research papers
Structure of internalin C from Listeria monocytogenes
aSchool of Biological and Chemical Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, England
*Correspondence e-mail: r.w.pickersgill@qmul.ac.uk
The Listeria monocytogenes has been determined at 2.0 Å resolution. Several observations implicate InlC in infection: inlC has the same transcriptional activator as other virulence genes, it is only present in pathogenic Listeria strains and an inlC deletion mutant is significantly less virulent. While the extended concave receptor-binding surfaces of the leucine-rich repeat (LRR) domains of internalins A and B have aromatic clusters involved in receptor binding, the corresponding surface of InlC is smaller, flatter and more hydrophilic, suggesting that InlC may be involved in weak or transient associations with receptors; this may help explain why no receptor has yet been discovered for InlC. In contrast, the Ig-like domain, to which the LRR domain is fused, has surface aromatics that may be of functional importance, possibly being involved in binding to the surface of the bacteria or in receptor binding.
of internalin C (InlC) from3D view: 1xeu
PDB reference: internalin C, 1xeu, r1zeusf
1. Introduction
Listeria monocytogenes is a Gram-positive food-borne bacterium capable of causing severe infections in immunocompromized individuals, giving rise to septicaemia, meningitis or meningoencephalitis. The bacterium induces its own uptake into cells with low phagocytic activity, using the `zipper' mechanism in which direct interaction between the bacterial surface proteins and the plasma membrane-bound host receptor proteins leads to invasion of the host cell. Two proteins that have been demonstrated to facilitate bacterial entry into human cells are internalins A and B (InlA and InlB). InlA, which is covalently linked to the surface of the bacterium as a result of its C-terminal LPXTG motif, typically promotes the infection of human enterocyte-like cell lines through interaction with the cellular receptor E-cadherin (Mengaud et al., 1996). InlB, which is non-covalently bound to the surface of the bacterium via an association of its GW domains with bacterial cell-wall lipoteichoic acids, has a wider host-cell spectrum (Braun et al., 1998; Parida et al., 1998) and promotes phagocytosis by activation of phosphatidylinositol-3-kinase (Ireton et al., 1996) in response to binding gC1q-R and a Met receptor tyrosine kinase (Braun & Cossart, 2000; Shen et al., 2000).
Most virulence genes in L. monocytogenes are in a cluster controlled by the transcriptional activator PrfA (Leimeister-Wachter et al., 1990; Mengaud et al., 1991). The inlAB operon is also regulated by additional promoters. The inlC gene was discovered in a prfA-enriched/PrfA-regulated gene-cluster knockout of L. monocytogenes (Engelbrecht et al., 1996), the result of a search for additional PrfA-regulated genes and their protein products. These authors showed that the inlC gene is transcribed into a monocistronic from a single promotor with typical consensus sequence for PrfA-binding and encodes a small secreted protein of 297 residues. The inlC deletion mutant showed significantly reduced virulence in an intravenous mouse model, suggesting that InlC contributes to infection. The authors argued that InlC may be important in the later stages of infection. Subsequently, it emerged that there are two promoters for inlC, a PrfA-independent promoter in addition to the previously discovered PrfA-dependent promoter (Luo et al., 2004); these authors suggest the PrfA-independent transcription of inlC may allow invasion of the bacteria into neighbouring host cells, whereas PrfA-dependent transcription has been shown to facilitate the action of InlA in cell invasion (Bergmann et al., 2002). Engelbrecht et al. (1998) suggest that the inlC gene has been transferred between invasive Listeria species by horizontal gene transfer. Several observations support the importance of inlC in infection: (i) inlC is PrfA-regulated and InlC promotes InlA-mediated cell invasion (Engelbrecht et al., 1998; Bergmann et al., 2002), (ii) the inlC gene is present in pathogenic but not in non-pathogenic Listeria (Engelbrecht et al., 1998) and (iii) the inlC deletion mutant has reduced virulence in a mouse model (Engelbrecht et al., 1998). In this paper, we report the of InlC and compare its surface characteristics with those of InlA and InlB. The surface of the Ig-like domain of internalin C is significantly richer in external hydrophobic residues compared with internalins A and B and these residues may be important for the function of InlC.
2. Materials and methods
2.1. Construction of the expression plasmid pET14b::inlC
The full coding sequence of internalin C (Engelbrecht et al., 1996) was amplified from a pQE-30::inlC expression vector using the following two oligonucleotide primers: forward (5′-CTTCATATGGAGAGTATTCAACGACCAACG-3′) and reverse (5′-CTTGGATCCTAATTCTTGATAGGTTGTG-3′). The forward and reverse incorporated NdeI and BamHI restriction sites at their 5′-ends, respectively (the restriction sites are indicated in bold). A Perkin–Elmer 9600 thermal cycler was used for the PCR using a total reaction volume of 50 µl with 25 cycles of amplification using 2.5 U of HotStar Taq polymerase (Qiagen). The PCR product (789 bp) was gel purified using a spin column (Qiagen) and ligated into the pGEM-T Easy (Promega) vector before subcloning into pET-14b. Use of the pGEM-T Easy vector increased ligation efficiency and enabled blue/white selection of transformants in Escherichia coli JM109 cells. Verification of the presence of the inlC gene sequence was accomplished using EcoRI restriction digest and the gene was subsequently sequenced. Construction of the His-tagged fusion protein expression plasmid was achieved by subcloning the inlC NdeI/BamHI fragment from the pGEM-T Easy construct into the analogous sites in pET-14b (Novagen), resulting in the plasmid construct pET-14b::inlC. This construct was transformed into the expression host, E. coli BL21 (DE3) pLysS (Novagen). An overnight culture was used to inoculate a 1 l shake flask of LB and overexpression of InlC was promoted after 2 h of pre-culture (A600 ≃ 0.5) by the addition of isopropyl thio-β-D-galactoside (IPTG) to a final concentration of 0.04 mM. 4 h after induction with IPTG, cells were harvested by centrifugation and the resulting cell pellet was solubilized in 20 ml 25 mM Tris–HCl pH 8.0 containing 1 M NaCl. Total soluble cell protein was isolated by sonication and clarification of the resulting solution from insoluble particulates by centrifugation.
2.2. Purification of internalin C
Purification of InlC to via a two-step purification procedure. The soluble crude protein extract described above was applied onto a Chelating Sepharose Fast-Flow (Pharmacia) column (4 × 1 ml) which had been charged with Ni2+ ions and equilibrated with 25 mM Tris–HCl buffer pH 8.0 containing 1.0 M NaCl and 10 mM imidazole. Nonspecifically bound proteins were eluted using 25 mM Tris–HCl buffer pH 8.0 containing 0.1 M NaCl and 50 mM imidazole. Bound protein, mainly comprising of histidine-tagged InlC, was eluted using 25 mM Tris–HCl buffer pH 8.0 containing 0.1 M NaCl and 500 mM imidazole in 1 ml fractions and assessed for protein content spectrophotometrically at 280 nm. The His tag was cleaved using thrombin. A Superdex-75 size-exclusion column equilibrated with 25 mM Tris–HCl buffer pH 8.0 containing 25 mM NaCl was used for the final purification step. SDS–PAGE revealed a single band migrating at approximately 30 kDa. The protein was concentrated to 20 mg ml−1 for crystallization.
was achieved2.3. Crystallization, data collection and structure determination
Crystallization trials utilized two commercially available screens, Hampton Research Crystal Screens I and II, and used the hanging-drop vapour-equilibration method with 2 µl drops. These crystals could be grown fairly reliably using stock reagents and the crystals diffracted to 2.5 Å resolution at room temperature using synchrotron radiation when mounted in a glass capillary. The crystals could be cryocooled by fast transfer through a solution corresponding to reservoir supplemented with 25% glycerol and these crystals were used for data collection at 100 K. Diffraction data to 2.5 Å, comprising 300 images each corresponding to a 0.5° rotation of the crystal, were recorded using crystal 1 and PX9.6 at SRS Daresbury (λ = 0.87 Å) equipped with an ADSC image plate. The diffraction data were processed using DENZO and SCALEPACK (Otwinowski & Minor, 1997) and the CCP4 program suite was used for subsequent calculations (Collaborative Computational Project, Number 4, 1994). The structure was solved by using MOLREP (Vagin & Teplyakov, 1997) and the structure of internalin B (Schubert et al., 2001) that had been solved by using the structure of the LRR domain (Marino et al., 1999). The 2.5 Å model of InlC was built using iterative cycles of building into σA-weighted 2Fobs − Fcalc maps using O (Jones et al., 1991) and using REFMAC (Murshudov et al., 1997). The final model was refined against subsequently collected 2.0 Å data and had an R factor of 21.1% (Rfree = 25.8%) and contains 263 residues (35–297) and 226 water molecules. Model stereochemistry was evaluated using the program PROCHECK (Laskowski et al., 1993). A total of 80.6% of residues are in the core regions of the Ramachandran plot, with no residues in disallowed regions. The data-collection and are summarized in Table 1.
‡Search models I and II were InlB and InlB truncated by the removal of one coil of the LRR domain, respectively. The molecular-replacement solution was similar for both models, but the was substantially more convincing for model II. §R = , where Fobs and Fcalc are the observed and calculated structure-factor amplitudes, respectively. The R factor and Rfree were calculated using the working (95% of the data) and test set (5% of the data), respectively. |
3. Results and discussion
3.1. Structure determination
Crystals grew as thin plates of maximum dimension 0.5 mm from the trial with reservoir containing 1.8 M magnesium sulfate, 0.1 M MES pH 6.5. The were consistent with P21212, which was confirmed by molecular-replacement results, with unit-cell parameters a = 58.78, b = 179.51, c = 42.26 Å, with the containing one InlC molecule and comprising approximately 65% solvent. Using the internalin domain of InlB (PDB code 1h6t ), the closest structure in the PDB, with 34% sequence identity to InlC, as the search model in gave a of only 0.223; however, if the third coil of the LRR domain was removed and a `shortened' InlB molecule produced then the correlation improved significantly to 0.318 (Table 1). The electron-density maps were sufficiently clear and lacking in bias to allow cycles of rebuilding and to give a final map in which all residues of InlC could be clearly seen. Subsequently, the structure was refined against the 2.0 Å data collected from a second crystal (over 30 crystals were screened to find two well diffracting ones) and the statistics for both refinements are presented in Table 1.
3.2. Overall structure
InlC comprises an α-helical cap, a leucine-rich repeat (LRR) and immunoglobulin-like (Ig-like) domains (Fig. 1) as revealed for other members of the internalin family (Schubert et al., 2001, 2002). The cap domain, residues 35–76, contains three α-helices and two very short β-strands. The LRR domain, residues 77–213, has right-handed superhelical architecture comprising six repeats, each with 22 residues per repeat except for coil three, which has 21 residues. A conserved pattern of internal aliphatic residues and an asparagine directed internally characterizes each repeat (Fig. 2). Each LRR tends to have leucine, isoleucine or valine at positions 5, 7, 12, 15, 18 and 21 of the coil, although some variation is possible. A typical coil comprises a five-residue β-strand and, less strictly, a five-residue 310-helix (Fig. 2a). In turn three the 310-helix is shortened and the aliphatic residue at position 21 is absent (Fig. 2b). The asparagine at position 10 appears to be important as it forms a buried asparagine ladder within the LRR domain. YopM, a LRR protein from Yersinia pestis, has 12 repeats of 20 residues and three of 22 residues (Evdokimov et al., 2001). There is a single 21-residue repeat in InlA that confers functionally important flexibility to the molecule by disrupting the β-sheet locally (Schubert et al., 2002). The deletion in InlC shortens the 310-helix and if it is of functional significance then it may be in flattening the putative receptor-binding surface. Residues 207–297 form an Ig-like domain, with afgc and deb β-sheets. In fact, because the domains are fused together, the last few residues of one domain are also the beginning of the next, as the residues form one contiguous structure.
3.3. Putative receptor-binding surface
The concave face of the β-sheet of the internalins has long been considered to be the binding surface by analogy with ribonuclease inhibitor protein (Kobe & Deisenhofer, 1995) and this has been demonstrated for InlA in its complex with E-cadherin (Schubert et al., 2002). Surface-exposed aromatic residues dominate the direct interactions of the InlA LRR with E-cadherin and aromatic residues have also been shown to be important in the interaction of the LRR domain of InlB with the Met receptor (Machner et al., 2003). The corresponding putative binding surface of InlC is flat compared with the concave surfaces of InlA and InlB, partially because InlC has the shortest LRR domain with only six repeats, compared with eight and 15 for InlB and InlA, respectively, but also because of the irregular nature of the 310-helices, including the truncated helix in repeat three. The surface of the LRR domain is principally hydrophilic with only two exposed hydrophobic residues, Phe146 on the fourth β-strand of the LRR domain and Phe182 on the fifth 310-helix of the LRR domain (Fig. 3). There is no obvious similarity in the pattern of hydrophobic residues or charged residues when the proposed binding surface is compared with those of InlA or InlB. Aromatic contacts are also important in binding the GW domains of InlB to glycosaminoglycans and gC1q-R (Marino et al., 2002).
3.4. Crystal packing
With few exceptions, the surface aromatic residues on the surface of InlC are either directly involved in crystal contacts or are very close to a crystal contact (Table 2; Fig. 4). The contacts therefore principally involve the Ig-like domain, with fewer contacts to the C-terminal part of the LRR domain and no contacts to the N-terminal part, mirroring the pattern of exposed aromatics (Fig. 3). The cap domain makes a contact with its twofold-related symmetry mate involving Pro42 and Leu68 (Fig. 4). Hydrogen bonds are involved in crystal contacts with molecules B and C (Table 2).
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3.5. Functional implications
The most similar structures in the PDB are InlB and InlA, with other structures with LLR domains also being detected by DALI (Holm & Sander, 1993; Table 3). Comparison of the structure of InlC with InlA and InlB reveals no commonality in their surface features; there is no conserved pattern of hydrophobic residues and no common charge distribution (Fig. 5), in agreement with the observation that InlC binds to a different receptor to those bound by InlA and InlB. The N-terminal end of the molecule and especially the Ig-like domain is where the aromatic residues are concentrated and this region might self-associate as seen in the crystal or it might associate with other proteins. It is possible that the Ig-like domain associates loosely with the bacterial cell wall. By analogy with InlA and InlB, the LRR domain would then be likely to interact with host proteins. The putative binding surface of the LRR domain of InlC is flat and predominantly hydrophilic; associations solely involving hydrophilic and charged residues have been less commonly seen at protein–protein interfaces and if involved in binding, this surface would be involved in relatively weak or transient associations with other molecules (Bahadur et al., 2004). The relatively flat surface of the β-sheet also suggests weak interactions. An involvement in transient interactions or interactions within the cytoplasm might explain why binding partners have yet to be discovered for InlC. Alternatively, the freely accessible Ig-like domain of InlC, unlike the Ig-like domains of InlA and B which are within larger multi-domain proteins, might itself constitute a potential receptor-binding site affording a new method of interaction between the small internalins and their host receptors.
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Acknowledgements
We are grateful to Fredi Engelbrecht and Werner Goebel (Universität Würzburg, Germany) for providing the original pQE-30::inlC construct. We acknowledge the use of the Synchrotron Radiation Source, Daresbury and the ESRF Grenoble. The Higher Education Funding Council for England and the Biotechnology and Biological Sciences Research Council supported this work.
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