- Email: [email protected]
Crystal Structure of LipL32, the Most Abundant Surface Protein of Pathogenic Leptospira spp.
J. Mol. Biol. (2009) 387, 1229–1238
doi:10.1016/j.jmb.2009.02.038
Available online at www.sciencedirect.com
Crystal Structure of LipL32, the Most Abundant Surface Protein of Pathogenic Leptospira spp. Julian P. Vivian 1,2 †, Travis Beddoe 1,2 ⁎†, Adrian D. McAlister 1 , Matthew C.J. Wilce 1 , Leyla Zaker-Tabrizi 1,2 , Sally Troy 1 , Emma Byres 1,2 , David E. Hoke 3 , Paul A. Cullen 3 , Miranda Lo 3 , Gerald L. Murray 3 , Ben Adler 2,3 and Jamie Rossjohn 1,2 ⁎ 1
The Protein Crystallography Unit, Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800, Australia 2
ARC Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton, Victoria 3800, Australia 3
Department of Microbiology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800, Australia Received 19 December 2008; received in revised form 10 February 2009; accepted 12 February 2009 Available online 21 February 2009 Edited by I. Wilson
Spirochetes of the genus Leptospira cause leptospirosis in humans and animals worldwide. Proteins exposed on the bacterial cell surface are implicated in the pathogenesis of leptospirosis. However, the biological role of the majority of these proteins is unknown; this is principally due to the lack of genetic systems for investigating Leptospira and the absence of any structural information on leptospiral antigens. To address this, we have determined the 2.0-Å-resolution structure of the lipoprotein LipL32, the most abundant outer-membrane and surface protein present exclusively in pathogenic Leptospira species. The extracellular domain of LipL32 revealed a compact, globular, “jelly-roll” fold from which projected an unusual extended β-hairpin that served as a principal mediator of the observed crystallographic dimer. Two acid-rich patches were also identified as potential binding sites for positively charged ligands, such as laminin, to which LipL32 has a propensity to bind. Although LipL32 shared no significant sequence identity to any known protein, it possessed structural homology to the adhesins that bind components of the extracellular matrix, suggesting that LipL32 functions in an analogous manner. Moreover, the structure provides a framework for understanding the immunological role of this major surface lipoprotein. © 2009 Elsevier Ltd. All rights reserved.
Keywords: Leptospira; LipL32; jelly-roll fold; outer-membrane protein
Introduction Leptospirosis is caused by infection with the spirochete bacterium, Leptospira. The organism has a broad host range and is thought to be the most widespread zoonotic agent worldwide. The main*Corresponding authors. The Protein Crystallography Unit, Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800, Australia. E-mail addresses: travis. [email protected]; jamie.rossjohn@med. monash.edu.au. † J.P.V. and T.B. contributed equally to this work. Abbreviations used: ECM, extracellular matrixc; SeMet, selenomethionine; SC, shape complementarity; PDB, Protein Data Bank.
tenance hosts involved in transmission to humans are mainly rodents, dogs, or cattle that harbor pathogenic Leptospira spp. in their proximal renal tubules, resulting in long-term urinary shedding of bacteria into the environment. Human infection occurs when leptospires enter via mucosal surfaces or broken skin following contact with infected animals or contaminated soil or water.1 Once in the body, leptospires are found in multiple organs and tissues. The disease ranges from mild symptoms to more serious complications including jaundice, pulmonary hemorrhage, and renal and hepatic failure, which may prove fatal.2–4 Pathogenic Leptospira spp., previously classified into the single species Leptospira interrogans, are now differentiated into at least 12 species, with L. interrogans and Leptospira borgpetersenii being the main causes of human disease worldwide. More than half
0022-2836/$ - see front matter © 2009 Elsevier Ltd. All rights reserved.
Crystal Structure of a Leptospiral Antigen
1230 of the 250 serovars of Leptospira are contained in these two species. The global importance of this disease is highlighted by estimates that leptospirosis causes severe complications for 1 million people annually.5–7 Immunity to Leptospira in humans is mediated primarily by antibodies directed towards lipopolysaccharide.8 This immunity is serovar specific and has led to a search for conserved cell surface proteins that might elicit cross-serovar immunity. One such candidate is LipL32, the most abundant outermembrane protein9 and the most abundant surfaceexposed protein.10 LipL32 is a lipoprotein, with the protein component remaining external yet anchored to the outer membrane through fatty acids covalently attached to the amino-terminal cysteine residue. There are several lines of evidence that suggest that LipL32 may be important in pathogenesis. The lipL32 gene is present only in pathogenic species, where it displays a high degree of conservation.11 Cellular assays indicate that LipL32 induces an inflammatory response in cultured renal cells12,13 and a promyelocytic cell line expressing Cd14. In vivo studies show that LipL32 is a major target of the antibody response in animals and humans and is expressed in the kidneys of infected animals.11,14,15 The function of LipL32 may be affected by posttranslational events such as proteolytic cleavage, with studies suggesting that the C-terminus of a subset of LipL32 proteins is shed while the N-terminus remains attached to the cell surface.9 LipL32 might be involved in the pathogenesis of leptospirosis through extracellular matrix (ECM) binding activity identified in two recent reports.15,16 Protein truncation studies were used to map this activity, with one report determining that the 72 C-terminal amino acids were sufficient for binding, while the other showed that the 85 C-terminal amino acids were sufficient and also inhibited binding to the intact protein. A LipL32 ortholog in the ubiquitous marine bacterium Pseudoalteromonas tunicata also showed C-terminal ECM binding activity and crossreacted with anti-LipL32 antiserum.16 These studies suggest that C-terminal elements within orthologous LipL32 proteins function as ECM-binding domains in diverse bacteria. Lastly, LipL32 has been shown in some studies to confer partial protection against infection in animal models.17–19 Interestingly, anti-LipL32 antibodies produced in response to naturally acquired infection do not appear to be protective. A recent study focused on LipL32 domains that are recognized during human infection and showed that the immunoglobulin M response was limited to a Cterminal domain during acute and convalescent phases.15 The immunoglobulin G response changed during infection, with most patient sera recognizing a central region of the protein during convalescence and 5 out of 12 patients responding to the C-terminus in the acute and convalescent phases. These findings suggest that differential immune recognition of LipL32 domains during infection and convalescence may be a factor in immunity to Leptospira. The lack of sequence similarity between LipL32 and any other functionally characterized protein has
hindered progress in determining structural domains that relate to function. Therefore, structural determination of LipL32 is important for future work in understanding the potential role of LipL32 in immunity to infection, characterization of the pro-inflammatory response, and interaction with ECM proteins. In this article, we describe the high-resolution crystal structure of LipL32, thereby providing fundamental insight into its role in infection and immunity.
Results Expression, purification, and crystallization of LipL32 To gain insight into the structure–function relationship of leptospiral antigens, we expressed the entire mature form of LipL32 in Escherichia coli and purified it to homogeneity (see Materials and Methods). While this domain produced extremely large crystals of defined morphology, they diffracted very poorly (approximately to 4 Å resolution) and the diffraction pattern exhibited diffuse thermal scattering, indicating potential disorder within the crystal lattice. Accordingly, we undertook a limited proteolysis approach in an attempt to “shave off” any flexible regions of LipL32. Of the proteases that Table 1. Data collection and refinement statistics
Data collection Space group Cell dimensions (Å) Resolution (Å) Total observations Unique observations Rsyma Rpimb I/σI Completeness (%) Redundancy Refinement Resolution Reflections in working set Rcrystc Rfreec Total protein atoms Total waters Bond lengths (Å) Bond angles (°) Ramachandran analysis (%) Most favored Additionally allowed Average B-factor (Å2) Main chain Side chain Waters
Native
KAuBr4
P3221 a = b = 125.9, c = 95.9 50–2.0 (2.11–2.0) 1,270,906 59,453 0.078 (0.724) 0.023 (0.273) 41.9 (2.4) 100 (100) 21.4 (20.3)
P3221 a = b = 124.7, c = 95.4 50–2.8 (2.95–2.80) 377,219 20,492 0.089 (0.822) 0.027 (0.311) 30.7 (2.1) 95.7 (77.7) 18.4 (9.9)
50–2.0 54,787 0.185 0.218 3520 447 0.011 1.314 97.3 2.7 47.5 48.8 53.5
Values in parentheses represent those of the highest-resolution shell. a Rsym = ∑(∣Ii − Imean∣)/∑(Ii). b Rpim = √(1/(n − 1)) · ∑(∣Ii − Imean∣)/∑(Ii). c Rcryst = ∑hkl∣∣Fo∣ − ∣Fc∣∣/∑hkl∣Fo∣ for all data excluding the 5% that comprised the Rfree used for cross-validation.
Crystal Structure of a Leptospiral Antigen
we investigated, cleavage with V8 protease yielded optimal results and provided a fragment that was more amenable for structural studies. The V8 protease cleaved LipL32 within the N-terminal tobacco etch virus protease recognition site and 11 residues from the C-terminus. This yielded a protein comprising 11 vector-encoded residues at the Nterminus and residues 2–242 of the mature LipL32, as confirmed by N-terminal sequencing and mass spectrometry (data not shown). The V8-cleaved LipL32 crystallized readily and diffracted to 2.0 Å resolution and belongs to space group P3221, with unit cell dimensions a = b = 125.9 Å and c = 95.9 Å, which is consistent with two LipL32 molecules per asymmetric unit (see Table 1). Given that sequence analysis suggested that there were no structural homologues of LipL32, we next sought to label the LipL32 with selenomethionine (Se-Met). However, the Se-Met-labeled LipL32 expressed extremely poorly (0.3 mg/l), and the resulting crystals were very small, thereby precluding structure determination via Se-Met multiwavelength anomalous diffraction phasing. Accordingly, we determined the structure of LipL32 via the single-isomorphous replacement method (see Materials and Methods), which resulted in a readily interpretable electron density map. The structure of LipL32 was subsequently refined to 2.0 Å resolution to an R-factor and Rfree of 18.5% and 21.8%, respectively. The model contains residues 6–242 of the mature protein, with residues 2–5 and 140–146 being uninterpretable in the electron density map and 447 water molecules. LipL32 crystallized as two protomers within the
1231 asymmetric unit, which were virtually identical with each other (rmsd, 0.47 Å over all Cα positions), and unless explicitly stated, structural analysis will be confined to one monomer. Overall structure of LipL32 LipL32 adopted a compact, globular fold of overall dimensions of ≈29 Å × 50 Å × 56 Å, is rich in β-sheet (38%), and possesses a few peripheral α-helices (17%), and the “top” and “bases” of LipL32 possess a number of meandering loops that serve to connect the major secondary structural elements (Fig. 1a). While these loops are generally long, the mobility of the loops is restricted via interloop contacts, and as such, the majority of the molecule displays limited mobility, with an average B-factor of ≈48Å2. The core of LipL32 comprised a mixed β-sheet that packed against an antiparallel β-sheet and adopted a “jelly-roll” fold of eight β-strands (β-3, β-5, β-6, β-8, β-9, β-10, β-11, and β-12) (Fig. 1b). The interior of this β-sandwich contained a large number of hydrophobic interactions, characterized by small aliphatic residues towards the top of the molecule and a cluster of aromatic residues at the base of the core. Prior to the core of the molecule is the N-terminal region (which represents the site of attachment to the bacterial outer membrane) that contains a β-hairpin that runs approximately orthogonal to and extends approximately 35Å from the βsheet of the jelly roll. On the other face of LipL32 are two C-terminal helical regions that interdigitate in an antiparallel manner. These two helices (residues 126– 134 and 230–241) pack against the core β-sheet
Fig. 1. Crystal structure of LipL32. (a) Cartoon representation of the LipL32 structure. The structure is color ramped from the N-terminus to the C-terminus. The secondary-structure elements of the protein are labeled. The structure comprises a two-strand N-terminal dimerization domain, with the β-1 (residues 11–20) and β-2 (residues 27–33) strands arranged in a β-hairpin. The core of the protein is a jelly roll composed of strands β-3 (residues 36–44), β-5 (residues 58–67), β-6 (residues 73–79), β-8 (residues 115–120), β-9 (residues 151–154), β-10 (residues 162–166), β-11 (residues 189–196), and β-12 (residues 205–214). LipL32 has three short parallel β-strands that form extensions to the core jelly roll including β-4 (residues 52–54), β-7 (residues 92–94), and β-13 (residues 225–227). Flanking the poles of the molecule are two sets of short helices, with helices α-1 (residues 96–100) and α-2 (residues 103–107) located opposite helices α-4 (residues 174–177) and α-5 (residues 182–184). Distal to the dimerization interface is a helical domain comprising α-3 (residues 126–134) and the C-terminal helix α-6 (residues 230–241). (b) Topology diagram of the LipL32 structure colored similarly to (a).
Crystal Structure of a Leptospiral Antigen
1232
Fig. 2. Representation of the electrostatic potential on the surface of LipL32. Two views of the LipL32 monomer with the charge distribution calculated by the Adaptive Poisson–Boltzmann Solver22 mapped onto the surface of the protein. For the purposes of modeling the charge distribution, the aspartate-rich loop (residues 140–146, sequence: KLDDDDD) that was not ordered in the crystal structure was modeled. The regions of electronegative and electropositive charge are labeled.
(predominantly via hydrophobic interactions) that is centered on Trp66 packing against Pro126, Ile129, and Ala240. Above and below the Trp residue are polar interactions that further serve to tether the helical region to the core of the molecule. Surface-exposed electrostatic or hydrophobic patches can often be indicative of regions that mediate protein–protein and/or protein–ligand interactions.20,21 Notably, LipL32 possessed a cluster of surface-exposed aromatic residues towards the base of the molecule, which included Phe112, His156, Tyr159, and Tyr198. Additionally, LipL32 possessed two patches of marked electronegative charge, with electronegative patch 1 dominated by residues Glu231, Glu232, Glu241, and Glu242, while patch 2 is dominated by Glu119 (Asp142, Asp143, Asp144, Asp145, and Asp146 modeled for electrostatic calculations), Asp147, and Asp148. Additionally, LipL32 possessed a region of positive charge that included residues Lys58, Lys59, Lys199, and Lys204 (Fig. 2). Accordingly, LipL32 possessed a compact globular fold with a number of surface-exposed features that indicated a potential capacity to bind other components or interact with itself. LipL32 as a dimer Although LipL32 was purified as a monomer by gel filtration, the two monomers within the asymmetric unit were observed to form a crystallographic dimer, with the monomers related by an approximate 2-fold rotation. This higher-order assembly is consistent with the propensity for LipL32 to oligomerize on the cell surface (data not shown). Although crystallographic dimers can often be an artifact of crystallization, the observed contacts within the crystalline lattice were suggestive of a biologically relevant LipL32 dimer, as was also suggested by the PISA web server‡.23 The interface ‡ http://www.ebi.ac.uk/msd-srv/prot_int/pistart. html
was extensive (circa 1250Å2), exhibited high shape complementarity (SC) (SC index = 0.66), and was characterized by a large number of hydrophobic interactions and only four hydrogen bonds (Table 2). The dimer is formed such that the N-terminal regions are close to the dyad axis, while the Cterminal helices are located at the external face of the dimer. There are two main sites of interaction at the dimer interface, mediated by the unusual β-hairpin and the base of the molecule (Fig. 3). Firstly, the β-hairpin that was observed to project from the core of the molecule wraps around its counterpart in the other protomer. While the top of the respective
Table 2. Interactions at the dimeric interface of LipL32 Chain A
Chain B
Interaction
Thr30 Thr82 Tyr159 Asn160 Gly6 Leu7 Lys11 Leu16 Ser17 Asp19 Ile21 Pro22 Val28 Thr30 Ile79 Pro81 Thr82 Gly83 Glu104 Ser107 Met108 Pro109 His110 Trp111 Phe112 Tyr159 Asn160 Pro200
Thr30 Tyr159 Thr82 Tyr159 Tyr159 Tyr159 Val28 Asp19, Val28 Asp19 Leu16, Ser17 Leu32 Leu16 Lys11, Leu16, Pro33 Asp19, Thr30 Tyr159 Phe112 Phe112, Tyr159 Phe112 Pro200 Phe112, Pro200 Pro200 Pro109, His110, Trp111 Pro109 Pro109 Pro81, Thr82, Gly83, Ser107 Gly6, Leu7, Pro8, Ile79, Thr82, Asn160 Tyr159, Asn160 Glu104, Met108
H-bond H-bond H-bond H-bond Nonbonded Nonbonded Nonbonded Nonbonded Nonbonded Nonbonded Nonbonded Nonbonded Nonbonded Nonbonded Nonbonded Nonbonded Nonbonded Nonbonded Nonbonded Nonbonded Nonbonded Nonbonded Nonbonded Nonbonded Nonbonded Nonbonded Nonbonded Nonbonded
Crystal Structure of a Leptospiral Antigen
1233
Fig. 3. Dimerization of LipL32. (a) and (b) are cartoon representations of the LipL32 dimer. (a) View of the LipL32 dimer looking perpendicularly to the dimer interaction surface. The monomers of LipL32 are colored green and brown. (b) Another view of the LipL32 dimer looking perpendicularly to the dimer interaction surface except that it is rotated 90° about the x-axis relative to (a). (c) View of the dimerization interface of LipL32. The figure is a surface representation of the LipL32 monomer with some of the important residues involved in dimer–dimer contacts labeled. The residues forming nonbonded contacts are colored green, and the atoms involved in forming hydrogen bonds are colored blue.
β-hairpins are not involved in mediating dimer contacts, the residues proximal and distal to the tip are involved in a number of van der Waals interactions, centered on Thr27. While Thr27 interacted with its equivalent residue, Ile21 packed against Leu32; Val28 packed against Leu16 and Pro33; Leu16 interacted with Asp19, the latter of which also forms van der Waals interactions with Ser17. Secondly, the base of the molecule, which is rich in aromatic residues, forms a cluster of hydrophobic contacts at the interface. Tyr159 wedged within a bulged region of β-strand 8, packing against Ile79, while the TyrOH H-bonds to the main chain of Thr82. The main chain of Tyr159 H-bonds to Asn160, the latter of which also forms van der Waals interactions with its counterpart. The hydrophobic cluster is extended with Phe112 interacting with Pro81, the main chain of Thr82, Gly83, and Ser107.
Taken together, the features at the dimer interface, namely, an extensive hydrophobic interface exhibiting high SC, suggest that the crystallographic LipL32 dimer may represent a biologically relevant LipL32 dimer. Structural comparisons While LipL32 possessed no significant sequence similarity to any protein in the Protein Data Bank (PDB), we undertook automated structural comparisons (DALI server) against the PDB database, as, often, structural conservation is stronger than sequence conservation. The DALI-based search revealed that LipL32 exhibited significant structural homology to proteins that also possess a jelly-roll fold despite the low level of sequence identity (circa 8–13%). Among the significant hits, LipL32 showed matches to a domain from calpain (Z score, 8.3;
1234
Crystal Structure of a Leptospiral Antigen
Fig. 4. Comparison of LipL32 with the structural homologue collagenase collagen-binding domain 3B from Clostridium histolyticum. Structural superposition of LipL32 with the collagen-binding domain 3B was performed with SSM.26 In (a) and (b), LipL32 is represented in cartoon format in green, and the collagen-binding domain 3B is represented in cartoon format in light pink. The two orthogonal views (a and b) show the structural similarity of the core β-sheets of the proteins. (c) Surface representation of collagen-binding domain 3B in the orientation depicted in (b). The residues comprising the hydrophobic surface shown to be important for collagen binding are highlighted in red. (d) Surface representation of LipL32 in the orientation depicted in (b). Highlighted in orange are residues that comprise the putative collagen binding site of LipL32. These residues form a hydrophobic surface positioned similarly to that of the structural homologue collagen-binding domain 3B.
rmsd, 3.5 Å over 124 Cα atoms) and collagenase (Z score, 6.0; rmsd, 2.5 Å over 87 Cα atoms). Interestingly, the collagenase domain binds the ECM component collagen,24 and the calpain domain is thought to localize calpain to the membrane.25 The ECM binding site of collagenase is characterized by hydrophophic interactions, with mutagenesis studies identifying a cluster of tyrosine residues, with a leucine, an arginine, and a threonine residue contributing to a hydrophobic surface.24 Structural superposition of LipL32 and the collagenase reveals a similarly located hydrophobic surface on LipL32, comprising residues Tyr62, Tyr151, Tyr198, Trp115, Leu53, Val54, and Arg117, as shown in Fig. 4. Accordingly, LipL32 possesses homology to structures that bind to ECM components. Recently, a sequence homologue of LipL32 has been reported from P. tunicata (PTD2-05920).16 This homologue, which shares 44% sequence identity with Leptospira LipL32, is from a marine bacterium that inhabits a primitive chordate (Ciona intestinalis) that has ECM components.27 The high level of sequence identity across the entire extracellular domain is indicative of P. tunicata PTD2-05920 possessing a similar fold, and moreover, as the electrostatic patches, including the
string of aspartate residues between 142 and 148 and the hydrophobic surface-exposed patches including residues Phe112, His156, Tyr159, and Tyr198, are conserved, it is likely to have similar binding properties to itself and other components. It has recently been shown that PTD2-05920 can bind ECM proteins.16
Discussion The array of cell-surface adhesion molecules found on pathogenic species of Leptospira (including LigA, LigB, and other lipoproteins) is of fundamental importance to our understanding and preventive treatment of leptospirosis. These molecules are key to the invasion and adhesion of bacteria to the host tissue, a process that is central to the pathogenesis of the organism. Furthermore, the cellsurface localization of these adhesins makes them ideal targets for vaccine development. Of these adhesins, LipL32 stands out as a prime candidate for vaccine development as it is the most abundant cellsurface protein in pathogenic Leptospira and is the major target of the immune system during human
Crystal Structure of a Leptospiral Antigen
infection. Despite the important role of LipL32, our current understanding of its functional role was unclear. To address this, we have determined the crystal structure of LipL32 to 2 Å resolution, the first crystal structure of a leptospiral adhesin. It has been shown recently that LipL32 has the capacity to interact in vitro with multiple ECM binding partners including plasma fibronectin, collagen IV, and laminin. 15,16 Fibronectin and laminin have defined cell- or bacteria-binding domains where collagen provides multiple sites of interactions along its triple-helical structures. 28 Electrostatic analysis of the LipL32 dimer has identified two noncontiguous electronegative patches arranged in tandem. It is tempting to speculate that these patches have a role in binding ECM components with sites of known complimentary charge, especially laminin. Similarly, a hydrophobic surface identified by comparison with the structural homologue collagen-binding protein 3B represents a putative site for collagen binding. Other studies have mapped ECM interaction sites to the Cterminal region of LipL32 comprising residues 166– 253. Mapping this C-terminal fragment onto the
1235 crystal structure of LipL32 reveals that it comprises the large loop between β-10 and β-11 (including helices 4 and 5) and the large loop region between βstrands 12 and 13. It also contains β-strands 11, 12, and 13 and helix 6 (Fig. 5a and b). As this C-terminal domain would be expected to have limited tertiary and secondary structure in isolation, the proposed site of interaction would, therefore, be a linear stretch of amino acids. There are three surfaceexposed regions spanning residues 168–185, 214– 222, and 230–242 that could mediate the interaction with ECM proteins. Linear peptides have previously been shown to inhibit binding to laminin by the Tp0751 adhesin from the spirochete Treponema pallidum, the causative agent of syphilis. 30 The observed affinity of LipL32 binding to ECM proteins is relatively weak with a Kd of ≈7–10 μM. Therefore, the existence of the observed dimer may be a means of increasing the avidity for the ECM proteins, through an increase in the local concentration of binding sites.16 The multiple ECM binding by LipL32 has also been shown in other leptospiral adhesins such as LigA, LigB, and the family of Len proteins.31,32 This
Fig. 5. Antibody and ECM-binding domains of LipL32. (a and b) Two views of the C-terminal fragment of LipL32 used in binding assays with ECM proteins. The LipL32 structure is shown in cartoon representation in green. The residues that were subcloned to produce a C-terminal fragment for binding assays (residues 166–253)15,16 are shown in orange. Analysis of this C-terminal fragment, with the benefit of the crystal structure of LipL32, suggests that, in all likelihood, this fragment would not fold as it does in the full-length protein. Therefore, binding of this C-terminal section to ECM proteins is likely to occur via linear segments. (c) Epitope 1. The LipL32 structure is shown in cartoon representation in green with residues 132–158 shown in pink.29 (d) Epitope 2. The LipL32 structure is shown in cartoon representation in green with residues 162–186 shown in pink.29
1236 redundancy in ECM adhesion would convey a selective advantage to Leptospira during the initial infection stage as 95% of human patients with leptospirosis produce antibodies against LipL32.14 Many bacterial adhesins can be effective vaccine candidates, such as Hap adhesin from Haemophilus influenzae and collagen adhesin from Staphylococcus aureus.33,34 In addition, peptide inhibitors of several bacterial pathogens such as Streptococcus mutans, Porphyromonas gingivalis, and Pseudomonas aeruginosa have been used to prevent attachment of these bacteria to the corresponding ligand.35–37 Various strategies have been used in these studies to inhibit adhesion. These include vaccination with recombinant protein, therapeutic administration of selected peptides, and passive administration of antibodies raised against peptide sequences that mediate adhesion.38 Recently, human antibody epitopes have been characterized for LipL32.29 They consist of residues 132–158 and 162–186 (Fig. 5c and d). The peptide epitope 162–186 corresponds to one of the proposed interaction loops of LipL32. In addition, this peptide also lies within the C-terminal domain, which is the most immunogenic region of LipL32.15 The crystal structure of LipL32 will facilitate future studies that aim to elucidate the interaction of Leptospira with host tissue components and its role in the pathogenesis of leptospirosis.
Materials and Methods Protein expression and purification DNA encoding residues 2–253 of mature LipL32 was amplified via PCR from L. interrogans serovar Lai strain L391 genomic DNA using the primers 5′-ATAGCGGCCGCAGGTGCTTTCGGTGGTCTG-3′ and 5′-GCCACCTTTCGGTACCTTTTTAACC-3′. The resultant PCR product was digested with NotI and KpnI restriction enzymes and cloned into modified pQE30 expression vector (Qiagen) containing a tobacco etch virus protease recognition site between the histidine tag and start of the mature protein. The recombinant protein was produced in E. coli BL21 (DE3) cells grown in LB broth supplemented with 100 μg/ml ampicillin, grown with shaking at 200 rpm at 37°C to an OD600 (optical density at 600 nm) of 0.5. At this point, IPTG was added to a concentration of 0.5 mM and the cells were grown for a further 4 h at 37 °C with shaking at 200 rpm. The cells were then harvested by centrifugation at 6000g for 15 min and resuspended in 20 mM Tris, pH 8.0, 300 mM NaCl, and 10 mM imidazole, pH 8.0, prior to lysis by French press. Cellular debris were cleared by centrifugation at 30,000g for 30 min, and the resultant supernatant was loaded onto a nickel-Sepharose column pre-equilibrated in 20 mM Tris, pH 8.0, and 300 mM NaCl. The column was washed with 6 column volumes of 20 mM Tris, pH 8.0, 300 mM NaCl, and 10 mM imidazole, pH 8.0, prior to elution of the bound protein with 5 × 1 column volumes of 20 mM Tris, pH 8.0, and 400 mM imidazole, pH 8.0. The eluted fractions were pooled, and further purification proceeded by gel-filtration chromatography using an S75 16/60 column equilibrated in 10 mM Tris, pH 8.0, and 150 mM NaCl. Fractions containing LipL32 were pooled, and the solution was
Crystal Structure of a Leptospiral Antigen
diluted 1:3 with 10 mM Tris, pH 8.0, prior to loading onto a Mono Q 5/50 (GE Healthcare) anion-exchange column. Pure LipL32 was collected from the unbound fraction. The protein was then dialyzed against 10 mM Tris, pH 8.0, and 150 mM NaCl prior to concentration to 10 mg/ml. This solution was used for crystallization trials. The protein was proteolyzed with V8 protease following elution from the size-exclusion column to improve the diffraction quality of the LipL32 crystals. V8 protease (Sigma) was added at a 1/20 ratio (w/w) to LipL32 for 80 min and then reapplied to the S75 16/60 column equilibrated in 10 mM Tris, pH 8.0, and 150 mM NaCl to separate V8 protease from Lipl32. The purified protein was concentrated to 10 mg/ml. Crystallization and X-ray diffraction data collection Initial crystallization trials centered on the full-length construct of LipL32, including the N-terminal His6 purification tag. Clusters of very thin needle-like crystals were produced using the hanging-drop vapor-diffusion method at 294 K. One μl of protein solution (10 mM Tris, pH 8.0, and 150 mM NaCl) was mixed with 1 μl of precipitant solution (2.0 M ammonium sulfate and 0.1 M Tris, pH 7.8) and suspended above 1 ml of precipitant solution. These crystals were improved by addition of 4% 1,4-butandiol; however, diffraction never exceeded 4 Å. Subsequent crystallization trials were based on purified LipL32 proteolyzed with V8 protease. The hanging-drop vapor-diffusion method was used with 1 μl of protein solution (10 mM Tris, pH 8.0, and 150 mM NaCl) mixed with 1 μl of precipitant solution (2.0 M sodium malonate, 0.1 M sodium cacodylate, pH 6.2, and 4% γ-butyrolactone). The drops were suspended above 1 ml of precipitant solution and incubated at 294 K. Typically, crystals appeared within 3 days and grew to dimensions 0.4 mm × 0.4 mm × 0.3 mm. The crystals were mounted in nylon loops and then flash-cooled in a nitrogen stream at 100 K without additional cryoprotectant. Native X-ray diffraction data were collected in-house in 0.25° oscillations on an R-AXIS IV++ detector at a distance of 150 mm. CuKα radiation was generated by a Rigaku RU-H3RHB rotating anode generator equipped with Osmic focusing mirrors (Auburn Hills, MI). Derivative crystals were produced by soaking in precipitant solution with 2 mM potassium tetrabromoaurate (III) added for 20 min. The crystals were then back-soaked 3 times in precipitant solution prior to flash-cooling. Derivative data were collected using 0.5° oscillations at beamline 3BM1 of the Australian Synchrotron from a single crystal mounted 350 mm from a Quantum 4 CCD detector. The crystals were of space group P3221, with unit cell dimensions a = b = 125.9 Å and c = 95.9 Å. The native data diffracted to beyond 2.0 Å, and the derivative, to 2.8 Å resolution. The data were processed and scaled with DENZO39 and SCALEPACK.39 The data collection statistics are summarized in Table 1. Structure determination and refinement The two gold atoms in the asymmetric unit were positioned from the anomalous diffraction signal using SHELXD.40 The initial heavy-atom positions were refined, and single-wavelength anomalous diffraction phases were calculated with BP3.41 Phase improvement was then performed with SOLOMON41 using solvent flipping. An initial model was traced into the resultant densitymodified map using TEXTAL.42,43 This model was
Crystal Structure of a Leptospiral Antigen
subsequently built and refined using the native data to 2.0 Å resolution with the automated package ARP/ wARP,44 which was able to successfully build ∼ 90% of the structure. The model was improved by iterative rounds of manual building in Coot45 and maximumlikelihood-based refinement in REFMAC.41,46 Strict noncrystallographic symmetry restraints were applied in the early rounds of refinement and were subsequently loosened and then removed for the final round of refinement. The refinement statistics are summarized in Table 1.
1237
9.
10. 11.
Accession code The atomic coordinates and observed structure factors have been deposited in the Research Collaboratory for Structural Bioinformatics PDB under accession code 2ZZ8.
12.
13.
Acknowledgements We thank the Australian Synchrotron staff for their assistance in data collection at the Australian Synchrotron, Australia. This work was supported by the Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics and the National Health and Medical Research Council (NHMRC) of Australia. J.R. is an Australian Research Council Federation Fellow, J.P.V. and G.L.M. are NHMRC Peter Doherty Fellows, T.B. is an NHMRC Career Development Award Fellow, and P.A.C. is an NHMRC C. J. Martin Fellow.
References 1. Levett, P. N. (2001). Leptospirosis. Clin. Microbiol. Rev. 14, 296–326. 2. Silva, J. J., Dalston, M. O., Carvalho, J. E., Setubal, S., Oliveira, J. M. & Pereira, M. M. (2002). Clinicopathological and immunohistochemical features of the severe pulmonary form of leptospirosis. Rev. Soc. Bras. Med. Trop. 35, 395–399. 3. Simpson, F. G., Green, K. A., Haug, G. J. & Brookes, D. L. (1998). Leptospirosis associated with severe pulmonary haemorrhage in Far North Queensland. Med. J. Aust. 169, 151–153. 4. Trevejo, R. T., Rigau-Perez, J. G., Ashford, D. A., McClure, E. M., Jarquin-Gonzalez, C., Amador, J. J. et al. (1998). Epidemic leptospirosis associated with pulmonary hemorrhage—Nicaragua, 1995. J. Infect. Dis. 178, 1457–1463. 5. Dolhnikoff, M., Mauad, T., Bethlem, E. P. & Carvalho, C. R. (2007). Pathology and pathophysiology of pulmonary manifestations in leptospirosis. Braz. J. Infect. Dis. 11, 142–148. 6. Dolhnikoff, M., Mauad, T., Bethlem, E. P. & Carvalho, C. R. (2007). Leptospiral pneumonias. Curr. Opin. Pulm. Med. 13, 230–235. 7. Isturiz, R. E., Torres, J. & Besso, J. (2006). Global distribution of infectious diseases requiring intensive care.Crit. Care Clin. 22, 469–488; ix. 8. Midwinter, A., Faine, S. & Adler, B. (1990). Vaccination of mice with lipopolysaccharide (LPS) and LPS-
14.
15.
16.
17.
18.
19.
20.
21.
derived immuno-conjugates from Leptospira interrogans. J. Med. Microbiol. 33, 199–204. Cullen, P. A., Cordwell, S. J., Bulach, D. M., Haake, D. A. & Adler, B. (2002). Global analysis of outer membrane proteins from Leptospira interrogans serovar Lai. Infect. Immun. 70, 2311–2318. Cullen, P. A., Xu, X., Matsunaga, J., Sanchez, Y., Ko, A. I., Haake, D. A. & Adler, B. (2005). Surfaceome of Leptospira spp. Infect. Immun. 73, 4853–4863. Haake, D. A., Chao, G., Zuerner, R. L., Barnett, J. K., Barnett, D., Mazel, M. et al. (2000). The leptospiral major outer membrane protein LipL32 is a lipoprotein expressed during mammalian infection. Infect. Immun. 68, 2276–2285. Naiman, B. M., Alt, D., Bolin, C. A., Zuerner, R. & Baldwin, C. L. (2001). Protective killed Leptospira borgpetersenii vaccine induces potent Th1 immunity comprising responses by CD4 and gammadelta T lymphocytes. Infect. Immun. 69, 7550–7558. Naiman, B. M., Blumerman, S., Alt, D., Bolin, C. A., Brown, R., Zuerner, R. & Baldwin, C. L. (2002). Evaluation of type 1 immune response in naive and vaccinated animals following challenge with Leptospira borgpetersenii serovar Hardjo: involvement of WC1(+) gammadelta and CD4 T cells. Infect. Immun. 70, 6147–6157. Flannery, B., Costa, D., Carvalho, F. P., Guerreiro, H., Matsunaga, J., Da Silva, E. D. et al. (2001). Evaluation of recombinant Leptospira antigen-based enzyme-linked immunosorbent assays for the serodiagnosis of leptospirosis. J. Clin. Microbiol. 39, 3303–3310. Hauk, P., Macedo, F., Romero, E. C., Vasconcellos, S. A., de Morais, Z. M., Barbosa, A. S. & Ho, P. L. (2008). LipL32, the major leptospiral lipoprotein: the Cterminus is the primary immunogenic domain and mediates interaction with collagen IV and plasma fibronectin. Infect. Immun. 76, 2642–2650. Hoke, D. E., Egan, S., Cullen, P. A. & Adler, B. (2008). LipL32 is an extracellular matrix-interacting protein of Leptospira spp. and Pseudoalteromonas tunicata. Infect. Immun. 76, 2063–2069. Branger, C., Chatrenet, B., Gauvrit, A., Aviat, F., Aubert, A., Bach, J. M. & Andre-Fontaine, G. (2005). Protection against Leptospira interrogans sensu lato challenge by DNA immunization with the gene encoding hemolysin-associated protein 1. Infect. Immun. 73, 4062–4069. Seixas, F. K., da Silva, E. F., Hartwig, D. D., Cerqueira, G. M., Amaral, M., Fagundes, M. Q. et al. (2007). Recombinant Mycobacterium bovis BCG expressing the LipL32 antigen of Leptospira interrogans protects hamsters from challenge. Vaccine, 26, 88–95. Seixas, F. K., Fernandes, C. H., Hartwig, D. D., Conceicao, F. R., Aleixo, J. A. & Dellagostin, O. A. (2007). Evaluation of different ways of presenting LipL32 to the immune system with the aim of developing a recombinant vaccine against leptospirosis. Can. J. Microbiol. 53, 472–479. Clements, C. S., Reid, H. H., Beddoe, T., Tynan, F. E., Perugini, M. A., Johns, T. G. et al. (2003). The crystal structure of myelin oligodendrocyte glycoprotein, a key autoantigen in multiple sclerosis. Proc. Natl Acad. Sci. USA, 100, 11059–11064. Marland, Z., Beddoe, T., Zaker-Tabrizi, L., Lucet, I. S., Brammananth, R., Whisstock, J. C. et al. (2006). Hijacking of a substrate-binding protein scaffold for use in mycobacterial cell wall biosynthesis. J. Mol. Biol. 359, 983–997.
Crystal Structure of a Leptospiral Antigen
1238 22. Holst, M. & Saied, F. (2003). Multigrid solution of the Poisson–Boltzmann equation. J. Comput. Chem. 14, 105–113. 23. Krissinel, E. & Henrick, K. (2007). Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797. 24. Wilson, J. J., Matsushita, O., Okabe, A. & Sakon, J. (2003). A bacterial collagen-binding domain with novel calcium-binding motif controls domain orientation. EMBO J. 22, 1743–1752. 25. Moldoveanu, T., Hosfield, C. M., Lim, D., Elce, J. S., Jia, Z. & Davies, P. L. (2002). A Ca(2+) switch aligns the active site of calpain. Cell, 108, 649–660. 26. Krissinel, E. & Henrick, K. (2004). Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions. Acta Crystallogr., Sect. D: Biol. Crystallogr. 60, 2256–2268. 27. Holmstrom, C., James, S., Neilan, B. A., White, D. C. & Kjelleberg, S. (1998). Pseudoalteromonas tunicata sp. nov., a bacterium that produces antifouling agents. Int. J. Syst. Bacteriol. 48, 1205–1212. 28. Cossart, P., Boquet, P., Normark, S. & Rappuoli, R. (2005). Cellular Microbiology, 2nd edit. ASM Press, Washington, D.C. 29. Lottersberger, J., Vanasco, N. B., Viarengo, G., Guerrero, S. A., Frank, R. & Tonarelli, G. G. (2007). Epitope mapping of Lipl32 protein of pathogenic leptospires. The 5th Meeting of the International Leptospirosis Society, Quito, Ecuador. 30. Cameron, C. E., Brouwer, N. L., Tisch, L. M. & Kuroiwa, J. M. (2005). Defining the interaction of the Treponema pallidum adhesin Tp0751 with laminin. Infect. Immun. 73, 7485–7494. 31. Choy, H. A., Kelley, M. M., Chen, T. L., Møller, A. K., Matsunaga, J. & Haake, D. A. (2007). Physiological osmotic induction of Leptospira interrogans adhesion: LigA and LigB bind extracellular matrix proteins and fibrinogen. Infect. Immun. 75, 2441–2450. 32. Stevenson, B., Choy, H. A., Pinne, M., Rotondi, M. L., Miller, M. C., Demoll, E. et al. (2007). Leptospira interrogans endostatin-like outer membrane proteins bind host fibronectin, laminin and regulators of complement. PLoS ONE, 2, e1188. 33. Liu, D. F., Mason, K. W., Mastri, M., Pazirandeh, M., Cutter, D., Fink, D. L. et al. (2004). The C-terminal fragment of the internal 110-kilodalton passenger domain of the Hap protein of nontypeable Haemophilus influenzae is a potential vaccine candidate. Infect. Immun. 72, 6961–6968. 34. Nilsson, I. M., Patti, J. M., Bremell, T., Hook, M. & Tarkowski, A. (1998). Vaccination with a recombinant
35.
36.
37.
38.
39.
40. 41. 42.
43.
44.
45. 46.
fragment of collagen adhesin provides protection against Staphylococcus aureus-mediated septic death. J. Clin. Invest. 101, 2640–2649. Cachia, P. J., Glasier, L. M., Hodgins, R. R., Wong, W. Y., Irvin, R. T. & Hodges, R. S. (1998). The use of synthetic peptides in the design of a consensus sequence vaccine for Pseudomonas aeruginosa. J. Pept. Res. 52, 289–299. Kelly, C. G., Younson, J. S., Hikmat, B. Y., Todryk, S. M., Czisch, M., Haris, P. I. et al. (1999). A synthetic peptide adhesion epitope as a novel antimicrobial agent. Nat. Biotechnol. 17, 42–47. Lee, K. K., Wong, W. Y., Sheth, H. B., Hodges, R. S., Paranchych, W. & Irvin, R. T. (1995). Use of synthetic peptides in characterization of microbial adhesins. Methods Enzymol. 253, 115–131. Kelly, C. G. & Younson, J. S. (2000). Anti-adhesive strategies in the prevention of infectious disease at mucosal surfaces. Expert Opin. Invest. Drugs, 9, 1711–1721. Otwinowski, Z., Borek, D., Majewski, W. & Minor, W. (2003). Multiparametric scaling of diffraction intensities. Acta Crystallogr., Sect. A: Found. Crystallogr. 59, 228–234. Schneider, T. R. & Sheldrick, G. M. (2002). Substructure solution with SHELXD. Acta Crystallogr., Sect. D: Biol. Crystallogr. 58, 1772–1779. The CCP4 suite: programs for protein crystallography. Acta Crystallogr., Sect. D: Biol. Crystallogr. 50. (1994)., 760–763. Ioerger, T. R., Holton, T., Christopher, J. A. & Sacchettini, J. C. (1999). TEXTAL: a pattern recognition system for interpreting electron density maps. Proc. Int. Conf. Intell. Syst. Mol. Biol. 130–137. Ioerger, T. R. & Sacchettini, J. C. (2003). TEXTAL system: artificial intelligence techniques for automated protein model building. Methods Enzymol. 374, 244–270. Cohen, S. X., Morris, R. J., Fernandez, F. J., Ben Jelloul, M., Kakaris, M., Parthasarathy, V. et al. (2004). Towards complete validated models in the next generation of ARP/wARP. Acta Crystallogr., Sect. D: Biol. Crystallogr. 60, 2222–2229. Emsley, P. & Cowtan, K. (2004). Coot: model-building tools for molecular graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 60, 2126–2132. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. (1997). Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr., Sect. D: Biol. Crystallogr. 53, 240–255.
doi:10.1016/j.jmb.2009.02.038
Available online at www.sciencedirect.com
Crystal Structure of LipL32, the Most Abundant Surface Protein of Pathogenic Leptospira spp. Julian P. Vivian 1,2 †, Travis Beddoe 1,2 ⁎†, Adrian D. McAlister 1 , Matthew C.J. Wilce 1 , Leyla Zaker-Tabrizi 1,2 , Sally Troy 1 , Emma Byres 1,2 , David E. Hoke 3 , Paul A. Cullen 3 , Miranda Lo 3 , Gerald L. Murray 3 , Ben Adler 2,3 and Jamie Rossjohn 1,2 ⁎ 1
The Protein Crystallography Unit, Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800, Australia 2
ARC Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton, Victoria 3800, Australia 3
Department of Microbiology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800, Australia Received 19 December 2008; received in revised form 10 February 2009; accepted 12 February 2009 Available online 21 February 2009 Edited by I. Wilson
Spirochetes of the genus Leptospira cause leptospirosis in humans and animals worldwide. Proteins exposed on the bacterial cell surface are implicated in the pathogenesis of leptospirosis. However, the biological role of the majority of these proteins is unknown; this is principally due to the lack of genetic systems for investigating Leptospira and the absence of any structural information on leptospiral antigens. To address this, we have determined the 2.0-Å-resolution structure of the lipoprotein LipL32, the most abundant outer-membrane and surface protein present exclusively in pathogenic Leptospira species. The extracellular domain of LipL32 revealed a compact, globular, “jelly-roll” fold from which projected an unusual extended β-hairpin that served as a principal mediator of the observed crystallographic dimer. Two acid-rich patches were also identified as potential binding sites for positively charged ligands, such as laminin, to which LipL32 has a propensity to bind. Although LipL32 shared no significant sequence identity to any known protein, it possessed structural homology to the adhesins that bind components of the extracellular matrix, suggesting that LipL32 functions in an analogous manner. Moreover, the structure provides a framework for understanding the immunological role of this major surface lipoprotein. © 2009 Elsevier Ltd. All rights reserved.
Keywords: Leptospira; LipL32; jelly-roll fold; outer-membrane protein
Introduction Leptospirosis is caused by infection with the spirochete bacterium, Leptospira. The organism has a broad host range and is thought to be the most widespread zoonotic agent worldwide. The main*Corresponding authors. The Protein Crystallography Unit, Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800, Australia. E-mail addresses: travis. [email protected]; jamie.rossjohn@med. monash.edu.au. † J.P.V. and T.B. contributed equally to this work. Abbreviations used: ECM, extracellular matrixc; SeMet, selenomethionine; SC, shape complementarity; PDB, Protein Data Bank.
tenance hosts involved in transmission to humans are mainly rodents, dogs, or cattle that harbor pathogenic Leptospira spp. in their proximal renal tubules, resulting in long-term urinary shedding of bacteria into the environment. Human infection occurs when leptospires enter via mucosal surfaces or broken skin following contact with infected animals or contaminated soil or water.1 Once in the body, leptospires are found in multiple organs and tissues. The disease ranges from mild symptoms to more serious complications including jaundice, pulmonary hemorrhage, and renal and hepatic failure, which may prove fatal.2–4 Pathogenic Leptospira spp., previously classified into the single species Leptospira interrogans, are now differentiated into at least 12 species, with L. interrogans and Leptospira borgpetersenii being the main causes of human disease worldwide. More than half
0022-2836/$ - see front matter © 2009 Elsevier Ltd. All rights reserved.
Crystal Structure of a Leptospiral Antigen
1230 of the 250 serovars of Leptospira are contained in these two species. The global importance of this disease is highlighted by estimates that leptospirosis causes severe complications for 1 million people annually.5–7 Immunity to Leptospira in humans is mediated primarily by antibodies directed towards lipopolysaccharide.8 This immunity is serovar specific and has led to a search for conserved cell surface proteins that might elicit cross-serovar immunity. One such candidate is LipL32, the most abundant outermembrane protein9 and the most abundant surfaceexposed protein.10 LipL32 is a lipoprotein, with the protein component remaining external yet anchored to the outer membrane through fatty acids covalently attached to the amino-terminal cysteine residue. There are several lines of evidence that suggest that LipL32 may be important in pathogenesis. The lipL32 gene is present only in pathogenic species, where it displays a high degree of conservation.11 Cellular assays indicate that LipL32 induces an inflammatory response in cultured renal cells12,13 and a promyelocytic cell line expressing Cd14. In vivo studies show that LipL32 is a major target of the antibody response in animals and humans and is expressed in the kidneys of infected animals.11,14,15 The function of LipL32 may be affected by posttranslational events such as proteolytic cleavage, with studies suggesting that the C-terminus of a subset of LipL32 proteins is shed while the N-terminus remains attached to the cell surface.9 LipL32 might be involved in the pathogenesis of leptospirosis through extracellular matrix (ECM) binding activity identified in two recent reports.15,16 Protein truncation studies were used to map this activity, with one report determining that the 72 C-terminal amino acids were sufficient for binding, while the other showed that the 85 C-terminal amino acids were sufficient and also inhibited binding to the intact protein. A LipL32 ortholog in the ubiquitous marine bacterium Pseudoalteromonas tunicata also showed C-terminal ECM binding activity and crossreacted with anti-LipL32 antiserum.16 These studies suggest that C-terminal elements within orthologous LipL32 proteins function as ECM-binding domains in diverse bacteria. Lastly, LipL32 has been shown in some studies to confer partial protection against infection in animal models.17–19 Interestingly, anti-LipL32 antibodies produced in response to naturally acquired infection do not appear to be protective. A recent study focused on LipL32 domains that are recognized during human infection and showed that the immunoglobulin M response was limited to a Cterminal domain during acute and convalescent phases.15 The immunoglobulin G response changed during infection, with most patient sera recognizing a central region of the protein during convalescence and 5 out of 12 patients responding to the C-terminus in the acute and convalescent phases. These findings suggest that differential immune recognition of LipL32 domains during infection and convalescence may be a factor in immunity to Leptospira. The lack of sequence similarity between LipL32 and any other functionally characterized protein has
hindered progress in determining structural domains that relate to function. Therefore, structural determination of LipL32 is important for future work in understanding the potential role of LipL32 in immunity to infection, characterization of the pro-inflammatory response, and interaction with ECM proteins. In this article, we describe the high-resolution crystal structure of LipL32, thereby providing fundamental insight into its role in infection and immunity.
Results Expression, purification, and crystallization of LipL32 To gain insight into the structure–function relationship of leptospiral antigens, we expressed the entire mature form of LipL32 in Escherichia coli and purified it to homogeneity (see Materials and Methods). While this domain produced extremely large crystals of defined morphology, they diffracted very poorly (approximately to 4 Å resolution) and the diffraction pattern exhibited diffuse thermal scattering, indicating potential disorder within the crystal lattice. Accordingly, we undertook a limited proteolysis approach in an attempt to “shave off” any flexible regions of LipL32. Of the proteases that Table 1. Data collection and refinement statistics
Data collection Space group Cell dimensions (Å) Resolution (Å) Total observations Unique observations Rsyma Rpimb I/σI Completeness (%) Redundancy Refinement Resolution Reflections in working set Rcrystc Rfreec Total protein atoms Total waters Bond lengths (Å) Bond angles (°) Ramachandran analysis (%) Most favored Additionally allowed Average B-factor (Å2) Main chain Side chain Waters
Native
KAuBr4
P3221 a = b = 125.9, c = 95.9 50–2.0 (2.11–2.0) 1,270,906 59,453 0.078 (0.724) 0.023 (0.273) 41.9 (2.4) 100 (100) 21.4 (20.3)
P3221 a = b = 124.7, c = 95.4 50–2.8 (2.95–2.80) 377,219 20,492 0.089 (0.822) 0.027 (0.311) 30.7 (2.1) 95.7 (77.7) 18.4 (9.9)
50–2.0 54,787 0.185 0.218 3520 447 0.011 1.314 97.3 2.7 47.5 48.8 53.5
Values in parentheses represent those of the highest-resolution shell. a Rsym = ∑(∣Ii − Imean∣)/∑(Ii). b Rpim = √(1/(n − 1)) · ∑(∣Ii − Imean∣)/∑(Ii). c Rcryst = ∑hkl∣∣Fo∣ − ∣Fc∣∣/∑hkl∣Fo∣ for all data excluding the 5% that comprised the Rfree used for cross-validation.
Crystal Structure of a Leptospiral Antigen
we investigated, cleavage with V8 protease yielded optimal results and provided a fragment that was more amenable for structural studies. The V8 protease cleaved LipL32 within the N-terminal tobacco etch virus protease recognition site and 11 residues from the C-terminus. This yielded a protein comprising 11 vector-encoded residues at the Nterminus and residues 2–242 of the mature LipL32, as confirmed by N-terminal sequencing and mass spectrometry (data not shown). The V8-cleaved LipL32 crystallized readily and diffracted to 2.0 Å resolution and belongs to space group P3221, with unit cell dimensions a = b = 125.9 Å and c = 95.9 Å, which is consistent with two LipL32 molecules per asymmetric unit (see Table 1). Given that sequence analysis suggested that there were no structural homologues of LipL32, we next sought to label the LipL32 with selenomethionine (Se-Met). However, the Se-Met-labeled LipL32 expressed extremely poorly (0.3 mg/l), and the resulting crystals were very small, thereby precluding structure determination via Se-Met multiwavelength anomalous diffraction phasing. Accordingly, we determined the structure of LipL32 via the single-isomorphous replacement method (see Materials and Methods), which resulted in a readily interpretable electron density map. The structure of LipL32 was subsequently refined to 2.0 Å resolution to an R-factor and Rfree of 18.5% and 21.8%, respectively. The model contains residues 6–242 of the mature protein, with residues 2–5 and 140–146 being uninterpretable in the electron density map and 447 water molecules. LipL32 crystallized as two protomers within the
1231 asymmetric unit, which were virtually identical with each other (rmsd, 0.47 Å over all Cα positions), and unless explicitly stated, structural analysis will be confined to one monomer. Overall structure of LipL32 LipL32 adopted a compact, globular fold of overall dimensions of ≈29 Å × 50 Å × 56 Å, is rich in β-sheet (38%), and possesses a few peripheral α-helices (17%), and the “top” and “bases” of LipL32 possess a number of meandering loops that serve to connect the major secondary structural elements (Fig. 1a). While these loops are generally long, the mobility of the loops is restricted via interloop contacts, and as such, the majority of the molecule displays limited mobility, with an average B-factor of ≈48Å2. The core of LipL32 comprised a mixed β-sheet that packed against an antiparallel β-sheet and adopted a “jelly-roll” fold of eight β-strands (β-3, β-5, β-6, β-8, β-9, β-10, β-11, and β-12) (Fig. 1b). The interior of this β-sandwich contained a large number of hydrophobic interactions, characterized by small aliphatic residues towards the top of the molecule and a cluster of aromatic residues at the base of the core. Prior to the core of the molecule is the N-terminal region (which represents the site of attachment to the bacterial outer membrane) that contains a β-hairpin that runs approximately orthogonal to and extends approximately 35Å from the βsheet of the jelly roll. On the other face of LipL32 are two C-terminal helical regions that interdigitate in an antiparallel manner. These two helices (residues 126– 134 and 230–241) pack against the core β-sheet
Fig. 1. Crystal structure of LipL32. (a) Cartoon representation of the LipL32 structure. The structure is color ramped from the N-terminus to the C-terminus. The secondary-structure elements of the protein are labeled. The structure comprises a two-strand N-terminal dimerization domain, with the β-1 (residues 11–20) and β-2 (residues 27–33) strands arranged in a β-hairpin. The core of the protein is a jelly roll composed of strands β-3 (residues 36–44), β-5 (residues 58–67), β-6 (residues 73–79), β-8 (residues 115–120), β-9 (residues 151–154), β-10 (residues 162–166), β-11 (residues 189–196), and β-12 (residues 205–214). LipL32 has three short parallel β-strands that form extensions to the core jelly roll including β-4 (residues 52–54), β-7 (residues 92–94), and β-13 (residues 225–227). Flanking the poles of the molecule are two sets of short helices, with helices α-1 (residues 96–100) and α-2 (residues 103–107) located opposite helices α-4 (residues 174–177) and α-5 (residues 182–184). Distal to the dimerization interface is a helical domain comprising α-3 (residues 126–134) and the C-terminal helix α-6 (residues 230–241). (b) Topology diagram of the LipL32 structure colored similarly to (a).
Crystal Structure of a Leptospiral Antigen
1232
Fig. 2. Representation of the electrostatic potential on the surface of LipL32. Two views of the LipL32 monomer with the charge distribution calculated by the Adaptive Poisson–Boltzmann Solver22 mapped onto the surface of the protein. For the purposes of modeling the charge distribution, the aspartate-rich loop (residues 140–146, sequence: KLDDDDD) that was not ordered in the crystal structure was modeled. The regions of electronegative and electropositive charge are labeled.
(predominantly via hydrophobic interactions) that is centered on Trp66 packing against Pro126, Ile129, and Ala240. Above and below the Trp residue are polar interactions that further serve to tether the helical region to the core of the molecule. Surface-exposed electrostatic or hydrophobic patches can often be indicative of regions that mediate protein–protein and/or protein–ligand interactions.20,21 Notably, LipL32 possessed a cluster of surface-exposed aromatic residues towards the base of the molecule, which included Phe112, His156, Tyr159, and Tyr198. Additionally, LipL32 possessed two patches of marked electronegative charge, with electronegative patch 1 dominated by residues Glu231, Glu232, Glu241, and Glu242, while patch 2 is dominated by Glu119 (Asp142, Asp143, Asp144, Asp145, and Asp146 modeled for electrostatic calculations), Asp147, and Asp148. Additionally, LipL32 possessed a region of positive charge that included residues Lys58, Lys59, Lys199, and Lys204 (Fig. 2). Accordingly, LipL32 possessed a compact globular fold with a number of surface-exposed features that indicated a potential capacity to bind other components or interact with itself. LipL32 as a dimer Although LipL32 was purified as a monomer by gel filtration, the two monomers within the asymmetric unit were observed to form a crystallographic dimer, with the monomers related by an approximate 2-fold rotation. This higher-order assembly is consistent with the propensity for LipL32 to oligomerize on the cell surface (data not shown). Although crystallographic dimers can often be an artifact of crystallization, the observed contacts within the crystalline lattice were suggestive of a biologically relevant LipL32 dimer, as was also suggested by the PISA web server‡.23 The interface ‡ http://www.ebi.ac.uk/msd-srv/prot_int/pistart. html
was extensive (circa 1250Å2), exhibited high shape complementarity (SC) (SC index = 0.66), and was characterized by a large number of hydrophobic interactions and only four hydrogen bonds (Table 2). The dimer is formed such that the N-terminal regions are close to the dyad axis, while the Cterminal helices are located at the external face of the dimer. There are two main sites of interaction at the dimer interface, mediated by the unusual β-hairpin and the base of the molecule (Fig. 3). Firstly, the β-hairpin that was observed to project from the core of the molecule wraps around its counterpart in the other protomer. While the top of the respective
Table 2. Interactions at the dimeric interface of LipL32 Chain A
Chain B
Interaction
Thr30 Thr82 Tyr159 Asn160 Gly6 Leu7 Lys11 Leu16 Ser17 Asp19 Ile21 Pro22 Val28 Thr30 Ile79 Pro81 Thr82 Gly83 Glu104 Ser107 Met108 Pro109 His110 Trp111 Phe112 Tyr159 Asn160 Pro200
Thr30 Tyr159 Thr82 Tyr159 Tyr159 Tyr159 Val28 Asp19, Val28 Asp19 Leu16, Ser17 Leu32 Leu16 Lys11, Leu16, Pro33 Asp19, Thr30 Tyr159 Phe112 Phe112, Tyr159 Phe112 Pro200 Phe112, Pro200 Pro200 Pro109, His110, Trp111 Pro109 Pro109 Pro81, Thr82, Gly83, Ser107 Gly6, Leu7, Pro8, Ile79, Thr82, Asn160 Tyr159, Asn160 Glu104, Met108
H-bond H-bond H-bond H-bond Nonbonded Nonbonded Nonbonded Nonbonded Nonbonded Nonbonded Nonbonded Nonbonded Nonbonded Nonbonded Nonbonded Nonbonded Nonbonded Nonbonded Nonbonded Nonbonded Nonbonded Nonbonded Nonbonded Nonbonded Nonbonded Nonbonded Nonbonded Nonbonded
Crystal Structure of a Leptospiral Antigen
1233
Fig. 3. Dimerization of LipL32. (a) and (b) are cartoon representations of the LipL32 dimer. (a) View of the LipL32 dimer looking perpendicularly to the dimer interaction surface. The monomers of LipL32 are colored green and brown. (b) Another view of the LipL32 dimer looking perpendicularly to the dimer interaction surface except that it is rotated 90° about the x-axis relative to (a). (c) View of the dimerization interface of LipL32. The figure is a surface representation of the LipL32 monomer with some of the important residues involved in dimer–dimer contacts labeled. The residues forming nonbonded contacts are colored green, and the atoms involved in forming hydrogen bonds are colored blue.
β-hairpins are not involved in mediating dimer contacts, the residues proximal and distal to the tip are involved in a number of van der Waals interactions, centered on Thr27. While Thr27 interacted with its equivalent residue, Ile21 packed against Leu32; Val28 packed against Leu16 and Pro33; Leu16 interacted with Asp19, the latter of which also forms van der Waals interactions with Ser17. Secondly, the base of the molecule, which is rich in aromatic residues, forms a cluster of hydrophobic contacts at the interface. Tyr159 wedged within a bulged region of β-strand 8, packing against Ile79, while the TyrOH H-bonds to the main chain of Thr82. The main chain of Tyr159 H-bonds to Asn160, the latter of which also forms van der Waals interactions with its counterpart. The hydrophobic cluster is extended with Phe112 interacting with Pro81, the main chain of Thr82, Gly83, and Ser107.
Taken together, the features at the dimer interface, namely, an extensive hydrophobic interface exhibiting high SC, suggest that the crystallographic LipL32 dimer may represent a biologically relevant LipL32 dimer. Structural comparisons While LipL32 possessed no significant sequence similarity to any protein in the Protein Data Bank (PDB), we undertook automated structural comparisons (DALI server) against the PDB database, as, often, structural conservation is stronger than sequence conservation. The DALI-based search revealed that LipL32 exhibited significant structural homology to proteins that also possess a jelly-roll fold despite the low level of sequence identity (circa 8–13%). Among the significant hits, LipL32 showed matches to a domain from calpain (Z score, 8.3;
1234
Crystal Structure of a Leptospiral Antigen
Fig. 4. Comparison of LipL32 with the structural homologue collagenase collagen-binding domain 3B from Clostridium histolyticum. Structural superposition of LipL32 with the collagen-binding domain 3B was performed with SSM.26 In (a) and (b), LipL32 is represented in cartoon format in green, and the collagen-binding domain 3B is represented in cartoon format in light pink. The two orthogonal views (a and b) show the structural similarity of the core β-sheets of the proteins. (c) Surface representation of collagen-binding domain 3B in the orientation depicted in (b). The residues comprising the hydrophobic surface shown to be important for collagen binding are highlighted in red. (d) Surface representation of LipL32 in the orientation depicted in (b). Highlighted in orange are residues that comprise the putative collagen binding site of LipL32. These residues form a hydrophobic surface positioned similarly to that of the structural homologue collagen-binding domain 3B.
rmsd, 3.5 Å over 124 Cα atoms) and collagenase (Z score, 6.0; rmsd, 2.5 Å over 87 Cα atoms). Interestingly, the collagenase domain binds the ECM component collagen,24 and the calpain domain is thought to localize calpain to the membrane.25 The ECM binding site of collagenase is characterized by hydrophophic interactions, with mutagenesis studies identifying a cluster of tyrosine residues, with a leucine, an arginine, and a threonine residue contributing to a hydrophobic surface.24 Structural superposition of LipL32 and the collagenase reveals a similarly located hydrophobic surface on LipL32, comprising residues Tyr62, Tyr151, Tyr198, Trp115, Leu53, Val54, and Arg117, as shown in Fig. 4. Accordingly, LipL32 possesses homology to structures that bind to ECM components. Recently, a sequence homologue of LipL32 has been reported from P. tunicata (PTD2-05920).16 This homologue, which shares 44% sequence identity with Leptospira LipL32, is from a marine bacterium that inhabits a primitive chordate (Ciona intestinalis) that has ECM components.27 The high level of sequence identity across the entire extracellular domain is indicative of P. tunicata PTD2-05920 possessing a similar fold, and moreover, as the electrostatic patches, including the
string of aspartate residues between 142 and 148 and the hydrophobic surface-exposed patches including residues Phe112, His156, Tyr159, and Tyr198, are conserved, it is likely to have similar binding properties to itself and other components. It has recently been shown that PTD2-05920 can bind ECM proteins.16
Discussion The array of cell-surface adhesion molecules found on pathogenic species of Leptospira (including LigA, LigB, and other lipoproteins) is of fundamental importance to our understanding and preventive treatment of leptospirosis. These molecules are key to the invasion and adhesion of bacteria to the host tissue, a process that is central to the pathogenesis of the organism. Furthermore, the cellsurface localization of these adhesins makes them ideal targets for vaccine development. Of these adhesins, LipL32 stands out as a prime candidate for vaccine development as it is the most abundant cellsurface protein in pathogenic Leptospira and is the major target of the immune system during human
Crystal Structure of a Leptospiral Antigen
infection. Despite the important role of LipL32, our current understanding of its functional role was unclear. To address this, we have determined the crystal structure of LipL32 to 2 Å resolution, the first crystal structure of a leptospiral adhesin. It has been shown recently that LipL32 has the capacity to interact in vitro with multiple ECM binding partners including plasma fibronectin, collagen IV, and laminin. 15,16 Fibronectin and laminin have defined cell- or bacteria-binding domains where collagen provides multiple sites of interactions along its triple-helical structures. 28 Electrostatic analysis of the LipL32 dimer has identified two noncontiguous electronegative patches arranged in tandem. It is tempting to speculate that these patches have a role in binding ECM components with sites of known complimentary charge, especially laminin. Similarly, a hydrophobic surface identified by comparison with the structural homologue collagen-binding protein 3B represents a putative site for collagen binding. Other studies have mapped ECM interaction sites to the Cterminal region of LipL32 comprising residues 166– 253. Mapping this C-terminal fragment onto the
1235 crystal structure of LipL32 reveals that it comprises the large loop between β-10 and β-11 (including helices 4 and 5) and the large loop region between βstrands 12 and 13. It also contains β-strands 11, 12, and 13 and helix 6 (Fig. 5a and b). As this C-terminal domain would be expected to have limited tertiary and secondary structure in isolation, the proposed site of interaction would, therefore, be a linear stretch of amino acids. There are three surfaceexposed regions spanning residues 168–185, 214– 222, and 230–242 that could mediate the interaction with ECM proteins. Linear peptides have previously been shown to inhibit binding to laminin by the Tp0751 adhesin from the spirochete Treponema pallidum, the causative agent of syphilis. 30 The observed affinity of LipL32 binding to ECM proteins is relatively weak with a Kd of ≈7–10 μM. Therefore, the existence of the observed dimer may be a means of increasing the avidity for the ECM proteins, through an increase in the local concentration of binding sites.16 The multiple ECM binding by LipL32 has also been shown in other leptospiral adhesins such as LigA, LigB, and the family of Len proteins.31,32 This
Fig. 5. Antibody and ECM-binding domains of LipL32. (a and b) Two views of the C-terminal fragment of LipL32 used in binding assays with ECM proteins. The LipL32 structure is shown in cartoon representation in green. The residues that were subcloned to produce a C-terminal fragment for binding assays (residues 166–253)15,16 are shown in orange. Analysis of this C-terminal fragment, with the benefit of the crystal structure of LipL32, suggests that, in all likelihood, this fragment would not fold as it does in the full-length protein. Therefore, binding of this C-terminal section to ECM proteins is likely to occur via linear segments. (c) Epitope 1. The LipL32 structure is shown in cartoon representation in green with residues 132–158 shown in pink.29 (d) Epitope 2. The LipL32 structure is shown in cartoon representation in green with residues 162–186 shown in pink.29
1236 redundancy in ECM adhesion would convey a selective advantage to Leptospira during the initial infection stage as 95% of human patients with leptospirosis produce antibodies against LipL32.14 Many bacterial adhesins can be effective vaccine candidates, such as Hap adhesin from Haemophilus influenzae and collagen adhesin from Staphylococcus aureus.33,34 In addition, peptide inhibitors of several bacterial pathogens such as Streptococcus mutans, Porphyromonas gingivalis, and Pseudomonas aeruginosa have been used to prevent attachment of these bacteria to the corresponding ligand.35–37 Various strategies have been used in these studies to inhibit adhesion. These include vaccination with recombinant protein, therapeutic administration of selected peptides, and passive administration of antibodies raised against peptide sequences that mediate adhesion.38 Recently, human antibody epitopes have been characterized for LipL32.29 They consist of residues 132–158 and 162–186 (Fig. 5c and d). The peptide epitope 162–186 corresponds to one of the proposed interaction loops of LipL32. In addition, this peptide also lies within the C-terminal domain, which is the most immunogenic region of LipL32.15 The crystal structure of LipL32 will facilitate future studies that aim to elucidate the interaction of Leptospira with host tissue components and its role in the pathogenesis of leptospirosis.
Materials and Methods Protein expression and purification DNA encoding residues 2–253 of mature LipL32 was amplified via PCR from L. interrogans serovar Lai strain L391 genomic DNA using the primers 5′-ATAGCGGCCGCAGGTGCTTTCGGTGGTCTG-3′ and 5′-GCCACCTTTCGGTACCTTTTTAACC-3′. The resultant PCR product was digested with NotI and KpnI restriction enzymes and cloned into modified pQE30 expression vector (Qiagen) containing a tobacco etch virus protease recognition site between the histidine tag and start of the mature protein. The recombinant protein was produced in E. coli BL21 (DE3) cells grown in LB broth supplemented with 100 μg/ml ampicillin, grown with shaking at 200 rpm at 37°C to an OD600 (optical density at 600 nm) of 0.5. At this point, IPTG was added to a concentration of 0.5 mM and the cells were grown for a further 4 h at 37 °C with shaking at 200 rpm. The cells were then harvested by centrifugation at 6000g for 15 min and resuspended in 20 mM Tris, pH 8.0, 300 mM NaCl, and 10 mM imidazole, pH 8.0, prior to lysis by French press. Cellular debris were cleared by centrifugation at 30,000g for 30 min, and the resultant supernatant was loaded onto a nickel-Sepharose column pre-equilibrated in 20 mM Tris, pH 8.0, and 300 mM NaCl. The column was washed with 6 column volumes of 20 mM Tris, pH 8.0, 300 mM NaCl, and 10 mM imidazole, pH 8.0, prior to elution of the bound protein with 5 × 1 column volumes of 20 mM Tris, pH 8.0, and 400 mM imidazole, pH 8.0. The eluted fractions were pooled, and further purification proceeded by gel-filtration chromatography using an S75 16/60 column equilibrated in 10 mM Tris, pH 8.0, and 150 mM NaCl. Fractions containing LipL32 were pooled, and the solution was
Crystal Structure of a Leptospiral Antigen
diluted 1:3 with 10 mM Tris, pH 8.0, prior to loading onto a Mono Q 5/50 (GE Healthcare) anion-exchange column. Pure LipL32 was collected from the unbound fraction. The protein was then dialyzed against 10 mM Tris, pH 8.0, and 150 mM NaCl prior to concentration to 10 mg/ml. This solution was used for crystallization trials. The protein was proteolyzed with V8 protease following elution from the size-exclusion column to improve the diffraction quality of the LipL32 crystals. V8 protease (Sigma) was added at a 1/20 ratio (w/w) to LipL32 for 80 min and then reapplied to the S75 16/60 column equilibrated in 10 mM Tris, pH 8.0, and 150 mM NaCl to separate V8 protease from Lipl32. The purified protein was concentrated to 10 mg/ml. Crystallization and X-ray diffraction data collection Initial crystallization trials centered on the full-length construct of LipL32, including the N-terminal His6 purification tag. Clusters of very thin needle-like crystals were produced using the hanging-drop vapor-diffusion method at 294 K. One μl of protein solution (10 mM Tris, pH 8.0, and 150 mM NaCl) was mixed with 1 μl of precipitant solution (2.0 M ammonium sulfate and 0.1 M Tris, pH 7.8) and suspended above 1 ml of precipitant solution. These crystals were improved by addition of 4% 1,4-butandiol; however, diffraction never exceeded 4 Å. Subsequent crystallization trials were based on purified LipL32 proteolyzed with V8 protease. The hanging-drop vapor-diffusion method was used with 1 μl of protein solution (10 mM Tris, pH 8.0, and 150 mM NaCl) mixed with 1 μl of precipitant solution (2.0 M sodium malonate, 0.1 M sodium cacodylate, pH 6.2, and 4% γ-butyrolactone). The drops were suspended above 1 ml of precipitant solution and incubated at 294 K. Typically, crystals appeared within 3 days and grew to dimensions 0.4 mm × 0.4 mm × 0.3 mm. The crystals were mounted in nylon loops and then flash-cooled in a nitrogen stream at 100 K without additional cryoprotectant. Native X-ray diffraction data were collected in-house in 0.25° oscillations on an R-AXIS IV++ detector at a distance of 150 mm. CuKα radiation was generated by a Rigaku RU-H3RHB rotating anode generator equipped with Osmic focusing mirrors (Auburn Hills, MI). Derivative crystals were produced by soaking in precipitant solution with 2 mM potassium tetrabromoaurate (III) added for 20 min. The crystals were then back-soaked 3 times in precipitant solution prior to flash-cooling. Derivative data were collected using 0.5° oscillations at beamline 3BM1 of the Australian Synchrotron from a single crystal mounted 350 mm from a Quantum 4 CCD detector. The crystals were of space group P3221, with unit cell dimensions a = b = 125.9 Å and c = 95.9 Å. The native data diffracted to beyond 2.0 Å, and the derivative, to 2.8 Å resolution. The data were processed and scaled with DENZO39 and SCALEPACK.39 The data collection statistics are summarized in Table 1. Structure determination and refinement The two gold atoms in the asymmetric unit were positioned from the anomalous diffraction signal using SHELXD.40 The initial heavy-atom positions were refined, and single-wavelength anomalous diffraction phases were calculated with BP3.41 Phase improvement was then performed with SOLOMON41 using solvent flipping. An initial model was traced into the resultant densitymodified map using TEXTAL.42,43 This model was
Crystal Structure of a Leptospiral Antigen
subsequently built and refined using the native data to 2.0 Å resolution with the automated package ARP/ wARP,44 which was able to successfully build ∼ 90% of the structure. The model was improved by iterative rounds of manual building in Coot45 and maximumlikelihood-based refinement in REFMAC.41,46 Strict noncrystallographic symmetry restraints were applied in the early rounds of refinement and were subsequently loosened and then removed for the final round of refinement. The refinement statistics are summarized in Table 1.
1237
9.
10. 11.
Accession code The atomic coordinates and observed structure factors have been deposited in the Research Collaboratory for Structural Bioinformatics PDB under accession code 2ZZ8.
12.
13.
Acknowledgements We thank the Australian Synchrotron staff for their assistance in data collection at the Australian Synchrotron, Australia. This work was supported by the Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics and the National Health and Medical Research Council (NHMRC) of Australia. J.R. is an Australian Research Council Federation Fellow, J.P.V. and G.L.M. are NHMRC Peter Doherty Fellows, T.B. is an NHMRC Career Development Award Fellow, and P.A.C. is an NHMRC C. J. Martin Fellow.
References 1. Levett, P. N. (2001). Leptospirosis. Clin. Microbiol. Rev. 14, 296–326. 2. Silva, J. J., Dalston, M. O., Carvalho, J. E., Setubal, S., Oliveira, J. M. & Pereira, M. M. (2002). Clinicopathological and immunohistochemical features of the severe pulmonary form of leptospirosis. Rev. Soc. Bras. Med. Trop. 35, 395–399. 3. Simpson, F. G., Green, K. A., Haug, G. J. & Brookes, D. L. (1998). Leptospirosis associated with severe pulmonary haemorrhage in Far North Queensland. Med. J. Aust. 169, 151–153. 4. Trevejo, R. T., Rigau-Perez, J. G., Ashford, D. A., McClure, E. M., Jarquin-Gonzalez, C., Amador, J. J. et al. (1998). Epidemic leptospirosis associated with pulmonary hemorrhage—Nicaragua, 1995. J. Infect. Dis. 178, 1457–1463. 5. Dolhnikoff, M., Mauad, T., Bethlem, E. P. & Carvalho, C. R. (2007). Pathology and pathophysiology of pulmonary manifestations in leptospirosis. Braz. J. Infect. Dis. 11, 142–148. 6. Dolhnikoff, M., Mauad, T., Bethlem, E. P. & Carvalho, C. R. (2007). Leptospiral pneumonias. Curr. Opin. Pulm. Med. 13, 230–235. 7. Isturiz, R. E., Torres, J. & Besso, J. (2006). Global distribution of infectious diseases requiring intensive care.Crit. Care Clin. 22, 469–488; ix. 8. Midwinter, A., Faine, S. & Adler, B. (1990). Vaccination of mice with lipopolysaccharide (LPS) and LPS-
14.
15.
16.
17.
18.
19.
20.
21.
derived immuno-conjugates from Leptospira interrogans. J. Med. Microbiol. 33, 199–204. Cullen, P. A., Cordwell, S. J., Bulach, D. M., Haake, D. A. & Adler, B. (2002). Global analysis of outer membrane proteins from Leptospira interrogans serovar Lai. Infect. Immun. 70, 2311–2318. Cullen, P. A., Xu, X., Matsunaga, J., Sanchez, Y., Ko, A. I., Haake, D. A. & Adler, B. (2005). Surfaceome of Leptospira spp. Infect. Immun. 73, 4853–4863. Haake, D. A., Chao, G., Zuerner, R. L., Barnett, J. K., Barnett, D., Mazel, M. et al. (2000). The leptospiral major outer membrane protein LipL32 is a lipoprotein expressed during mammalian infection. Infect. Immun. 68, 2276–2285. Naiman, B. M., Alt, D., Bolin, C. A., Zuerner, R. & Baldwin, C. L. (2001). Protective killed Leptospira borgpetersenii vaccine induces potent Th1 immunity comprising responses by CD4 and gammadelta T lymphocytes. Infect. Immun. 69, 7550–7558. Naiman, B. M., Blumerman, S., Alt, D., Bolin, C. A., Brown, R., Zuerner, R. & Baldwin, C. L. (2002). Evaluation of type 1 immune response in naive and vaccinated animals following challenge with Leptospira borgpetersenii serovar Hardjo: involvement of WC1(+) gammadelta and CD4 T cells. Infect. Immun. 70, 6147–6157. Flannery, B., Costa, D., Carvalho, F. P., Guerreiro, H., Matsunaga, J., Da Silva, E. D. et al. (2001). Evaluation of recombinant Leptospira antigen-based enzyme-linked immunosorbent assays for the serodiagnosis of leptospirosis. J. Clin. Microbiol. 39, 3303–3310. Hauk, P., Macedo, F., Romero, E. C., Vasconcellos, S. A., de Morais, Z. M., Barbosa, A. S. & Ho, P. L. (2008). LipL32, the major leptospiral lipoprotein: the Cterminus is the primary immunogenic domain and mediates interaction with collagen IV and plasma fibronectin. Infect. Immun. 76, 2642–2650. Hoke, D. E., Egan, S., Cullen, P. A. & Adler, B. (2008). LipL32 is an extracellular matrix-interacting protein of Leptospira spp. and Pseudoalteromonas tunicata. Infect. Immun. 76, 2063–2069. Branger, C., Chatrenet, B., Gauvrit, A., Aviat, F., Aubert, A., Bach, J. M. & Andre-Fontaine, G. (2005). Protection against Leptospira interrogans sensu lato challenge by DNA immunization with the gene encoding hemolysin-associated protein 1. Infect. Immun. 73, 4062–4069. Seixas, F. K., da Silva, E. F., Hartwig, D. D., Cerqueira, G. M., Amaral, M., Fagundes, M. Q. et al. (2007). Recombinant Mycobacterium bovis BCG expressing the LipL32 antigen of Leptospira interrogans protects hamsters from challenge. Vaccine, 26, 88–95. Seixas, F. K., Fernandes, C. H., Hartwig, D. D., Conceicao, F. R., Aleixo, J. A. & Dellagostin, O. A. (2007). Evaluation of different ways of presenting LipL32 to the immune system with the aim of developing a recombinant vaccine against leptospirosis. Can. J. Microbiol. 53, 472–479. Clements, C. S., Reid, H. H., Beddoe, T., Tynan, F. E., Perugini, M. A., Johns, T. G. et al. (2003). The crystal structure of myelin oligodendrocyte glycoprotein, a key autoantigen in multiple sclerosis. Proc. Natl Acad. Sci. USA, 100, 11059–11064. Marland, Z., Beddoe, T., Zaker-Tabrizi, L., Lucet, I. S., Brammananth, R., Whisstock, J. C. et al. (2006). Hijacking of a substrate-binding protein scaffold for use in mycobacterial cell wall biosynthesis. J. Mol. Biol. 359, 983–997.
Crystal Structure of a Leptospiral Antigen
1238 22. Holst, M. & Saied, F. (2003). Multigrid solution of the Poisson–Boltzmann equation. J. Comput. Chem. 14, 105–113. 23. Krissinel, E. & Henrick, K. (2007). Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797. 24. Wilson, J. J., Matsushita, O., Okabe, A. & Sakon, J. (2003). A bacterial collagen-binding domain with novel calcium-binding motif controls domain orientation. EMBO J. 22, 1743–1752. 25. Moldoveanu, T., Hosfield, C. M., Lim, D., Elce, J. S., Jia, Z. & Davies, P. L. (2002). A Ca(2+) switch aligns the active site of calpain. Cell, 108, 649–660. 26. Krissinel, E. & Henrick, K. (2004). Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions. Acta Crystallogr., Sect. D: Biol. Crystallogr. 60, 2256–2268. 27. Holmstrom, C., James, S., Neilan, B. A., White, D. C. & Kjelleberg, S. (1998). Pseudoalteromonas tunicata sp. nov., a bacterium that produces antifouling agents. Int. J. Syst. Bacteriol. 48, 1205–1212. 28. Cossart, P., Boquet, P., Normark, S. & Rappuoli, R. (2005). Cellular Microbiology, 2nd edit. ASM Press, Washington, D.C. 29. Lottersberger, J., Vanasco, N. B., Viarengo, G., Guerrero, S. A., Frank, R. & Tonarelli, G. G. (2007). Epitope mapping of Lipl32 protein of pathogenic leptospires. The 5th Meeting of the International Leptospirosis Society, Quito, Ecuador. 30. Cameron, C. E., Brouwer, N. L., Tisch, L. M. & Kuroiwa, J. M. (2005). Defining the interaction of the Treponema pallidum adhesin Tp0751 with laminin. Infect. Immun. 73, 7485–7494. 31. Choy, H. A., Kelley, M. M., Chen, T. L., Møller, A. K., Matsunaga, J. & Haake, D. A. (2007). Physiological osmotic induction of Leptospira interrogans adhesion: LigA and LigB bind extracellular matrix proteins and fibrinogen. Infect. Immun. 75, 2441–2450. 32. Stevenson, B., Choy, H. A., Pinne, M., Rotondi, M. L., Miller, M. C., Demoll, E. et al. (2007). Leptospira interrogans endostatin-like outer membrane proteins bind host fibronectin, laminin and regulators of complement. PLoS ONE, 2, e1188. 33. Liu, D. F., Mason, K. W., Mastri, M., Pazirandeh, M., Cutter, D., Fink, D. L. et al. (2004). The C-terminal fragment of the internal 110-kilodalton passenger domain of the Hap protein of nontypeable Haemophilus influenzae is a potential vaccine candidate. Infect. Immun. 72, 6961–6968. 34. Nilsson, I. M., Patti, J. M., Bremell, T., Hook, M. & Tarkowski, A. (1998). Vaccination with a recombinant
35.
36.
37.
38.
39.
40. 41. 42.
43.
44.
45. 46.
fragment of collagen adhesin provides protection against Staphylococcus aureus-mediated septic death. J. Clin. Invest. 101, 2640–2649. Cachia, P. J., Glasier, L. M., Hodgins, R. R., Wong, W. Y., Irvin, R. T. & Hodges, R. S. (1998). The use of synthetic peptides in the design of a consensus sequence vaccine for Pseudomonas aeruginosa. J. Pept. Res. 52, 289–299. Kelly, C. G., Younson, J. S., Hikmat, B. Y., Todryk, S. M., Czisch, M., Haris, P. I. et al. (1999). A synthetic peptide adhesion epitope as a novel antimicrobial agent. Nat. Biotechnol. 17, 42–47. Lee, K. K., Wong, W. Y., Sheth, H. B., Hodges, R. S., Paranchych, W. & Irvin, R. T. (1995). Use of synthetic peptides in characterization of microbial adhesins. Methods Enzymol. 253, 115–131. Kelly, C. G. & Younson, J. S. (2000). Anti-adhesive strategies in the prevention of infectious disease at mucosal surfaces. Expert Opin. Invest. Drugs, 9, 1711–1721. Otwinowski, Z., Borek, D., Majewski, W. & Minor, W. (2003). Multiparametric scaling of diffraction intensities. Acta Crystallogr., Sect. A: Found. Crystallogr. 59, 228–234. Schneider, T. R. & Sheldrick, G. M. (2002). Substructure solution with SHELXD. Acta Crystallogr., Sect. D: Biol. Crystallogr. 58, 1772–1779. The CCP4 suite: programs for protein crystallography. Acta Crystallogr., Sect. D: Biol. Crystallogr. 50. (1994)., 760–763. Ioerger, T. R., Holton, T., Christopher, J. A. & Sacchettini, J. C. (1999). TEXTAL: a pattern recognition system for interpreting electron density maps. Proc. Int. Conf. Intell. Syst. Mol. Biol. 130–137. Ioerger, T. R. & Sacchettini, J. C. (2003). TEXTAL system: artificial intelligence techniques for automated protein model building. Methods Enzymol. 374, 244–270. Cohen, S. X., Morris, R. J., Fernandez, F. J., Ben Jelloul, M., Kakaris, M., Parthasarathy, V. et al. (2004). Towards complete validated models in the next generation of ARP/wARP. Acta Crystallogr., Sect. D: Biol. Crystallogr. 60, 2222–2229. Emsley, P. & Cowtan, K. (2004). Coot: model-building tools for molecular graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 60, 2126–2132. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. (1997). Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr., Sect. D: Biol. Crystallogr. 53, 240–255.