substitutions within the L1 region disrupted PM localization of the BteA effector without hampering its cytotoxic activity during B. bronchiseptica infection of HeLa cells. The LRT-mediated targeting of BteA to the cytosolic leaflet of the PM of host cells is, therefore, dispensable for effector cytotoxicity. Bacterial toxins and effectors localize to specific compartments within the host cell environment to access their intracellular targets and enhance the signaling specificity and efficacy. Various membrane-targeting strategies have evolved to trap the catalytic domain or adaptor domain at the proximity of the membrane-anchored substrates (1Varela-Chavez C. Blondel A. Popoff M.R. Bacterial intracellularly active toxins: Membrane localisation of the active domain.Cell Microbiol. 2020; 22e13213Crossref PubMed Scopus (4) Google Scholar). Some of the proteins insert directly into the membrane as integral membrane proteins through transmembrane domains, such as the Salmonella type III secretion system (T3SS) effector SteD (2Bayer-Santos E. Durkin C.H. Rigano L.A. Kupz A. Alix E. Cerny O. Jennings E. Liu M. Ryan A.S. Lapaque N. Kaufmann S.H.E. Holden D.W. The Salmonella effector SteD mediates MARCH8-dependent ubiquitination of MHC II molecules and inhibits T cell activation.Cell Host Microbe. 2016; 20: 584-595Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Others undergo covalent lipid modification, such as Salmonella T3SS effector proteins SspH2 and SseI that are S-palmitoylated on a conserved cysteine residue within their N-terminal domains by a specific subset of host–cell palmitoyltransferases (3Hicks S.W. Charron G. Hang H.C. Galan J.E. Subcellular targeting of Salmonella virulence proteins by host-mediated S-palmitoylation.Cell Host Microbe. 2011; 10: 9-20Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Nevertheless, a significant part of bacterial toxins and effectors possess a dedicated membrane localization domain (MLD) that binds phospholipids. For example, recruitment of the Legionella pneumophila effector DrrA to the Legionella-containing vacuole, where it AMPylates Rab1, is mediated by a phosphatidylinositol 4-phosphate (PI(4)P)-binding domain. This domain is characterized by a deep electropositive binding pocket and surrounding membrane-penetrating leucine residues (4Brombacher E. Urwyler S. Ragaz C. Weber S.S. Kami K. Overduin M. Hilbi H. Rab1 guanine nucleotide exchange factor SidM is a major phosphatidylinositol 4-phosphate-binding effector protein of Legionella pneumophila.J. Biol. Chem. 2009; 284: 4846-4856Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 5Del Campo C.M. Mishra A.K. Wang Y.H. Roy C.R. Janmey P.A. Lambright D.G. Structural basis for PI(4)P-specific membrane recruitment of the Legionella pneumophila effector DrrA/SidM.Structure. 2014; 22: 397-408Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Another domain termed 4HBM for four-helix bundle MLD is shared by multiple bacterial toxins, including Pasteurella multocida mitogenic toxin, multifunctional-autoprocessing RTX toxins, and large clostridial glucosyltransferase toxins exemplified by Clostridium difficile toxin A and toxin B and Clostridium sordellii lethal toxin (TcsL) (1Varela-Chavez C. Blondel A. Popoff M.R. Bacterial intracellularly active toxins: Membrane localisation of the active domain.Cell Microbiol. 2020; 22e13213Crossref PubMed Scopus (4) Google Scholar, 6Kamitani S. Kitadokoro K. Miyazawa M. Toshima H. Fukui A. Abe H. Miyake M. Horiguchi Y. Characterization of the membrane-targeting C1 domain in Pasteurella multocida toxin.J. Biol. Chem. 2010; 285: 25467-25475Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 7Geissler B. Tungekar R. Satchell K.J. Identification of a conserved membrane localization domain within numerous large bacterial protein toxins.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 5581-5586Crossref PubMed Scopus (59) Google Scholar, 8Geissler B. Ahrens S. Satchell K.J. Plasma membrane association of three classes of bacterial toxins is mediated by a basic-hydrophobic motif.Cell Microbiol. 2012; 14: 286-298Crossref PubMed Scopus (32) Google Scholar). The phospholipid-binding site of 4HBM is located at the apex of the structure, the so-called bundle tip, which is formed by two protruding loops, loop 1 (L1) connecting helices 1 and 2, and loop 3 (L3) in between helices 3 and 4. Indeed, the positively charged and hydrophobic residues within L1 and L3 are key for lipid-binding and membrane localization of 4HBM as revealed by their mutagenesis (8Geissler B. Ahrens S. Satchell K.J. Plasma membrane association of three classes of bacterial toxins is mediated by a basic-hydrophobic motif.Cell Microbiol. 2012; 14: 286-298Crossref PubMed Scopus (32) Google Scholar, 9Varela Chavez C. Haustant G.M. Baron B. England P. Chenal A. Pauillac S. Blondel A. Popoff M.R. The tip of the four N-terminal alpha-helices of Clostridium sordellii lethal toxin contains the interaction site with membrane phosphatidylserine facilitating small GTPases glucosylation.Toxins (Basel). 2016; 8: 90Crossref PubMed Scopus (10) Google Scholar). Furthermore, the proper localization of Pasteurella multocida mitogenic toxin and TcsL toxins also proved to be critical for both toxin activities and TcsL cytotoxicity (6Kamitani S. Kitadokoro K. Miyazawa M. Toshima H. Fukui A. Abe H. Miyake M. Horiguchi Y. Characterization of the membrane-targeting C1 domain in Pasteurella multocida toxin.J. Biol. Chem. 2010; 285: 25467-25475Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 10Craven R. Lacy D.B. Clostridium sordellii lethal-toxin autoprocessing and membrane localization activities drive GTPase glucosylation profiles in endothelial cells.mSphere. 2016; 1: e00012-e00015Crossref PubMed Scopus (9) Google Scholar). The cytotoxic effector BteA is injected into the host cells by a T3SS of classical Bordetella species, Bordetella pertussis, and Bordetella bronchiseptica (11Kamanova J. Bordetella type III secretion injectosome and effector proteins.Front. Cell. Infect. Microbiol. 2020; 10: 466Crossref PubMed Scopus (14) Google Scholar). These bacteria colonize the ciliated epithelia of the respiratory tract of diverse mammals and cause respiratory illness with differing symptoms, duration, and severity. The strictly human-adapted B. pertussis is the primary causative agent of pertussis or whooping cough, a contagious respiratory illness of humans that remains one of the least controlled vaccine-preventable infectious diseases (12Mattoo S. Cherry J.D. Molecular pathogenesis, epidemiology, and clinical manifestations of respiratory infections due to Bordetella pertussis and other Bordetella subspecies.Clin. Microbiol. Rev. 2005; 18: 326-382Crossref PubMed Scopus (862) Google Scholar). The B. bronchiseptica species, on the other hand, infects a broad range of mammals and causes infections ranging from lethal pneumonia to asymptomatic and chronic respiratory carriage (12Mattoo S. Cherry J.D. Molecular pathogenesis, epidemiology, and clinical manifestations of respiratory infections due to Bordetella pertussis and other Bordetella subspecies.Clin. Microbiol. Rev. 2005; 18: 326-382Crossref PubMed Scopus (862) Google Scholar, 13Goodnow R.A. Biology of Bordetella bronchiseptica.Microbiol. Rev. 1980; 44: 722-738Crossref PubMed Google Scholar). The activity of T3SS of B. bronchiseptica is required for persistent colonization of the lower respiratory tract of rats, mice, and pigs, presumably because of the actions of BteA effector (14Yuk M.H. Harvill E.T. Miller J.F. The BvgAS virulence control system regulates type III secretion in Bordetella bronchiseptica.Mol. Microbiol. 1998; 28: 945-959Crossref PubMed Scopus (142) Google Scholar, 15Pilione M.R. Harvill E.T. The Bordetella bronchiseptica type III secretion system inhibits gamma interferon production that is required for efficient antibody-mediated bacterial clearance.Infect. Immun. 2006; 74: 1043-1049Crossref PubMed Scopus (55) Google Scholar, 16Nicholson T.L. Brockmeier S.L. Loving C.L. Register K.B. Kehrli Jr., M.E. Shore S.M. The Bordetella bronchiseptica type III secretion system is required for persistence and disease severity but not transmission in swine.Infect. Immun. 2014; 82: 1092-1103Crossref PubMed Scopus (21) Google Scholar). In tissue culture, however, BteA actions account for rapid cell death that is nonapoptotic and caspase-1 independent (17Stockbauer K.E. Foreman-Wykert A.K. Miller J.F. Bordetella type III secretion induces caspase 1-independent necrosis.Cell Microbiol. 2003; 5: 123-132Crossref PubMed Scopus (54) Google Scholar). Remarkably, compared with the high BteA–mediated cytotoxicity of B. bronchiseptica, the cytotoxicity of BteA of B. pertussis is strongly attenuated because of the insertion of an extra alanine at position 503, which may represent an evolutionary adaptation of B. pertussis (18Bayram J. Malcova I. Sinkovec L. Holubova J. Streparola G. Jurnecka D. Kucera J. Sedlacek R. Sebo P. Kamanova J. Cytotoxicity of the effector protein BteA was attenuated in Bordetella pertussis by insertion of an alanine residue.PLoS Pathog. 2020; 16e1008512Crossref PubMed Scopus (12) Google Scholar). Nevertheless, the role of BteA and T3SS activity in the pathophysiology of human pertussis remains to be established. The 69-kDa effector protein BteA exhibits modular architecture, consisting of two functional domains, an N-terminal localization/lipid raft targeting domain of ∼130 amino acid residues and a cytotoxic C-terminal domain of ∼528 amino acid residues without any known structural homologs (19French C.T. Panina E.M. Yeh S.H. Griffith N. Arambula D.G. Miller J.F. The Bordetella type III secretion system effector BteA contains a conserved N-terminal motif that guides bacterial virulence factors to lipid rafts.Cell Microbiol. 2009; 11: 1735-1749Crossref PubMed Scopus (41) Google Scholar). The N-terminal domain of BteA is sufficient to target GFP to lipid rafts of HeLa cell plasma membrane (PM) and was therefore termed lipid raft targeting (LRT) domain (19French C.T. Panina E.M. Yeh S.H. Griffith N. Arambula D.G. Miller J.F. The Bordetella type III secretion system effector BteA contains a conserved N-terminal motif that guides bacterial virulence factors to lipid rafts.Cell Microbiol. 2009; 11: 1735-1749Crossref PubMed Scopus (41) Google Scholar). Homologous domains were identified by sequence similarity searches in a number of known and putative virulence factors of other bacteria, including a T3SS effector Plu4750 and a multifunctional-autoprocessing RTX toxin Plu3217 of Photorhabdus luminescens which as LRT targeted GFP to lipid rafts (19French C.T. Panina E.M. Yeh S.H. Griffith N. Arambula D.G. Miller J.F. The Bordetella type III secretion system effector BteA contains a conserved N-terminal motif that guides bacterial virulence factors to lipid rafts.Cell Microbiol. 2009; 11: 1735-1749Crossref PubMed Scopus (41) Google Scholar). The structure of LRT domain, described by LRT helices (A-A')-B-C-E, displays an overall tertiary fold similar to 4HBM (20Geissler B. Bacterial toxin effector-membrane targeting: Outside in, then back again.Front. Cell. Infect. Microbiol. 2012; 2: 75Crossref PubMed Scopus (14) Google Scholar, 21Yahalom A. Davidov G. Kolusheva S. Shaked H. Barber-Zucker S. Zarivach R. Chill J.H. Structure and membrane-targeting of a Bordetella pertussis effector N-terminal domain.Biochim. Biophys. Acta Biomembr. 2019; 1861: 183054Crossref PubMed Scopus (8) Google Scholar). Interestingly, the topology of the LRT domain tip is different from that of 4HBM, being formed by the loop region L1, connecting helices A and B, and the capping perpendicular helix D, connecting helices C and E (21Yahalom A. Davidov G. Kolusheva S. Shaked H. Barber-Zucker S. Zarivach R. Chill J.H. Structure and membrane-targeting of a Bordetella pertussis effector N-terminal domain.Biochim. Biophys. Acta Biomembr. 2019; 1861: 183054Crossref PubMed Scopus (8) Google Scholar). The in vivo membrane-targeting mechanism of LRT domain and its contribution to the cytotoxicity of BteA effector remains poorly characterized. The recombinant LRT domain binds phosphatidylinositol 4,5-bisphosphate (PIP2)-containing nanodisks suggesting that BteA may associate with the host PM and lipid rafts through phospholipid interaction (21Yahalom A. Davidov G. Kolusheva S. Shaked H. Barber-Zucker S. Zarivach R. Chill J.H. Structure and membrane-targeting of a Bordetella pertussis effector N-terminal domain.Biochim. Biophys. Acta Biomembr. 2019; 1861: 183054Crossref PubMed Scopus (8) Google Scholar). Interestingly, ectopically expressed BteA of B. bronchiseptica remains cytotoxic even upon deletion of the LRT domain or the removal of the first 200 N-terminal amino acids, while its cytotoxicity is diminished upon deletion of the last 14 C-terminal amino acid residues (19French C.T. Panina E.M. Yeh S.H. Griffith N. Arambula D.G. Miller J.F. The Bordetella type III secretion system effector BteA contains a conserved N-terminal motif that guides bacterial virulence factors to lipid rafts.Cell Microbiol. 2009; 11: 1735-1749Crossref PubMed Scopus (41) Google Scholar, 22Kuwae A. Momose F. Nagamatsu K. Suyama Y. Abe A. BteA secreted from the Bordetella bronchiseptica type III secetion system induces necrosis through an actin cytoskeleton signaling pathway and inhibits phagocytosis by macrophages.PLoS One. 2016; 11e0148387Crossref PubMed Scopus (13) Google Scholar). In this work, we provide a mechanical insight into in vivo membrane targeting of the LRT domain and its contribution to BteA cytotoxicity during B. bronchiseptica infection. To understand the membrane-targeting mechanism of BteA effector, the lipid-binding properties of its N-terminal LRT domain comprising 130 amino acid residues were tested in a protein–lipid overlay assay. The purified recombinant glutathione-S-transferase (GST)-tagged LRT domain of B. pertussis BteA (LRT) was used to probe commercial lipid strips. These have been spotted with 100 pmol of different lipids, and bound LRT was detected using an anti-GST antibody. In contrast to GST alone, which displayed no binding affinity for lipids, LRT protein preferentially interacted with negatively charged lipids, with a preference for PIP2 and phosphatidic acid (PA), as shown in Figure 1A and Fig. S1A. The LRT binding depended on the spotted lipid concentration as further corroborated using home-made lipid arrays spotted with a concentration gradient of 10, 100, and 1000 pmol of various lipids or cholesterol per spot (Fig. 1B). Interestingly, as also shown in Figure 1, A and B and Fig. S1A, the GST-tagged full-length BteA protein of B. pertussis in a complex with its cognate chaperone BtcA (BteA/BtcA) exhibited similar, although, somehow stronger binding of negatively charged lipids than LRT alone. Nevertheless, the lipid-binding abilities of the full-length BteA protein might have been affected by the presence of BtcA, which co-purified with the BteA. In contrast to LRT protein, we were unable to produce soluble BteA without its cognate chaperone BtcA in Escherichia coli. To avoid the limitations of the protein–lipid overlay assay and experiment-to-experiment variability, we next analyzed LRT interaction with a native phospholipid bilayer by surface plasmon resonance (SPR). Four types of large unilamellar vesicles consisting of (i) phosphatidylcholine (PC) only, or the mixture of (ii) phosphatidylserine with PC (PS/PC, molar ratio 20:80), (iii) PA with PC (PA/PC, molar ratio 5:95), and (iv) PIP2 with PC (PIP2/PC, molar ratio 5:95) were prepared and captured on a neutravidin-coated sensor chip. The recombinant proteins were then serially diluted and injected over the immobilized liposome surface to monitor their binding. While GST alone did not interact with any of the lipid vesicles (Fig. S1B), the concentration-dependent binding curves of LRT protein revealed its interaction with all four membrane surfaces with the typical association and dissociation phases of the sensograms (Fig. 1C). The binding affinities of LRT to lipid vesicles were next calculated from steady-state binding data as the global fitting of the binding curves to several kinetic models did not provide satisfactory results in terms of χ2 and residual statistics. The near-equilibrium values (Req), which were taken from the end of the association phase of the individual binding curves, were plotted as a function of the LRT concentration (Fig. 1D), and the equilibrium dissociation constant (KD) was determined by nonlinear least-squares analysis of the binding isotherm. As shown in Figure 1D, the binding of LRT to PC vesicles was not saturable (up to 500 nM), indicating a nonspecific interaction of the LRT with the charge-neutral phospholipid bilayer. In contrast, the negatively charged lipid surfaces conferred the binding of LRT in a saturable manner, with apparent KD values of 1225 ± 245 for PS-enriched vesicles and 492 ± 86 nM and 446 ± 65 nM for PA- and PIP2-containing vesicles, respectively. These results, thus, demonstrate that the LRT motif has a direct affinity for negatively charged lipid surfaces in vitro. It was next important to test for the role of negatively charged lipids in guiding the localization of LRT and full-length BteA effector in vivo. To this end, the cellular distribution of ectopically expressed GFP-tagged LRT and BteA proteins of B. pertussis was analyzed by live-cell fluorescence microscopy in Saccharomyces cerevisiae WT and mutant strains that harbored specific defects in biosynthesis pathways for PS, PA, and PIP2. As illustrated in Figure 2A, when LRT–GFP was expressed in WT strain of S. cerevisiae BY4742, it readily associated with the PM, similarly to its previously reported localization in HeLa cells (19French C.T. Panina E.M. Yeh S.H. Griffith N. Arambula D.G. Miller J.F. The Bordetella type III secretion system effector BteA contains a conserved N-terminal motif that guides bacterial virulence factors to lipid rafts.Cell Microbiol. 2009; 11: 1735-1749Crossref PubMed Scopus (41) Google Scholar). The full-length BteA–GFP protein also exhibited peripheral localization in WT strain, although its distribution was patchier (Fig. 2A). Upon expression in the cho1Δ mutant harboring a deletion of PS synthase Cho1, however, both proteins were distributed throughout the cytoplasm without any PM localization (Fig. 2A). In the same manner, GFP-tagged PS-specific binding protein, GFP-Lact-C2, localized to the PM of WT strain and dispersed throughout the cytoplasm in the cho1Δ mutant (Fig. 2A). In contrast, no difference in the localization of PIP2-specific binding protein tagged with GFP, GFP-2xPH(PLCδ), was observed between both strains (Fig. S2A). These results demonstrate that both LRT and BteA proteins respond specifically to a decreased level of PS in the cho1Δ mutant and point to the importance of PS in their localization. To address the role of PA, LRT–GFP and full-length BteA–GFP proteins were expressed in the dgk1Δ mutant of S. cerevisiae, which harbors a deletion of diacylglycerol kinase responsible for PA synthesis from diacylglycerol (23Han G.S. O'Hara L. Siniossoglou S. Carman G.M. Characterization of the yeast DGK1-encoded CTP-dependent diacylglycerol kinase.J. Biol. Chem. 2008; 283: 20443-20453Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). As shown in Figure 2B, however, no change in the localization of analyzed proteins or commonly used GFP-tagged PA sensor, GFP-Spo2051–91 (24Nakanishi H. de los Santos P. Neiman A.M. Positive and negative regulation of a SNARE protein by control of intracellular localization.Mol. Biol. Cell. 2004; 15: 1802-1815Crossref PubMed Scopus (140) Google Scholar), was observed between the dgk1Δ mutant and parental WT strain BY4741 of S. cerevisiae. These data suggest no depletion of PA levels in the dgk1Δ mutant in the used conditions and/or reduced specificity of the commonly used PA sensor. To test for the role of PIP2, a thermosensitive mutant of the phosphatidylinositol 4-phosphate 5-kinase Mss4 (mss4ts), which generates PIP2 from PI(4)P was used. To confirm the decreased PM levels of PIP2 in the mss4ts mutant at the restrictive temperature, PIP2 levels were visualized by GFP-2xPH(PLCδ). As shown in Figure 2C, GFP-2xPH(PLCδ), indeed, relocated from the PM to cell cytosol after the shift to the restrictive temperature (38 °C, 1 hod). In contrast, the localization of PS-specific probe, GFP-Lact-C2, was unaltered, showing that depletion of PIP2 did not merely disrupt the PM integrity (Fig. S2B). As further shown in Figure 2C, LRT-GFP and BteA-GFP proteins localized to the PM of the mss4ts strain at the permissive temperature (25 °C), although the association of BteA–GFP was poor when compared with the parental WT strain SEY6210 (see Fig. S2C for comparison at lower magnification). Importantly, at the restrictive temperature (38 °C), BteA-GFP was entirely cytoplasmic localized (Fig. 2C and Fig. S2C), whereas LRT–GFP partially relocated from the PM to the internal puncta, as highlighted by the white arrows in Figure 2C. Collectively, although the contribution of PA to localization of BteA effector in vivo is unclear, our data demonstrate that PS and PIP2 levels influence the proper localization of LRT and BteA proteins. Having shown that negatively charged phospholipids PS and PIP2 guide the localization of BteA effector in vivo, we further analyzed the structural determinants of LRT that confer its membrane interaction. The LRT protein bound to lipid vesicles composed of PC only (Fig. 1C), suggesting that it associates with lipid membranes at least partly via hydrophobic interactions. Indeed, as depicted in Figure 3A, several hydrophobicity patches, including a protruding hydrophobic leucine 51 (L51) residue, can be visualized on the surface of the LRT structure (aa 29–121, PDB code: 6RGN, (21Yahalom A. Davidov G. Kolusheva S. Shaked H. Barber-Zucker S. Zarivach R. Chill J.H. Structure and membrane-targeting of a Bordetella pertussis effector N-terminal domain.Biochim. Biophys. Acta Biomembr. 2019; 1861: 183054Crossref PubMed Scopus (8) Google Scholar)). To test for the role of the L51 residue in the binding of LRT to lipid membranes, the residue was replaced by asparagine (L51N) or phenylalanine (L51F), and the capacity of these mutants to interact with lipidic surfaces was evaluated in vitro by SPR and in vivo by fluorescence microscopy. As shown in Figure 3B and Fig. S3A, the substitution of hydrophobic L51 by hydrophilic asparagine decreased the binding of LRT–L51N protein to phospholipid bilayers, regardless of the charge and composition of the immobilized lipid vesicles. In contrast, the replacement of the hydrophobic side chain of L51 by a more hydrophobic aromatic ring of phenylalanine augmented the lipid-binding of LRT–L51F mutant protein. The importance of the L51 hydrophobic side chain in membrane targeting was further corroborated in vivo by fluorescence microscopy. As shown in Figure 3C, the L51N variant of LRT–GFP fusion protein was distributed throughout the cytoplasm upon ectopic expression in S. cerevisiae cells. In contrast, the WT protein preferentially localized to the cell periphery. Besides, the introduction of L51F mutation into LRT–GFP fusion protein enhanced its peripheral localization (Fig. 3C). The change of localization for both mutant proteins proved to be significant (p < 0.0001, unpaired two-tailed t test, n = 10, compared with LRT-WT) as determined by PM indexes of the proteins. These were calculated as the ratios of the highest intensity value measured at the cell periphery and cell interior, which yielded 1.8 ± 0.6 for LRT-WT, and 0.8 ± 0.1 and 5.7 ± 1.7 for LRT–L51N and LRT–51F mutant proteins, respectively (see Experimental procedures for more details) (Fig. 3C). Importantly, as shown in Fig. S3B by immunoblot analysis, the L51 substitutions within the LRT segment did not affect the GFP-fusion protein stability in yeast, and thus, the change in LRT localization was not related to protein degradation. These data, therefore, demonstrate that the hydrophobic moiety of the L51 residue is required for efficient interaction of LRT with the phospholipid bilayer. We reasoned that besides LRT–L51 hydrophobic interaction with lipid membrane, also electrostatic interactions with negatively charged phospholipid headgroups contribute to membrane binding. Indeed, the structure of LRT revealed a four-helix bundle protein that consists of a large number of positively charged arginine and lysine amino acid residues (Fig. 4, A and B). To determine critical amino acid residues, a set of LRT mutant proteins harboring charge-reversal substitutions within loop L1 (R50E + H52E + H53E, LRT-L1), helix B (R59E + K62E + R66E, LRT-hB), and helix D (K99E + R100E, LRT-hD) was first constructed, and the capacity of the mutant proteins to interact with the immobilized lipid vesicles was evaluated by SPR. As shown in Figure 4C, the LRT–L1 and LRT–hD mutant proteins exhibited almost complete loss of binding to lipid membranes, regardless of their composition. In contrast, the interaction of LRT–hB with the immobilized PIP2-enriched vesicles was only slightly reduced while completely abolished for the PS-containing liposomes. Hence, the positively charged residues within the loop L1 and helices B and D are required for the interaction with negatively charged membranes, whereas the positively charged side chains at the tip of the LRT structure seem to confer the specificity for PIP2. To corroborate these data in vivo, the localization of mutant GFP-tagged LRT proteins was analyzed by fluorescence microscopy in S. cerevisiae and HeLa cells upon LRT ectopic expression. As shown in Figure 4D, charge reversal substitutions within loop L1 (R50E + H52E + H53E, LRT-GFP-L1), helix B (R59E + K62E + R66E, LRT-GFP-hB), and helix D (K99E + R100E, LRT-GFP-hD) resulted in cytoplasmic localization of the mutant proteins in S. cerevisiae cells. In transiently transfected HeLa cells, as compared with LRT–GFP, the mutant proteins also predominantly localized to cell cytosol and accumulated in cell nuclei by passive diffusion through nuclear pore complexes (Fig. 4E), although slight PM localization of LRT–GFP–hB was still noticeable. Again, no change in GFP-fusion protein stability was observed in yeast and HeLa cells, as revealed by immunoblot analysis and shown in Fig. S4, A and B. To gain more insight into residue specificity, we further performed glutamic acid and/or alanine mutagenesis screens of arginine, lysine, and histidine residues within the LRT motif. As determined by PM indexes of the respective variants and shown in Figure 5 and Fig. S5 and Table S1, substitutions of positively charg
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