Crystal Structure and Standardized Geometric Analysis of InlJ, a Listerial Virulence Factor and Leucine-Rich Repeat Protein with a Novel Cysteine Ladder

doi:10.1016/j.jmb.2008.01.100

Journal of Molecular Biology

Volume 378, Issue 1, 18 April 2008, Pages 87-96

https://doi.org/10.1016/j.jmb.2008.01.100 Get rights and content

Abstract

We report on the crystal structure of the internalin domain of InlJ, a virulence-associated surface protein of Listeria monocytogenes, at 2.7-Å resolution. InlJ is a member of the internalin family of listerial cell surface proteins characterized by a common N-terminal domain. InlJ bears 15 leucine-rich repeats (LRRs), the same number as in InlA, the prototypical internalin family member. The LRRs of InlJ differ from those of other internalins by having 21, rather than 22, residues and by replacing 1 LRR-defining hydrophobic residue with a conserved cysteine. These cysteines stack to form an intramolecular ladder and regular hydrophobic interactions in consecutive repeats. Analyzing the curvature, twist, and lateral bending angles of InlJ and comparing these with several other LRR proteins, we provide a systematic geometric comparison of LRR protein structures (http://bragi2.helmholtz-hzi.de/Angulator/). These indicate that both cysteine and asparagine ladders stabilize the LRR fold, whereas substitutions in some repeat positions are more likely than others to induce changes in LRR geometry.

Introduction

Listeria monocytogenes, the causative agent of listeriosis in humans, is a facultative intracellular pathogen readily transmitted by contaminated food.¹ Its ability to induce its own uptake into intestinal cells, proliferate locally, and spread throughout the host has been traced to distinct members of its substantial set of surface-exposed cell wall-associated virulence factors.2, 3

Internalins belong to a multigene family exclusive to Listeria species, distinguished by variable numbers of leucine-rich repeats (LRRs).⁴ Structurally, the LRR domain forms a superhelix, the overall bending and twisting geometry of which is dictated by the consensus sequence of the particular protein and by the amino acid sequence of the individual repeat.⁵ The availability of extended concave surfaces combined with a rigid scaffold makes LRR proteins ideal protein–protein interaction partners, a feature shared with other repeat proteins.6, 7, 8 LRR proteins are found in plants, yeast, numerous metazoans, and bacteria.⁹ A family of 25 LRR-containing genes in L. monocytogenes is unique among known Gram-positive bacterial genomes.4, 10, 11

Two members of the family of listerial internalins, InlA and InlB, have been particularly well studied both functionally12, 13 and structurally.14, 15, 16, 17, 18 Both are major virulence factors of L. monocytogenes, and, in particular, both are invasion proteins that enable bacterial uptake by normally non-phagocytic host cells.2, 12 The crystal structures of the functional domains of InlA,14, 17 InlB,15, 16 InlC,¹⁹ InlE,²⁰ and InlH [Protein Data Bank (PDB) entry code 1H6U] have been published, as well as those of InlA in complex with its human receptor E-cadherin14, 17 and InlB in complex with its receptor c-Met.¹⁸

The family of 7 listerial proteins containing the typical internalin superdomain has recently been extended by the identification of another 6 members⁴, including InlI and InlJ, encoded by the genes inlI (lmo0333) and inlJ (lmo2821), respectively.²¹ All 13 members share the N-terminal “internalin” domain,¹⁶ consisting of an α-helical cap, an LRR, and an Ig-like interrepeat domain. A C-terminal LPXTG motif suggests that InlJ undergoes sortase-mediated C-terminal cleavage and covalent anchoring to the cell wall peptidoglycan.²² Between the internalin domain and the C-terminal cell surface anchor, InlJ, bears four mucin binding protein (MucBP) repeats.²¹

The inlJ gene is conserved in pathogenic Listeria species.²¹ An in-frame deletion of inlJ significantly reduces bacterial loads in the liver and spleen of wild-type mice after intravenous infection. With the use of a transgenic mouse model for food-borne listeriosis,²³ oral infection with the ΔinlJ strain was shown to result in reduced bacterial levels in the intestine, mesenteric lymph nodes, liver, and spleen.²¹ InlJ is thus a bona fide virulence factor. However, a potential receptor has not been identified as yet.

The LRR consensus sequence of InlJ deviates from that of all other internalin family members in that each repeat comprises 21, instead of the standard 22, residues. In addition, a hydrophobic residue in one of the LRR-defining positions, immediately following the β-strand, is replaced by a cysteine in InlJ. This results in a novel subtype of the LRR motif. InlJ bears a total of 14 cysteine residues in 15 LRRs—unequalled by any other LRR protein structure. We have crystallized the functional domain of InlJ and determined its structure at 2.7-Å resolution. Furthermore, we have systematically analyzed the geometry of the InlJ LRR domain, allowing us to compare its molecular architecture with that of other listerial internalin family proteins, as well as other bacterial and eukaryotic LRR proteins.

Section snippets

Overall structure of InlJ_34–508

The internalin domain of InlJ (residues 34–508, hereinafter referred to as InlJ) was produced in Escherichia coli, purified, and crystallized as described in Materials and Methods. Data for crystals with cubic symmetry (space group I23) were collected at beamline X11 (EMBL Outstation, DESY, Hamburg, Germany). The structure was solved by molecular replacement using fragments of InlA and InlB as search models and complemented manually (for details, see Materials and Methods). Difficulties in

Protein expression, purification, and crystallization

The InlJ functional domain (cap, LRR, and interrepeat domains, residues 34–508, lacking an N-terminal signal peptide and the C-terminal MucBP repeat region) was produced as an N-terminal glutathione S-transferase fusion protein using the vector pETM30 (EMBL Protein Expression and Purification Facility) in E. coli BL21 CodonPlus cells (Invitrogen). Protein production was induced by 0.1 mM IPTG in cells at an A₆₀₀ of 0.8 in LB medium with 30 μg/mL of kanamycin and 34 μg/mL of chloramphenicol and

Acknowledgements

Funding by the Deutsche Forschungsgemeinschaft as part of Priority Program 1150 (SCHU 1560/1-1 and 1-2) to W.D.S is gratefully acknowledged. We thank Karl-Heinz Genther for helpful discussions on the geometry calculations and Hartmut Niemann for helpful comments on the manuscript. We also thank Santosh Panjikar and Matthew Groves (EMBL) for help with data collection and technical support, and we gratefully acknowledge beam time at beamlines X11 and BW7A (EMBL, DESY, Hamburg, Germany), BL1

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Leucine rich repeats (LRRs) occurring in tandem are 20–29 amino acids long. Eleven LRR types have been recognized. Sequence features of LRRs from viruses were investigated using over 600 LRR proteins from 89 species. Directly before, metagenome data of nucleo-cytoplasmic large dsDNA viruses (NCLDVs) have been published; the 2,074 NCLDVs encode 199,021 proteins. From the NCLDVs 547 LRR proteins were identified and 502 were used for analysis. Various variants of known LRR types were identified in viral LRRs. A comprehensive analysis of TpLRR and FNIP that belong to an LRR type was first performed. The repeating unit lengths (RULs) in five types are 19 residues which is the shortest among all LRRs. The RULs of eight LRR types including FNIP are one to five residues shorter than those of the known, corresponding LRR types. The conserved hydrophobic residues such as Leu, Val or Ile in the consensus sequences are frequently substituted by cysteine at one or two positions. Four unique LRR motifs that are different from those identified previously are observed. The present study enhances the previous result. An evolutionary scenario of short or unique LRR was discussed.
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The twelve β strands form a parallel β sheet that defines the concave surface of the structure, whereas the eleven variable fragments define the convex surface (Figure 1B). The curvature of the β sheet is similar to that of the internalins, a family of LRR proteins from Listeria (Bublitz et al., 2008). The LRR superstructure is stabilized by an extensive hydrophobic core comprising conserved, closely packed (iso)leucines; it is also stabilized by inter-repeat backbone-backbone and backbone-side chain (i.e., the asparagine ladder) hydrogen bonds (Figure 1B).
SDS22 is an ancient regulator of protein phosphatase-1 (PP1). Our crystal structure of SDS22 shows that its twelve leucine-rich repeats adopt a banana-shaped fold that is shielded from solvent by capping domains at its extremities. Subsequent modeling and biochemical studies revealed that the concave side of SDS22 likely interacts with PP1 helices α5 and α6, which are distal from the binding sites of many previously described PP1 interactors. Accordingly, we found that SDS22 acts as a “third” subunit of multiple PP1 holoenzymes. The crystal structure of SDS22 also revealed a large basic surface patch that enables binding of a phosphorylated form of splicing factor BCLAF1. Taken together, our data provide insights into the formation of PP1:SDS22 and the recruitment of additional interaction proteins, such as BCLAF1.
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Instead, the homologous residues located internally on the loops that follow β1, β2 and β3 are hydrophobic (Val91, Ile111, and Val135), and add to the strongly nonpolar character of the pocket. This finding is reminiscent of the results of Bublitz et al., who identified that the LRR could be internally stabilized by a non-Asn residue ladder [15]. In the structures of other internalin family members, the LRR regions are followed by an Ig-like segment that protrudes from the LRR but is stably associated to it and is often referred to as the inter-repeat region [20].
Listeria monocytogenes is a human pathogen that employs a wide variety of virulence factors in order to adhere to, invade, and replicate within target cells. Internalins play key roles in processes ranging from adhesion to receptor recognition and are thus essential for infection. Recently, InlK, a surface-associated internalin, was shown to be involved in Listeria's ability to escape from autophagy by recruitment of the major vault protein (MVP) to the bacterial surface. Here, we report the structure of InlK, which harbors four domains arranged in the shape of a “bent arm”. The structure supports a role for the “elbow” of InlK in partner recognition, as well as of two Ig-like pedestals intercalated by hinge regions in the projection of InlK away from the surface of the bacterium. The unusual fold and flexibility of InlK could be essential for MVP binding and concealment from recognition by molecules involved in the autophagic process.
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Journal of Molecular Biology

Crystal Structure and Standardized Geometric Analysis of InlJ, a Listerial Virulence Factor and Leucine-Rich Repeat Protein with a Novel Cysteine Ladder

Abstract

Introduction

Section snippets

Overall structure of InlJ34–508

Protein expression, purification, and crystallization

Acknowledgements

J. Mol. Biol.

Trends Biochem. Sci.

Trends Biochem. Sci.

Prog. Biophys. Mol. Biol.

Trends Microbiol.

Cell

Cell

Mol. Cell

J. Mol. Biol.

Cell

Biochim. Biophys. Acta

Curr. Opin. Struct. Biol.

J. Mol. Biol.

Curr. Opin. Struct. Biol.

J. Biol. Chem.

J. Biol. Chem.

J. Struct. Biol.

Structure

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

Infect. Genet. Evol.

Microbes Infect.

Methods Enzymol.

Food-related illness and death in the United States

Emerging Infect. Dis.

Listeria monocytogenes: a multifaceted model

Nat. Rev. Microbiol.

Subversion of cellular functions by Listeria monocytogenes

J. Pathol.

Overall structure of InlJ_34–508