Structure and biochemical analysis of a secretin pilot protein
2005; Springer Nature; Volume: 24; Issue: 6 Linguagem: Inglês
10.1038/sj.emboj.7600610
ISSN1460-2075
AutoresPaula I. Lario, Richard A. Pfuetzner, Elizabeth A. Frey, Louise Creagh, Charles A. Haynes, Anthony T. Maurelli, N.C.J. Strynadka,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoArticle10 March 2005free access Structure and biochemical analysis of a secretin pilot protein Paula I Lario Paula I Lario Department of Biochemistry, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Richard A Pfuetzner Richard A Pfuetzner Department of Biochemistry, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Elizabeth A Frey Elizabeth A Frey Department of Biochemistry, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Louise Creagh Louise Creagh Biotechnology Laboratory, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Charles Haynes Charles Haynes Biotechnology Laboratory, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Anthony T Maurelli Anthony T Maurelli Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD, USA Search for more papers by this author Natalie C J Strynadka Corresponding Author Natalie C J Strynadka Department of Biochemistry, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Paula I Lario Paula I Lario Department of Biochemistry, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Richard A Pfuetzner Richard A Pfuetzner Department of Biochemistry, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Elizabeth A Frey Elizabeth A Frey Department of Biochemistry, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Louise Creagh Louise Creagh Biotechnology Laboratory, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Charles Haynes Charles Haynes Biotechnology Laboratory, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Anthony T Maurelli Anthony T Maurelli Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD, USA Search for more papers by this author Natalie C J Strynadka Corresponding Author Natalie C J Strynadka Department of Biochemistry, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Author Information Paula I Lario1, Richard A Pfuetzner1, Elizabeth A Frey1, Louise Creagh2, Charles Haynes2, Anthony T Maurelli3 and Natalie C J Strynadka 1 1Department of Biochemistry, University of British Columbia, Vancouver, BC, Canada 2Biotechnology Laboratory, University of British Columbia, Vancouver, BC, Canada 3Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD, USA *Corresponding author. Department of Biochemistry & Molecular Biology, University of British Columbia, 2146 Health Sciences Mall, Vancouver, BC, Canada V6T 1Z3. Tel.: +1 604 822 0789; Fax: +1 604 822 5227; E-mail: [email protected] The EMBO Journal (2005)24:1111-1121https://doi.org/10.1038/sj.emboj.7600610 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The ability to translocate virulence proteins into host cells through a type III secretion apparatus (TTSS) is a hallmark of several Gram-negative pathogens including Shigella, Salmonella, Yersinia, Pseudomonas, and enteropathogenic Escherichia coli. In common with other types of bacterial secretion apparatus, the assembly of the TTSS complex requires the preceding formation of its integral outer membrane secretin ring component. We have determined at 1.5 Å the structure of MxiM28–142, the Shigella pilot protein that is essential for the assembly and membrane association of the Shigella secretin, MxiD. This represents the first atomic structure of a secretin pilot protein from the several bacterial secretion systems containing an orthologous secretin component. A deep hydrophobic cavity is observed in the novel 'cracked barrel' structure of MxiM, providing a specific binding domain for the acyl chains of bacterial lipids, a proposal that is supported by our various lipid/MxiM complex structures. Isothermal titration analysis shows that the C-terminal domain of the secretin, MxiD525–570, hinders lipid binding to MxiM. Introduction Shigella flexneri invades colonic epithelial cells and causes bacillary dysentery, a severe form of gastroenteritis, which leads to over 1 million deaths per year worldwide (Philpott et al, 2000). This cell invasion process requires a functional type III secretion system (TTSS), encoded by the mxi and spa genes in the Shigella virulence plasmid (Parsot et al, 1995). The secretion and subsequent translocation of proteins (known as effectors) into the cytoplasm of host cells by the TTSS is a process essential for virulence of Shigella as well as many other Gram-negative bacterial pathogens of mammals and plants. While much is known about the type and function of virulence effector proteins delivered to host cells by the TTSS, the detailed molecular mechanisms of secretion and translocation remain poorly understood (Tampakaki et al, 2004). Protein secretion in general is required for different aspects of the bacterial life cycle including organelle biogenesis, nutrient acquisition, as well as delivery of virulence effectors into the host cell (Thanassi and Hultgren, 2000). The latter is a particularly challenging task for Gram-negative bacteria because secreted proteins must cross three barriers: the inner or cytoplasmic membrane, the periplasmic space, and the outer membrane (OM). Multiple protein secretory pathways have evolved in these microorganisms to carry out this difficult yet important process. These pathways are found to differ not only in the means by which the secreted proteins reach the cell surface, but also in the types and organization of the structural components involved in the secretion process. In spite of this diversity, the secretin superfamily of proteins appears to be the one, if not the only, structural component shared among several apparently unrelated protein secretion pathways: TTSS, the type II secretion system, and the type IV pilus biogenesis pathway (Thanassi and Hultgren, 2000). Secretins are integral membrane proteins that are synthesized in the bacterial cytoplasm and exported to the periplasm by the sec-dependent pathway. They are found to associate into large and highly stable oligomers of 12–14 subunits in the bacterial OM (Linderoth et al, 1997; Burghout et al, 2004; Collins et al, 2004). A proposed role as an OM protein-conducting channel has been supported by electron microscopy (EM) studies of PulD (Nouwen et al, 1999) and several other representative secretins (Koster et al, 1997; Crago and Koronakis, 1998; Tamano et al, 2000; Blocker et al, 2001), which showed that they adopt a ring-like structure with a central pore ranging from 2 to 10 nm in diameter, wide enough to accommodate completely unfolded or partially folded proteins (Koster et al, 1997; Nouwen et al, 2000; Blocker et al, 2001). Construction of the various bacterial multiprotein secretory complexes is believed to occur in a highly regulated manner, and accessory proteins are often present to ensure specific structural components are correctly localized and adopt their functional conformation. In particular, a specialized class of proteins known as 'pilots' mediate the assembly of the homo-oligomeric secretin ring. Previous studies have demonstrated that these pilot proteins bind and directly affect the stability, localization, and/or multimerization states of their associated secretins (Drake et al, 1997; Crago and Koronakis, 1998; Daefler and Russel, 1998; Shevchik and Condemine, 1998; Schuch and Maurelli, 1999; Burghout et al, 2004). EM studies of the type II secretion apparatus, PulS-PulD, indicate that the membrane-embedded PulD secretin complex is flanked by protein spokes (Nouwen et al, 1999). These radial spokes have been attributed to the secretin pilot protein, PulS, implying a key structural role in anchoring PulD to the OM. To date, PulS of Klebsiella oxytoca, OutS of Erwinia chrysanthemi, PilP of Pseudomonas aeruginosa, InvH of Salmonella, YscW of Yersinia, and MxiM of Shigella have been identified as pilots for the secretins PulD, OutD, PilQ, InvG, YscC, and MxiD, respectively. Although these pilots are all relatively small (∼15 kDa) and are all predicted to be covalently attached to a lipid moiety at their N-termini, very little sequence identity is observed (typically in the range of 17–19%). This is perhaps not surprising given that pilots typically bind to the C-terminal tail of their associated secretin, the most sequence-divergent region among the otherwise well-conserved family (Shevchik et al, 1997; Daefler and Russel, 1998; Schuch and Maurelli, 2001). The pilot protein MxiM from the Shigella TTSS is a 142-residue lipoprotein, which binds and affects several features of the secretin MxiD, including its stability in the periplasm, OM association, as well as assembly into multimeric structures (Schuch and Maurelli, 2001). A deficiency in MxiM results in a complete loss of TTSS function and virulence, presumably due to the lack of and/or improper assembly of the key secretin component MxiD (Schuch et al, 1999). Previous lipid labeling and fractionation studies have indicated that MxiM is anchored to the inner leaflet of the OM via its N-terminal lipid moiety (Schuch and Maurelli, 1999). Its peripheral membrane localization suggests that MxiM assists in secretin ring formation by anchoring the MxiD to the OM. In this work, we present the crystal structure of MxiM from S. flexneri, the first high-resolution structural data for a secretin pilot protein from any system. The structure reveals a pseudo-β-barrel fold that presents a narrow and deep hydrophobic cavity for the binding of specific lipids. Our isothermal titration calorimetry (ITC) experiments show that MxiM binds lipids and its cognate secretin MxiD. Collectively, our findings provide a foundation for understanding the structural and functional roles of secretin pilots in assembly of the secretin complex, an essential component in the secretion apparatus from various protein secretion pathways. Results and discussion Purification, biochemical characterization, and crystallization of MxiM28–142 To provide sufficient quantities of soluble MxiM necessary for crystallization, a recombinant form excluding the N-terminal signal sequence (residues 1–23) but including a cleavable N-terminal hexahistidine tag was used. Mass spectrometry analysis of the purified protein after thrombin-mediated cleavage of the histidine tag reported a mass of 12 888.0 Da, indicating that further processing had occurred resulting in the N-terminal deletion mutant MxiM28–142 (calculated molecular mass of 12 886.9 Da). MxiM and other secretin pilots encode at their N-termini the classic LXGC sequence motif characteristic of the lipidation site of many bacterial lipoproteins (covalently bound to the cysteine residue) (Wu and Tokunaga, 1986). Biochemical studies have shown that MxiM is both lipid modified at Cys24 of the LXGC motif and is membrane associated (Allaoui et al, 1992; Schuch and Maurelli, 1999). Importantly for this study, binding and assembly of the secretin/pilot complex occurs even in the absence of the lipid anchor (Schuch and Maurelli, 2001). While the lipidation of MxiM has been shown to be important for specifically targeting MxiM to the OM as well as stability to heat denaturation of the membrane-embedded pilot/secretin complex (Schuch and Maurelli, 1999), studies with an unlipidated G23R mutant show that this periplasmic form also associates with MxiD oligomers although the resulting complex is less heat resistant than those formed with lipidated MxiM (Schuch and Maurelli, 2001). Recently, studies of the Yersinia secretin pilot, YscW, have shown that a putatively unlipidated construct (YscWΔlip) putatively still binds to YscC (as evidenced by altered expression levels in coexpression systems with YscW) but apparently fails to promote YscC secretin oligomer formation and localization to the OM (Burghout et al, 2004). While these apparently contrasting results may suggest fundamental differences in pilot function between the two species, we note that in the Yersinia study, no evidence was provided for the translocation of the unlipidated YscW construct to the periplasm (which would obviously be a critical step for subsequent YscC oligomerization and OM localization). Static light scattering experiments in the presence of 0.5 mM dodecylmaltoside (DDM) show that MxiM28–142 has a measured mass of 15 kDa, indicating that the protein is predominately monomeric in solution. These results are consistent with size exclusion chromatography experiments where the elution position relative to molecular weight standards on a superdex 75 column (Amersham) also suggests a monomeric state. MxiM could only be crystallized in the presence of maltoside-based detergents or 1-monohexanoyl-2-hydroxy-sn-glycero-3-phosphate as described in Materials and methods. The crystals are monoclinic (C2) with a monomer of MxiM observed in the asymmetric unit also consistent with our static light scattering and size exclusion results. Unique fold of Shigella MxiM We have determined the crystal structure of the MxiM secretin pilot from S. flexneria to 1.5 Å. The structure was solved using the method of multiwavelength anomalous diffraction (MAD) with a single crystal of selenomethionine (Se-Met)-labeled protein. Data collection and refinement statistics are presented in Table I. Table 1. Data collection and refinement statistics Se MAD Native +Lipida Data set Peak Remote Inflection Data collection Wavelength (Å) 0.9794 0.8981 0.9796 1.00 1.5418 Resolution range (Å)b 20–1.7 (1.76–1.70) 20–1.7 (1.76–1.70) 20–1.7 (1.76–1.70) 20–1.50 (1.53–1.50) 42–1.87 (1.94–1.87) Total observations 46 694 46 586 47 370 73 722 75 315 Unique reflections 23 037 23 083 23 220 18 209 9566 Completeness (%)b 90.0 (65.6) 90.3 (65.7) 90.3 (62.8) 96.5 (100.0) 97.6 (94.4) Rmerge (%)b,c, b,c 4.8 (20.9) 5.0 (24.3) 4.8 (22.2) 4.4 (42.1) 6.4 (32.7) 〈I/σI〉 24.1 (3.5) 20.7 (2.9) 25.7(3.5) 16.5 (3.0) 21.5 (4.4) Refinement Resolution range (Å)b 20–1.5 (1.52–1.50) 42–1.87 (1.89–1.87) Rworkb,d, b,d 22.0 (25) 20.6 (26) Rfreeb,e, b,e 25.1 (32) 24.9 (31) Average B-factor (Å2) Main chain 23.0 31.9 Side chain 25.2 33.7 R.m.s. deviation Bond lengths (Å) 0.013 0.0014 Angles (deg) 1.5 1.5 a Anionic lipid, 1-hexanoyl-2-hydroxy-sn-glycerol-3-phosphate. b Values in parentheses correspond to the highest resolution shell. c Rmerge=∑∣(Ihkl)−〈I〉∣/∑(Ihkl), where Ihkl is the integrated intensity of a given reflection. d Rwork=(∑∣Fo−Fc∣)/(∑Fo), where Fo and Fc are observed and calculated structure factors. e A total of 5% of reflections were excluded from the refinement to calculate Rfree. MxiM is a conical-shaped structure with average dimensions of 40 × 30 × 30 Å (Figure 1A). The 10 β-strands present in the structure curve to form a β-sheet (Figure 1B). This curved β-sheet resembles a β-barrel structure except that the barrel is discontinuous due to the presence of the α-helix, F1. In addition, this pseudo-barrel structure is closed at one end in part due to the presence of a disulfide bridge between loops L2 and L4 that tethers the N-terminus of helix F1 to the loop L2 (Figure 1). The fold we observe for MxiM is considered unique based on a DALI search of the existing structural database (Holm and Sander, 1995). Figure 1.Structure of MxiM. (A) A ribbon representation of the protein with the β-structure shown in blue and the helical domains shown in magenta. (B) The fold topology of MxiM is shown where the β-strands of the antiparallel β-sheet are shown as arrows and labeled A–H. The α-helix is shown as a rectangle and labeled F1. The dotted line represents the hydrogen bonding interactions between the β-strands and the dashed line represents the single disulfide bond in the structure. Download figure Download PowerPoint It is difficult to predict if pilots from other species or from other systems will have a similar fold as that observed for MxiM. The lack of sequence identity among secretin pilots was found to extend to a lack of similarity in predicted secondary structure. A number of algorithms were utilized, including Jpred (Cuff et al, 1998), PredictProtein (Rost and Liu, 2003), and nnPredict (Zhang and Zhang, 2000), to analyze the sequences of the known secretin pilots, resulting in a wide variety of predicted secondary structures. MxiM and the type III secretin pilot YscW from Yersinia (Burghout et al, 2004) are predicted to be composed of primarily β-structure in accordance with our structural observations, while sequences of other pilots generate a variety of secondary structure predictions. There is clearly significant precedent for proteins with similar function to adopt strikingly similar structural folds despite very low or undetectable sequence identity and despite misleading secondary structure prediction profiles (the type III secretion chaperones are a case in point; Luo et al, 2001; Birtalan et al, 2002). Structural characterization of functional orthologs of MxiM will enable us to understand fully the similarities among this sequence diverse family of proteins. Hydrophobic ligand-binding cavity and membrane association The 'cracked barrel' motif of MxiM creates a dramatic cleft in the center of the protein, ∼8 Å wide and 20 Å deep (Figure 2A and B). The cavity is entirely hydrophobic in nature and is notably rich in aromatic groups nearer the surface of the protein. Aromatic groups are commonly observed at the protein/membrane interface (Saez-Cirion et al, 2003; Gambhir et al, 2004) and suggest a possible role of membrane association for these conserved amino acids at the MxiM surface. Other observations that suggest that the hydrophobic cavity of MxiM is likely in close proximity to the OM include the observed charge localization surrounding this area of the protein. Although the overall electrostatic surface of the MixM structure is for the most part evenly distributed with positive and negative charges, there is one exception in a concentration of positively charged surfaces (involving residues K34, K82, K121, K118, and R140) observed near the mouth of the hydrophobic cavity (Figure 2C). These basic amino acids could plausibly interact with negatively charged lipid phosphate groups present on the inner leaflet of the OM. Figure 2.Hydrophobic cavity of MxiM. (A) Stereoscopic representation showing the side chains of the residues that line the hydrophobic cavity. Atoms are colored according to type: carbon atoms are shown in gray, nitrogen atoms are colored blue, oxygen atoms are colored red, and sulfur atoms are colored yellow. The protein backbone trace is represented by the cyan colored worm. (B) GRASP (Nicholls et al, 1991) surface representation of MxiM. Polar and hydrophobic areas are in gray and green, respectively. (C) The electrostatic surface of MxiM is mapped on to the molecular surface as calculated using the program GRASP. The basic residues at the lip of the pore are labeled. Positive and negatively charged areas are blue and red, respectively. All three panels are shown with a similar view. Download figure Download PowerPoint Given the dimensions and nature of the hydrophobic pore, its functional role could serve to bind a single fatty acid tail of a membrane phospholipid. Within the putative lipid-binding cavity of MxiM, we observe additional Fo−Fc difference density at 3σ that is not accounted for after the amino-acid sequence had been traced in the electron density maps. Contouring the electron density map at a slightly lower cutoff (2.5σ) indicated that this density was continuous (Figure 3A) and can be accommodated by the lipid tail of the DDM detergent required for ordered crystal formation of MxiM. When the lipid tail of DDM (11 carbon acyl chain) was included in the structure, both the R and Rfree crystallographic factors were lower than the model refined without the lipid tail. The lipid tail of DDM forms several hydrophobic interactions with the side chains of residues lining the pore including W36, I38, W54, F83, L111, I128, and L138. There is no evidence for the maltoside head group of DDM at the top of the cavity, indicating that it does not form stabilizing interactions with the protein surface and thus is likely highly mobile (disordered) in our electron density maps (Figure 3A). Supporting our structural data of DDM binding to MxiM, we observed that the diffraction quality of MxiM crystals was dramatically affected by the presence of detergent. Crystals soaked in precipitant solutions lacking DDM retained their crystal habit (no observed cracking) but lost all diffraction. As there is no other evidence for the presence of ordered detergent in this high-resolution crystal structure (1.5 Å), it is plausible that the presence of the detergent tail in the cavity is important for stabilizing the unique fold of MxiM by maximizing hydrophobic packing within the core of the protein. It is tempting to speculate that in vivo, the natural N-terminal lipidation on MxiM could serve an analogous stabilizing role for the protein structure before association of the pilot with the MxiD secretin and OM. Furthermore, this intramolecular interaction could provide a regulatory mechanism to prevent the premature association of unbound pilot monomers to the membrane. Such a lipid-switch mechanism has been previously characterized for the myristoylated protein recoverin (Ames et al, 1999). Although our construct lacks the lipidation site, our structure suggests that the N-terminus is localized sufficiently near the lipid-binding cavity to potentially facilitate such an interaction. Figure 3.Representations of the electron density within the hydrophobic core of MxiM. (A) Electron density for the MxiM structure excluding the detergent acyl chain in the refinement. Green contours represent the Fo−Fc map contoured at 2.5σ and blue represents the weighted 2Fo−Fc contoured at 1σ. The Fo−Fc map represents electron density present in the structure that is not accounted for by the model. The refined position of the acyl chain is shown in the figure. (B) Refined MxiM–lipid (1-monohexanoyl-2-hydroxy-sn-glycerol-3-phosphate) crystal structure. Blue contours represent the weighted 2Fo−Fc contoured at 1σ. These figures are orientated such that the top is at the opening of the cavity. Download figure Download PowerPoint The proposed role of MxiM interacting directly with phospholipids on the inner leaflet of the OM (via the observed hydrophobic cavity) is an attractive hypothesis. The proposal is consistent with the observation that both lipidated and unlipidated MxiM can stabilize MxiD oligomers (Schuch and Maurelli, 1999, 2001). Further, one can envisage that disruption of the membrane via the extraction and binding of lipid molecules by MxiM could facilitate the subsequent insertion of the associated secretin MxiD. Our observations also provide the basis for an additional anchoring role of MxiM (along with the N-terminal lipidation) to optimally stabilize the large secretin ring once inserted into the membrane. Lipid binding to MxiM To investigate further the role of lipid binding to MxiM, co-crystallization experiments with MxiM and a variety of lipids were carried out. The size and quality of the MxiM crystals improved with the presence of the negatively charged single-chain lipid 1-hexanoyl-2-hydroxy-sn-glycerol-3-phosphate (Avanti #857119) in the crystallization buffer. High-resolution data (1.87 Å) were obtained for these lipid–MxiM cocrystals using a rotating anode X-ray source. The crystal structure of the lipid/pilot complex unambiguously identifies the presence of the short-chain (C-5) lipid in the hydrophobic cavity (Figure 3B). The structure shows that the binding cavity selectively holds a single chain of lipid, with the close packing of hydrophobic groups around the lipid tail sterically prohibiting the binding of a second lipid chain or bound water molecules (Figure 4A and B). The lipid makes close contacts with the same residues as that observed in the complex with DDM, but the geometry of the aliphatic chain differs slightly due to the shorter length of the lipid, which results in the inclusion of its carbonyl group deep within the nonpolar cavity (Figure 4B). Figure 4.Molecular surface representations performed using PyMol (DeLano, 2004), where the surface is colored according to atom type: nitrogen atoms are in blue, oxygen atoms in red, and carbon atoms in green. Stick representation of the protein is gray with coloring to emphasize the different residue types: polar residues (gray), hydrophobic residues (green), acidic residues (red), and basic residues (blue). (A) The refined position of the lipid (1-monohexanoyl-2-hydroxy-sn-glycerol-3-phosphate) is represented in ball and stick where the carbon atoms are shown in yellow and the polar atoms are colored according to atom type. (B) Zoomed view of the hydrophobic cavity in (A). (C) Shown in cyan are the carbon atoms of 1-palmitoyl-2-palmitoleoyl-glycerol-3-phospho-rac-1-glycerol (PG) which is modeled into the cavity. (D) Top view of the cavity opening showing the differing positions of the head groups of the bound lipid (yellow) and the modeled PG (cyan). Download figure Download PowerPoint In order to investigate the binding of MxiM28–142 with various OM lipids in solution, we performed ITC studies where MxiM28–142 (in 20 mM HEPES, pH 7.5) was titrated with four different negatively charged lipids ((1) 1,2-dioctanoyl-sn-glycero-3-phosphate (Figure 5); (2) 1,2-dioctanoyl-sn-glycero-3-phospho-rac-(1-glycerol); (3) 1,2-dihexanoyl-sn-glycero-3-phosphate; (4) 1,2-dihexanoyl-sn-glycero-3-phospho-rac-(1-glycerol)). The binding constants obtained from these studies range in value from 2 × 104 to 1 × 106 M−1 at 25°C, indicating that MxiM28–142 does indeed bind lipids with varying specificity (where lipid (1) K=7 (±3) × 105 M−1, n=0.6 (±1), ΔH=−5.0 (±0.6); (2) K=2 (±1) × 105 M−1, n=0.60 (±0.05), ΔH=−6 (±1); (3) K of <104 M−1, n and ΔH indeterminate; (4) K=2 (±1) × 104 M−1, n=0.40 (±0.05), ΔH=−4.9 (±0.9)). Due to the uncertainty regarding the presence of residual bound detergent in the lipid-binding cavity of MxiM, the reported binding constants are either Ka or K′ (apparent equilibrium constants). Nevertheless, these ITC results confirm that longer octyl chain lipids bind to MxiM with a more favorable binding constant: K between 105 and 106 M−1 at 25°C. Shorter hexyl chain lipids displayed weaker binding, with the binding affinity of 1,2-dihexanoyl-sn-glycero-3-phosphate being below the detection limit of the instrument. In our structure, the most extensive hydrophobic interactions observed between the lipid tail and MxiM originate from the five carbons at the terminal end of either one of the two acyl tails of the lipid. The second acyl tail of the lipid is therefore free to interact with a second MxiM molecule. This may explain our regressed binding stoichiometry, which was less than unity for all lipids studied. The hydrophobic interaction between the alkyl tail and MxiM would likely be quite similar among the various lipids studied, and thus the observed differences in binding are likely a result of the ability to position simultaneously the lipid head groups in an optimal manner to interact with polar and charged amino acids at the surface of the lipid-binding cavity, an ability that is obviously a direct consequence of varying lipid tail lengths. The negatively charged head group of the lipid used in our structural study (1-hexanoyl-2-hydroxy-sn-glycerol-3-phosphate) has the weakest electron density in the lipid/pilot complex, likely due to the positioning of the polar head group within the hydrophobic neck of the cavity, a restriction imposed by the shorter length of the lipid chain (Figure 4A and B). Although the extremely hydrophobic and aggregative nature of the longer chain lipids prohibited analysis in solution and in our structures, further modeling based on our current lipid complexes indicates that optimal length of acyl chain for the observed binding cavity would be ∼15 carbon atoms, in line with that of the lipids that make up the Gram-negative bacterial OM (the naturally abundant phosphatidylglycerol (PG) lipids such as 1-palmitoyl-2-palmitoleoyl-glycerol-3-phospho-rac-1-glycerol modeled in Figure 4C and D). This length of acyl chain allows for the optimal positioning of the negatively charged head group with the adjacent positively charged surfaces in the MxiM pilot. Figure 5.Analysis of the lipid, 1,2-dioctanoyl-sn-gylcero-3-phosphate, binding to MxiM using ITC, at 298 K. (Top) Raw titration data showing the heat response resulting from each 10 μl injection of 0.24 mM lipid into the ITC cell containing 24.6 μM MxiM in 20 mM HEPES at pH 7.5. (Bottom) Peak area normalized to the moles of lipid added and corrected for the heat of dilution (squares), and nonlinear least squares fit (line) to a single-site bimolecular interaction model. Download figure Download PowerPoint The presence of the deep hydroph
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