• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of embojLink to Publisher's site
EMBO J. Mar 24, 2004; 23(6): 1245–1256.
Published online Mar 18, 2004. doi:  10.1038/sj.emboj.7600142
PMCID: PMC381414

Structural basis for host recognition by the Haemophilus influenzae Hia autotransporter

Abstract

Haemophilus influenzae is an important human pathogen that initiates infection by colonizing the upper respiratory tract. The H. influenzae Hia autotransporter is an adhesive protein that promotes adherence to respiratory epithelial cells. Hia adhesive activity resides in two homologous binding domains, called HiaBD1 and HiaBD2. These domains interact with the same host cell receptor, but bind with different affinities. In this report, we describe the crystal structure of the high-affinity HiaBD1 binding domain, which has a novel trimeric architecture with three-fold symmetry and a mushroom shape. The subunit constituents of the trimer are extensively intertwined. The receptor-binding pocket is formed by an acidic patch that is present on all three faces of the trimer, providing potential for a multivalent interaction with the host cell surface, analogous to observations with the trimeric tumor necrosis factor superfamily of proteins. Hia is a novel example of a bacterial trimeric adhesin and may be the prototype member of a large family of bacterial virulence proteins with a similar architecture.

Keywords: adhesion, autotransporter, crystal structure, homotrimer, microbial pathogenesis

Introduction

Adherence to host tissues is a fundamental early step in the pathogenesis of most bacterial infections. The process of adherence requires specialized bacterial proteins called adhesins, which recognize specific host cell receptors and mediate stable interaction with the host cell surface (Hultgren et al, 1993; St Geme, 1997a).

In order to mediate interaction with host cells, adhesins must be presented on the bacterial surface in an active binding conformation. In Gram-negative bacteria, surface localization requires that the protein be translocated across the inner and outer membranes. In general, surface localization occurs via one of five different secretion pathways, distinguished at least in part by the mechanism of translocation across the outer membrane and designated types I–V (Stathopoulos et al, 2000; Thanassi and Hultgren, 2000).

Proteins secreted by the type V pathway are referred to as autotransporters (Henderson et al, 2000). Typically, autotransporters are expressed as precursor proteins with three basic functional domains, including an N-terminal signal peptide, an internal passenger domain, and a C-terminal translocator domain (β domain) (Henderson et al, 1998). The signal peptide directs export of the precursor protein across the inner membrane via the Sec machinery and is then cleaved by signal peptidase I. Subsequently, the β domain inserts into the outer membrane and forms a β-barrel structure with a central channel, allowing extrusion of the passenger domain across the membrane (Shannon and Fernandez, 1999; Veiga et al, 2002). Once on the surface of the organism, the passenger domain is usually cleaved from the translocator domain and released extracellularly. In some cases, the passenger domain is cleaved but remains cell associated.

Haemophilus influenzae is a human-specific pathogen and a major source of morbidity worldwide, producing systemic diseases such as meningitis and sepsis and localized respiratory tract diseases such as otitis media, bronchitis, and pneumonia (Turk, 1984; St Geme, 1993; Moxon and Murphy, 2000). The pathogenesis of H. influenzae disease begins with colonization of the nasopharynx, and adherence to the respiratory epithelium represents an essential early step in the colonization process (Murphy et al, 1987; St Geme, 1997b). A high-molecular-mass protein called Hia is the predominant adhesin in a subset of nonencapsulated strains (Barenkamp and St Geme, 1996; St Geme et al, 1998). A homolog of Hia called Hsf is ubiquitous in encapsulated isolates and is the major nonpilus adhesin in these strains (St Geme et al, 1996).

In previous work, we established that Hia is a member of the autotransporter family (Figure 1) (St Geme and Cutter, 2000). In addition, we found that Hia remains uncleaved at the C terminus and fully cell associated, an unusual fate for autotransporters (St Geme and Cutter, 2000). More recently, we discovered that Hia is an example of a variant subfamily of autotransporters characterized by an unusually small translocator domain that undergoes trimer formation (Surana et al, 2004). Examination of a series of GST–Hia fusion proteins and Hia deletion derivatives demonstrated that the Hia passenger domain contains two homologous binding domains, called HiaBD1 and HiaBD2 (Laarmann et al, 2002). These two binding domains recognize the same host cell receptor but bind with differing affinities. HiaBD1 is defined by residues 541–714 and has a Kd of 0.05–0.1 nM, while HiaBD2 is defined by amino acids 50–374 and has a Kd of 1–2 nM. The identity of the host cell receptor is not known.

Figure 1
Schematic diagram of the primary sequence of Hia. The Hia domain organization is indicated: signal peptide (SP; residues 1–49), passenger domain (residues 50–1022), and β domain (residues 1023–1098). The black boxes in ...

In this study, we report that the HiaBD1 binding domain has a novel trimeric architecture with three-fold symmetry. Using site-directed mutagenesis, we show that the HiaBD1 receptor-binding domain is located in an acidic pocket present on all three faces of the trimer, suggesting potential for a multivalent interaction with the host cell surface, analagous to observations with the trimeric tumor necrosis factor superfamily of proteins. Hia is a novel example of a bacterial trimeric adhesin and may be the prototype member of a large subfamily of autotransporter proteins with a similar trimeric architecture.

Results and discussion

Structure determination

In earlier work, we discovered that the high-affinity binding activity of Hia resides in the region defined by amino acids 541–714, referred to as HiaBD1. As judged by size-exclusion chromatography, purified HiaBD1 (Hia541–714) forms stable oligomers (likely globular hexamers or elongated trimers), mimicking purified Hia50–779 (full-length passenger domain). Native HiaBD1 crystallized in the monoclinic space group P21 with two trimers per asymmetric unit and diffracted to a resolution of 2.1 Å. Given that HiaBD1 contains no methionine residues, use of the multiple anomalous dispersion (MAD) phasing method at and around the selenium edge was not possible. An exhaustive search for heavy metal derivatives was performed and was unsuccessful. Ultimately, site-directed mutagenesis was performed, and two methionines were introduced into HiaBD1 at sites that do not disturb adhesive function (data not shown). Subsequently, selenomethionine-substituted HiaBD1 (SeMetHiaBD1) crystals were generated, allowing phasing using the MAD method. The SeMetHiaBD1 protein crystallized in the trigonal space group P321 with two molecules per asymmetric unit and diffracted to a resolution of 2.7 Å. The expected four selenium sites in the asymmetric unit were located using the programs SOLVE, SnB, and CNS. Experimental phases followed by solvent flipping (CNS) provided an electron density map of excellent quality (Figure 2A). The model was built using the program O and was first refined against the SeMetHiaBD1 data to a resolution of 3.0 Å. The refined model was then positioned into the native data by molecular replacement and was refined against the native data to a resolution of 2.1 Å. The final model has an R/Rfree of 21.7/25.4% (Table I). The present model contains residues 548–705 of chain A, residues 549–705 of chains B, E, and F, residues 550–706 of chain C, residues 549–706 of chain D, and 488 water molecules.

Figure 2
Structure of the Hia monomer. (A) Representative region (αB helix and β9 strand) of the experimental phased electron density at 3 Å resolution. The electron density results from a map calculated using MAD phases after solvent flipping ...
Table 1
Crystallographic data and summary of refinement

Molecular architectures of the HiaBD1 subunit and of the functional trimer

The HiaBD1 subunit contains three well-defined structural domains, designated Domain 1 (residues 548–585), Domain 2 (residues 586–653), and Domain 3 (residues 654–705) (Figure 2B–D). Submission of the coordinates of the full-length HiaBD1 and/or individual domains to the DALI server revealed no known structures with significant similarity, indicating a novel protein fold.

Domain 1 is a four-stranded antiparallel β sheet, which forms a slightly concave sheet structure. One side of the sheet is composed of hydrophobic residues, while the other side consists primarily of polar residues. Domain 2 is a globular domain with mixed structure, containing four short β strands (β5–β8) and four helices (αA–B and 310a–b). This domain protrudes laterally and forms a knob. Domain 3 is an all β domain and consists of five long β strands (β9–β13) and one short β strand (β9′). These strands are twisted and segregate into three distinct subdomains, including a β hairpin formed by the antiparallel β9/β9′ and β10 strands, a connector formed by the β11 strand, and a β hairpin formed by the antiparallel β12 and β13 strands. There are very few macromolecular contacts between these subdomains, and Domain 3 appears to hold together via association with other HiaBD1 subunits (Figure 2B).

As shown in Figure 3A and B, the crystal structure of purified HiaBD1 reveals that individual subunits are assembled into a trimer, forming a mushroom-shaped structure with a broad stem at the N-terminal end and an elongated cap at the C-terminal end. The trimer has dimensions of 80 Å in length and 45 Å in width. At least three pieces of evidence argue that the trimer detected by crystallography represents the physiologic state of HiaBD1. First, in other work, we have discovered that the Hia C-terminal translocator domain of Hia forms a stable trimer in the bacterial outer membrane (Surana et al, 2004). Second, based on size-exclusion chromatography, purified HiaBD1 and SeMetHiaBD1 form oligomers in solution. Third, while HiaBD1 and SeMetHiaBD1 crystallize in different space groups, in both crystal forms the arrangement of the protein subunits is very similar, with head-to-head trimers.

Figure 3
Structure of the HiaBD1 trimer. (A) Stereo ribbon diagram (Carson, 1997) of the HiaBD1 trimer. The three subunits are shown in green, yellow, and red. The subunit in green is the same orientation as in Figure 2D. For clarity, only selected secondary structural ...

All three domains in HiaBD1 participate in the trimer interface (Figures 2C and and3C).3C). The total buried area between subunits within the trimer is approximately 15 240 Å2, with Domain 1 contributing 2580 Å2, Domain 2 contributing 4440 Å2, and Domain 3 contributing 8220 Å2. Of the available solvent-accessible surface area, approximately 25% of Domain 1, 24% of Domain 2, and 49% of Domain 3 is used for trimer formation (Figure 3C), suggesting that trimer formation may be required for protein stability. The Domain 1 β strands in one subunit interact with the corresponding β strands in both neighboring subunits to form an almost perfect triangle (the stem of the mushroom). The Domain 2 β5 strand in one subunit forms a small β sheet together with the Domain 1 β4 strand in a second subunit and the Domain 2 β8 strand in a third subunit, while the αB helix in one subunit interacts with the αB helix in the other subunits to form a tight hydrophobic core (Figure 3C). The Domain 3 β strands in one subunit interdigitate with the Domain 3 β strands in the other two subunits, resulting in extensive subunit–subunit interactions and a very compact structure (Figures 2B and and3C).3C). Each of the Domain 3 subdomains from one subunit contributes to a separate side of the three-sided tapering mushroom cap. In addition, the Domain 3 region holds the trimer together through an extensive hydrophobic/aromatic core consisting primarily of phenylalanine and tryptophan residues.

In considering the surface of HiaBD1, several structural features are notable. The concave β sheet of Domain 1 forms a shallow groove that runs parallel to the three-fold axis of the trimer (Figure 3B). Domain 2 forms a knob that extends laterally from the structure, into the solvent in vitro and into the extracellular milieu in vivo (Figure 3A and B). This knob consists of the αA helix, the 310a helix, and the loop structures between β6 and β8 (Figure 2D). Domains 2 and 3 form a major groove that runs diagonal to the three-fold axis of the trimer, just underneath the knob (Figure 3B). This groove is lined with charged residues, including R651, K661, R674, K682, R697, and R698 (Figure 3B).

Structural relationship of HiaBD1 and HiaBD2 and identification of repeating structural units

The Hia passenger domain contains a secondary binding domain defined by residues 50–374 and designated HiaBD2. HiaBD1 and HiaBD2 share sequence homology and interact with the same host cell receptor. With our knowledge of the structure of HiaBD1, we re-examined an alignment of the amino-acid sequences of HiaBD1 and HiaBD2. As shown in Figure 4A, residues 585–705 of HiaBD1 (Domains 2 and 3) share striking homology with residues 50–166 of HiaBD2, with 44% identity and 76% similarity overall. This high level of homology suggests that the 585–706 region of HiaBD1 (HiaBDp in Figure 1) and the 50–166 region of HiaBD2 (HiaBDs in Figure 1) have very similar structures.

Figure 4
Modular architecture of the Hia passenger domain. (A) Location of identical, conserved, and nonconserved residues between HiaBD1 and HiaBD2 on the surface of HiaBD1. A sequence alignment is provided at the bottom of the panel. Color coding is indicated. ...

Interestingly, sequence homology between HiaBD1 and the remainder of the Hia passenger domain is not limited to amino acids 50–166. Additional analysis revealed that residues 641–705 encompassing the β8 strand and the αB helix of Domain 2 and all of Domain 3 could be aligned with two other stretches of 67 and 64 residues within the Hia passenger domain, namely residues 250–316 (D3b) and 359–422 (D3c) (Figures 1 and and4B).4B). Among the 67 residues in D3b, 18 (27%) are identical and 43 (63%) are conserved relative to the corresponding residues in Domains 2 and 3 of HiaBD1. Similarly, among the 64 amino acids in D3c, 11 (19%) are identical and 42 (65%) are conserved relative to the corresponding amino acids in HiaBD1. Search of the Swiss-Prot data base using the motif [LVMI]-X(3)-G-W-X(7,10)-[TN]-X(6,9)-V-X(5)-V-X-F-X-[GSA] returns 80 hits belonging to 42 sequences, 38 of which are Hia, Hsf, or NhhA sequences from different strains. Both Hsf from H. influenzae and NhhA from Neisseria meningitidis are known close homologs of Hia. Extending this motif to include residues in the β12 and β13 strands of Domain 3 results in a marked decrease in the number of hits and restricts them to Hia, Hsf, and NhhA sequences. Thus, the D3 homology is restricted to Hia, Hsf, and NhhA.

These observations indicate that the Hia passenger domain has a modular structure with repeating Domain 3-like regions.

Identification of the receptor-binding region

In an effort to identify the receptor-binding region of the HiaBD1 structure, we began by generating a GST fusion protein that contains Hia581–714, which encompasses Domains 2 and 3 and lacks all but five residues in Domain 1. Following cleavage of the GST moiety, Hia581–714 was purified and examined for adherence activity. Based on size-exclusion chromatography, Hia581–714 formed oligomers in solution, analogous to purified HiaBD1 and Hia50–779. In assays assessing cell binding activity and capacity to inhibit adherence by bacteria expressing wild-type Hia, Hia581–714 was indistinguishable from HiaBD1 (data not shown). Thus, Domain 1 is not required for HiaBD1-mediated adherence.

With this information in mind, we focused primarily on Domains 2 and 3 and generated site-specific mutations in a series of surface-exposed residues (Table II and Figure 5A). We concentrated on residues lining potential binding pockets and introduced the mutations into pHMW8-7ΔBS2, then expressed the mutant proteins in Escherichia coli. Western analysis of outer membrane preparations confirmed that the mutant proteins were present in the bacterial outer membrane at wild-type levels (data not shown). Examination of bacterial adherence revealed that the D618K, A620R, and V656R mutations resulted in complete abrogation of Hia-mediated bacterial adherence. As depicted in Figure 5A, residues D618 and A620 are located on the underside of the Domain 2 knob, forming a roof over an acidic patch containing E668 and E678. Residue V656 is adjacent to the same acidic patch.

Figure 5
Identification of the receptor-binding region of HiaBD1. (A) Left: Location of the residues mutated in this study (see Table II). Mutations in residues in cyan do not affect adherence, while mutations in residues in magenta result in reduced adhesive ...
Table 2
Adherence activity of HiaBD1 point mutants

To extend these results, we produced GST fusions of the mutated HiaBD1 proteins. After cleavage of the GST moiety, the HiaBD1 derivatives were purified and then examined for their capacity to inhibit Hia-mediated adherence. Consistent with previous results, purified wild-type HiaBD1 was capable of complete inhibition (<5% of adherence observed when monolayers were untreated) (Figure 5B). In contrast, purified HiaBD1/D618K, HiaBD1/A620R, and HiaBD1/V656R had minimal effect on Hia-mediated adherence (70–100% of adherence observed when monolayers were untreated) (Figure 5B). Interestingly, purified HiaBD1/N617R, HiaBD1/E668K, and HiaBD1/E678A also had minimal effect on Hia-mediated adherence (Figure 5B), suggesting that N617, E668, and E678 also contribute to the Hia receptor-binding region. Control proteins including HiaBD1/K597E, HiaBD1/D600R, HiaBD1/L606K, HiaBD1/A610R, and HiaBD1/K630D all behaved like wild-type HiaBD1 (Figure 5B).

To confirm that the loss of adhesive activity of HiaBD1/D618K, HiaBD1/A620R, HiaBD1/V656R, HiaBD1/N617R, HiaBD1/E668K, and HiaBD1/E678A was not due to global changes in protein folding and disruption of the trimer, we assessed susceptibility to limited trypsin proteolysis. As shown in Figure 5C, wild-type HiaBD1 was completely resistant to trypsin in a 30 min assay using 15 μg of purified HiaBD1 and 250 ng of trypsin, indicating a stable structure. Similarly, all of the adhesive and nonadhesive HiaBD1 derivatives were resistant to trypsin treatment (Figure 5C). As a control, we introduced a mutation at F681, which lies within the β11 strand in Domain 3 and is involved in subunit–subunit interactions with both of the other subunits in the trimer. As predicted, purified HiaBD1/F681E was completely degraded by trypsin, suggesting an unstable trimer and an unfolded protein (Figure 5C). Similarly, a derivative of HiaBD1 with a deletion of residues 672–682 in the core (HiaBD1Δ6) was completely degraded by trypsin (Figure 5C).

Together, these results indicate that the HiaBD1 receptor-binding domain is an acidic pocket formed by N617, D618, A620, V656, E668, and E678. The entire binding pocket is contained within a single monomer, and thus the trimer has three identical binding sites related by a 120o rotation.

Conclusion

Adherence to host tissues is a fundamental step in the pathogenesis of most infectious diseases and is mediated by specialized microbial factors called adhesins. In this study, we have characterized the structure of the HiaBD1 binding domain of the H. influenzae Hia adhesin, an autotransporter protein that mediates efficient adherence to the respiratory epithelium and facilitates H. influenzae colonization of the upper respiratory tract. HiaBD1 has a novel trimeric architecture with three-fold symmetry and three identical receptor-binding pockets.

The HiaBD1 subunit fold is inherently unstable by itself and is stabilized by trimerization, with each domain in each subunit lending complementary secondary structures to the other subunits. Most striking is the extensive interdigitation between Domain 3 β strands, an arrangement that appears to be repeated four times along the Hia passenger domain (designated D3a, D3b, D3c, and D3d in Figure 1). Moreover, mutations in the core of the trimer interface such as F681E destabilize the trimer and eliminate adhesive activity. Thus, trimerization appears to be an essential structural feature of a functional Hia passenger domain.

In most autotransporters, the C-terminal translocator domain is approximately 300 residues in length and forms a β barrel with 12–14 transmembrane β strands. However, the Hia C-terminal translocator domain is much smaller, consisting of only 76 amino acids (Surana et al, 2004). Similarly, the YadA autotransporter expressed by Yersinia spp has a C-terminal translocator domain that contains only 70 amino acids (Roggenkamp et al, 2003). Hia and YadA appear to define a new subfamily of autotransporters with a distinctive C-terminal translocator domain that trimerizes. Based on homology analysis, at least 28 proteins belong to this subfamily, including proteins expressed by Salmonella enteritidis, N. meningitidis, Moraxella catarrhalis, and pathogenic E. coli, among other pathogens (Table III). Members of this subfamily may share a similar Hia-like architecture.

Table 3
Proteins identified as having a C terminus similar to the Hia C terminus

The fact that both the translocator domain and the passenger domain are trimeric in this class of trimeric autotransporters has implications regarding the mechanism of translocation of the passenger domain. Indeed, three passenger domains must translocate through a central trimeric β-domain pore to form an extensively intertwined trimer. We would like to propose that, to produce a functional trimeric adhesion domain, the passenger domain of each subunit must be translocated in a concerted fashion and that as these domains are extruded from the pore, folding and trimerization must occur coincidently, perhaps providing the driving force that pulls the unfolded passenger domain across the pore.

Site-directed mutagenesis established that residues N617, D618, A620, V656, E668, and E678 contribute to the receptor-binding pocket of HiaBD1. Mutations of D618, A620, and V656 abrogated adhesive activity in purified HiaBD1 and also in full-length Hia expressed on the surface of bacteria. In contrast, mutations of N617, E668, and E678 abolished adhesive activity in purified HiaBD1 but had little effect on Hia expressed by bacteria. Residues D618, A620, and V656 form the rim of the binding pocket, while residues N617, E668, and E678 lie behind the rim or at the base of the pocket. One possibility is that N617, E668, and E678 are sites of secondary contact with the Hia receptor; as a consequence, mutations of these residues may result in only a modest decrease in HiaBD1 binding affinity, an effect that can be overcome by the cooperative effect of multiple molecules of Hia on the surface of an organism, at least under the conditions of our adherence assay. Based on alignment of the amino-acid sequences of Hia proteins from nine different strains of H. influenzae, it appears that the HiaBD1 receptor-binding pocket is highly conserved, with absolute conservation of D618, A620, V656, E668, and E678. Comparison of the HiaBD1 and HiaBD2 primary and secondary binding domains reveals absolute conservation of A620, V656, E668, and E678. In contrast, HiaBD1 has an aspartic acid at position 618, while HiaBD2 has a glutamine at the corresponding position, perhaps accounting in part for the differences in HiaBD1 and HiaBD2 binding affinity.

The observation that a single HiaBD1 trimer contains three identical receptor-binding pockets is reminiscent of the tumor necrosis factor (TNF) superfamily of proteins, which play a critical role in several fundamental cellular processes, including apoptosis, survival, differentiation, and proliferation (Aggarwal, 2003). TNFα, TNFβ, and all other members of the TNF family are trimeric proteins with three spatially distinct but equivalent receptor-binding sites that recognize members of the TNF receptor superfamily (Locksley et al, 2001). In general, each trimer interacts with three receptor monomers in a final 3:3 complex (Banner et al, 1993; Locksley et al, 2001). In the case of Hia, it is possible that a single HiaBD1 trimer interacts with three separate receptor molecules or with three related domains on one receptor. A multivalent interaction with the host cell surface increases avidity and may stabilize bacterial adherence, allowing organisms to overcome physical forces in the respiratory tract such as coughing, sneezing, and mucociliary activity.

Based on comparison of the Hia sequences from diverse strains, the most variable portion of the HiaBD1 structure is the knob that protrudes laterally on all three faces (residues 599–619 and 631–636). This observation suggests that the knob may be a major target of the host immune response, perhaps in part because of its overt accessibility to the immune system. Accordingly, it is possible that strain-to-strain antigenic variation in this region of Hia has occurred to evade the immune response, analogous to the hypervariable regions that protrude from the N. gonorrhoeae pilin protein (Parge et al, 1995).

Hia is a major nonpilus adhesin of nonencapsulated H. influenzae (Barenkamp and St Geme, 1996; St Geme et al, 1998). A homolog of Hia called Hsf is ubiquitous in encapsulated isolates of H. influenzae and is the major nonpilus adhesin in these strains (St Geme et al, 1996). The HiaBD1 and HiaBD2 domains are highly conserved in Hsf. Thus, the receptor-binding pocket identified in HiaBD1 represents an attractive template for design of binding inhibitors that target encapsulated H. influenzae and an important subset of nonencapsulated H. influenzae. More broadly, knowledge of the HiaBD1 structure may facilitate development of novel antimicrobials with activity against other pathogens that express homotrimeric autotransporters, perhaps by disrupting trimerization.

Experimental procedures

Bacterial strains and plasmids

E. coli strains included BL21(DE3), XL-1 Blue, and DL41, and have been described previously (Sambrook and Russel, 2001).

The plasmid pHMW8-7 contains wild-type hia from H. influenzae strain 11 in pT7-7. The plasmid pHMW8-7ΔBS2 has been described previously and is a derivative of pHMW8-7 with a deletion of coding sequence for Hia residues 114–127, effectively eliminating the adhesive function of the HiaBD2 domain. The plasmid pGEX6P-1WT1BD has been described previously and contains coding sequence for Hia residues 541–714 cloned into the BamH1/EcoRI sites of pGEX6P-1 (Amersham), allowing production of GST–HiaBD1. The plasmid pGEX6P-HiaBD1(I560M/I657M) is a derivative of pGEX6P-1WT1BD and encodes a GST fusion protein containing HiaBD1 with methionine residues inserted in place of I560 and I657. pGEX6P-HiaBD1(I560M/I657M) was produced for crystallization to determine the structure of HiaBD1 using the multiwavelength anomalous diffraction (MAD) method. Adhesive activity of HiaBD1(I560M/I657M) is similar to that of wild-type HiaBD1 (data not shown), indicating that mutations of I560 and I657 to methionine do not affect receptor binding.

Recombinant DNA methods

DNA ligations, restriction endonuclease digestions, gel electrophoresis, and polymerase chain reactions were performed according to standard techniques. Plasmids were introduced into E. coli by electroporation (Ausubel et al, 1994).

Site-directed mutagenesis reactions were performed using standard protocols and Stratagene site-directed mutagenesis kits. QuikChange was used for mutations in pGEX6P-1WT1BD, while QuikChange XL was used for mutations in pHMW8-7ΔBS2. Mutants plasmids were initially transformed in E. coli XL-1 Blue, and nucleotide sequencing was performed. Plasmid clones with the correct nucleotide sequence were then transformed into E. coli BL21 (DE3) and E. coli DL41.

Protein production and purification

In order to produce native HiaBD1 and most HiaBD1 mutants, pGEX-6P-1WT1BD derivatives were introduced into E. coli BL21(DE3), and the resulting strains were incubated at 37°C in LB medium containing 100 μg/ml ampicillin. In order to produce selenomethionine-substituted HiaBD1 (SeMetHiaBD1), the plasmid pGEX6P-HiaBD1(I560M/I657M) was introduced into E. coli DL41, and the resulting strain was incubated at 37°C in LeMaster medium containing selenomethionine and 100 μg/ml ampicillin (Lemaster and Richards, 1985). All cultures were incubated to an OD600=0.4–0.5, then supplemented with isopropyl-β-D-thiogalactopyranoside at a final concentration of 0.1 mM and shifted to 30°C. After incubation for another 4–5 h, bacteria were harvested by centrifugation at 6600 g, then resuspended in 25 ml lysis buffer (5 mM EDTA, 1 mM Pefabloc SC, 1:100 protease inhibitor mix (Roche Molecular Biochemicals) in PBS) and lysed using a French pressure cell. Cell fragments were removed by centrifugation at 10 000 g, Triton X-100 was added to a final concentration of 1%, and GST fusion proteins were isolated by affinity chromatography using glutathione–Sepharose 4B beads (Amersham Pharmacia Biotech). Following extensive washing with Tris-buffered saline (TBS), the HiaBD1 portion of fusion proteins was cleaved from GST by adding 80 U/ml PreScission protease (Amersham Pharmacia Biotech) and 1 mM dithiothreitol to the beads and incubating overnight at 4°C. The HiaBD1 protein was separated from the GST moiety by centrifugation at 1000 g for 5 min. At this step, isolated proteins were analyzed for purity by SDS–polyacrylamide gel electrophoresis and staining with Coomassie blue. Protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad #500-0006). For crystallization, native HiaBD1 and SeMetHiaBD1 were further purified using a gel filtration column (Sephacryl S300 26/60 column, Amersham Pharmacia Biotech) equilibrated in a buffer containing 20 mM HEPES (pH 7.0), 100 mM NaCl, 1 mM DTT, and 10% glycerol. This step was necessary to produce crystals. The highly pure fractions (about 95%) from a single peak were combined and concentrated to approximately 10 mg/ml.

Crystallization of HiaBD1 and data collection

The native HiaBD1 crystals were grown at room temperature by vapor diffusion in hanging drops against a reservoir solution containing 25% PEG 4000, 0.2 M MgCl2, and with a 100 mM buffer ranging from pH 3.8 to 10. In the acidic condition, crystals formed as well-shaped triangular plates, but these crystals did not diffract well. When using Tris–HCl (pH 8.5), thin plate crystals were formed within 18 h and grew for a week. Single crystals (typical dimensions 0.3 × 0.4 × 0.05 mm) were cryoprotected gradually to a final solution containing 30% PEG 4000, 0.2 M MgCl2 100 mM Tris–HCl (pH 8.5), and 25% glycerol, and the crystals were flash-frozen in liquid nitrogen. Crystals were in monoclinic space group P21 with cell dimension a=83.58 Å, b=86.2 Å, c=89.12 Å, and β=99.08°. Crystals contained six molecules per asymmetric unit and diffracted to a resolution of 2.8 Å in the laboratory and 2.1 Å at the synchrotron (APS, beamline 19BM). Crystals of selenomethionyl HiaBD1 (SeMetHiaBD1) were grown under the same conditions as the native crystals. However, these crystals were not stable. With further extensive screening efforts, crystals of selenomethionyl HiaBD1 were grown in a solution containing 1.4 M Na-citrate and 100 mM HEPES (pH 7). Long hexagonal needle or hexagonal tube crystals formed within 24 h and grew for a week. Single crystals (typical dimensions 0.1 × 0.6 × 0.025 mm) were cryoprotected in a solution containing 1.45 M Na-citrate and 25% glycerol, and the crystals were flash-frozen in liquid nitrogen. Crystals were in trigonal space group P321 with cell dimension a=b=84.61 and c=154.25. Crystals contained two molecules per asymmetric unit, with solvent contents of 72%, and diffracted to a resolution of 2.7 Å at the synchrotron (SRS Daresbury, beamline 14-2).

Structure determination and refinement

The SeMetHiaBD1 monomer contains two methionines. Accordingly, four selenium sites were expected in the asymmetric unit. The four sites were found using the programs SOLVE (Terwilliger and Berendzen, 1999), SnB2.2 (Smith et al, 1998), and CNS (Brünger et al, 1998), and were then employed in phasing using the MAD phasing method followed by density modification (solvent flipping), as implemented by the program CNS. The resulting 3.0 Å electron density map was readily interpretable, into which approximately 60% of the secondary structural elements could be built as poly-ala fragments using the program O (Jones et al, 1991). The complete asymmetric unit was generated using NCS operators derived from Se sites. From this partial model, an NCS mask was constructed using the program MAMA (Kleywegt and Jones, 1999). Several cycles of combining calculated phases from the minimized partial model with MAD experimental phases followed by NCS averaging and solvent flipping (program CNS) resulted in electron density maps that allowed assignment of the remaining poly-ala chain. As phases continued to improve, side chains were gradually introduced. Most of the side chains could also be placed unambiguously into density. The resulting atomic model was refined (program CNS) against 3.0 Å data using conjugate gradient minimization. A random 5% of the data were removed prior to refinement and constituted the test set for crossvalidation. When R/Rfree reached 0.30/0.32, the model was used as the search model to solve the native crystal structure using the molecular replacement method (Navaza and Saludjian, 1997). For molecular replacement, we used only the model from residues 580–704, since the density and the model of this portion were better defined than those of residues 550–579. The final model was refined against the 2.1 Å resolution native dataset. During the final rounds of refinement, solvent molecules (water) were added. The final model, with R/Rfree factors of 21.7/25.4, includes 1370 atoms of residues 548–705 of chain A, 549–705 of chains B, E, and F, 550–706 of chain C, 549–706 of chain D, and 488 water molecules in the asymmetric unit (see Table I for refinement statistics). The model has been deposited in the pdb (entry code 1S7M).

Quantitative adherence assays

Chang epithelial cells (Wong–Kilbourne derivative, clone 1-5c-4, human conjunctiva; ATCC CCL20.2) were maintained in minimal essential medium (MEM) supplemented with nonessential amino acids and 10% (vol/vol) fetal calf serum (FCS) and were cultivated at 37°C in 5% CO2. Adherence assays were performed as described previously (St Geme et al, 1993). Briefly, cells were seeded into 24-well tissue culture plates at a density of 1.8 × 105 cells/well and were incubated overnight. Bacteria were inoculated into broth and allowed to grow to a density of ~2 × 109 colony-forming units (CFU) per ml. Approximately 1–2 × 107 CFU were inoculated into each well, and plates were gently centrifuged at 165 g for 5 min. After incubation at 37°C in 5% CO2 for 25 min, monolayers were rinsed with PBS to remove nonadherent bacteria, treated with trypsin–EDTA, then resuspended in BHI broth. Suspensions were plated on agar to yield the number of adherent CFU per monolayer. Adherence was calculated by dividing the number of adherent CFU per monolayer by the number of inoculated CFU.

For adherence inhibition assays, monolayers were preincubated with 100 nM of purified protein for 1.5 h at 37°C in 5% CO2 (Laarmann et al, 2002). Following rinsing with PBS to remove unbound protein, E. coli expressing pHMW8-7 was inoculated onto monolayers, and adherence was quantitated as described above.

The results of quantitative adherence assays were complemented by qualitative assays in which samples were stained with Giemsa and examined by light microscopy, as described previously (St Geme and Falkow, 1990).

Limited trypsin proteolyis

To assess susceptibility to trypsin of HiaBD1 derivatives, 15 μg of purified protein was incubated with 250 ng of sequencing grade modified trypsin (Sigma #1-521-187) in trypsin digestion buffer (50 mM Tris–HCl pH 7.5, 0.1 M NaCl, 1 mM EDTA). The reaction was allowed to proceed for 30 min at 37°C. After boiling for 5 min in Laemmli buffer containing 5% β-mercaptoethanol, a 25 μl aliquot of the reaction was resolved on a 10% SDS–polyacrylamide gel.

Acknowledgments

This work was supported by Wellcome Trust grant 070001 and NIH grant RO1-AI49950 to GW, by NIH grant RO1-AI44167 to JWS, and by NIH training grant T32-HL07873 to SEC. SEC is a member of the Medical Scientist Training Program at Washington University School of Medicine. We thank the staff of beamline 19BM of the Structural Biology Center at APS (Argonne National Laboratory), beamline 19ID of ESRF, and beamline 14.2 of Daresbury Synchrotron Light Source for help during data collection.

References

  • Aggarwal BB (2003) Signalling pathways of the TNF superfamily: a double-edged sword. Nat Rev Immunol 3: 745–756. [PubMed]
  • Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (1994) Current Protocols in Molecular Biology. John Wiley & Sons, Inc.
  • Banner DW, D'Arcy A, Janes W, Gentz R, Schoenfeld HJ, Broger C, Loetscher H, Lesslauer W (1993) Crystal structure of the soluble human 55 kd TNF receptor–human TNF beta complex: implications for TNF receptor activation. Cell 73: 431–445. [PubMed]
  • Barenkamp SJ, St Geme JW III (1996) Identification of a second family of high-molecular-weight adhesion proteins expressed by non-typable Haemophilus influenzae. Mol Microbiol 19: 1215–1223. [PubMed]
  • Brünger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nilges M, Pannu NS, Read RJ, Rice LM, Simonson T, Warren GL (1998) Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr D 54 (Part 5): 905–921. [PubMed]
  • Carson M (1997) Ribbons. Methods Enzymol 277: 493–505. [PubMed]
  • Henderson IR, Cappello R, Nataro JP (2000) Autotransporter proteins, evolution and redefining protein secretion. Trends Microbiol 8: 529–532. [PubMed]
  • Henderson IR, Navarro-Garcia F, Nataro JP. (1998) The great escape: structure and function of the autotransporter proteins. Trends Microbiol 6: 370–378. [PubMed]
  • Hultgren SJ, Abraham S, Caparon M, Falk P, St Geme JW III, Normark S (1993) Pilus and nonpilus bacterial adhesins: assembly and function in cell recognition. Cell 73: 887–901. [PubMed]
  • Jones TA, Zou JY, Cowan SW, Kjeldgaard (1991) Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A 47 (Part 2): 110–119. [PubMed]
  • Kleywegt GJ, Jones TA (1999) Software for handling macromolecular envelopes. Acta Crystallogr D 55 (Part 4): 941–944. [PubMed]
  • Laarmann S, Cutter D, Juehne T, Barenkamp SJ, St Geme JW (2002) The Haemophilus influenzae Hia autotransporter harbours two adhesive pockets that reside in the passenger domain and recognize the same host cell receptor. Mol Microbiol 46: 731–743. [PubMed]
  • Lemaster DM, Richards FM (1985) H-15N heteronuclear NMR studies of Escherichia coli thioredoxin in samples isotopically labeled by residue type. Biochemistry 24: 7263–7268. [PubMed]
  • Locksley RM, Killeen N, Lenardo MJ (2001) The TNF and TNF receptor superfamilies: integrating mammalian biology. Cell 104: 487–501. [PubMed]
  • Moxon ER, Murphy TF (2000) Haemophilus influenzae. In Principles and Practice of Infectious Diseases, Mandell GL, Bennett JE, Dolin R (eds) pp 2369–2376. Philadelphia: Churchill Livingstone.
  • Murphy TF, Bernstein JM, Dryja DM, Campagnari AA, Apicella MA (1987) Outer membrane protein and lipooligosaccharide analysis of paired nasopharyngeal and middle ear isolates in otitis media due to nontypable Haemophilus influenzae: pathogenetic and epidemiological observations. J Infect Dis 156: 723–731. [PubMed]
  • Navaza J, Saludjian P (1997) AMoRe: an automated molecular replacement program package. Methods Enzymol 276: 581–594.
  • Nicholls A, Sharp KA, Honig B (1991) Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Protein Struct Funct Genet 11: 281–296. [PubMed]
  • Parge HE, Forest KT, Hickey MJ, Christensen DA, Getzoff ED, Tainer JA (1995) Structure of the fibre-forming protein pilin at 2.6 A resolution. Nature 378: 32–38. [PubMed]
  • Roggenkamp A, Ackermann N, Jacobi CA, Truelzsch K, Hoffmann H, Heesemann J (2003) Molecular analysis of transport and oligomerization of the Yersinia enterocolitica adhesin YadA. J Bacteriol 185: 3735–3744. [PMC free article] [PubMed]
  • Sambrook J, Russel DW (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Press.
  • Shannon JL, Fernandez RC (1999) The C-terminal domain of the Bordetella pertussis autotransporter BrkA forms a pore in lipid bilayer membranes. J Bacteriol 181: 5838–5842. [PMC free article] [PubMed]
  • Smith GD, Nagar B, Rini JM, Hauptman HA, Blessing RH (1998) The use of SnB to determine an anomalous scattering substructure. Acta Crystallogr D 54 (Part 5): 799–804. [PubMed]
  • Stathopoulos C, Hendrixson DR, Thanassi DG, Hultgren SJ, St Geme JW III, Curtiss R III (2000) Secretion of virulence determinants by the general secretory pathway in Gram-negative pathogens: an evolving story. Microbes Infect 2: 1061–1072. [PubMed]
  • St Geme JW III (1993) Nontypeable Haemophilus influenzae disease: epidemiology, pathogenesis, and prospects for prevention. Infect Agents Dis 2: 1–16. [PubMed]
  • St Geme JW III (1997a) Bacterial adhesins: determinants of microbial colonization and pathogenicity. Adv Pediatr 44: 43–72. [PubMed]
  • St Geme JW III (1997b) Insights into the mechanism of respiratory tract colonization by nontypable Haemophilus influenzae. Pediatr Infect Dis J 16: 931–935. [PubMed]
  • St Geme JW III, Cutter D (2000) The Haemophilus influenzae Hia adhesin is an autotransporter protein that remains uncleaved at the C terminus and fully cell associated. J Bacteriol 182: 6005–6013. [PMC free article] [PubMed]
  • St Geme JW III, Cutter D, Barenkamp SJ (1996) Characterization of the genetic locus encoding Haemophilus influenzae type b surface fibrils. J Bacteriol 178: 6281–6287. [PMC free article] [PubMed]
  • St Geme JW III, Falkow S (1990) Haemophilus influenzae adheres to and enters cultured human epithelial cells. Infect Immun 58: 4036–4044. [PMC free article] [PubMed]
  • St Geme JW III, Falkow S, Barenkamp SJ (1993) High-molecular-weight proteins of nontypable Haemophilus influenzae mediate attachment to human epithelial cells. Proc Natl Acad Sci USA 90: 2875–2879. [PMC free article] [PubMed]
  • St Geme JW III, Kumar VV, Cutter D, Barenkamp SJ (1998) Prevalence and distribution of the hmw and hia genes and the HMW and Hia adhesins among genetically diverse strains of nontypeable Haemophilus influenzae. Infect Immun 66: 364–368. [PMC free article] [PubMed]
  • Surana NK, Cutter D, Barenkamp SJ, St Geme JW III (2004) The Haemophilus influenzae Hia autotransporter contains an unusually short, trimeric translocator domain. online version: 2004; 10.1074/jbc/M311496200. [PubMed]
  • Terwilliger TC, Berendzen J (1999) Automated MAD and MIR structure solution. Acta Crystallogr D 55 (Part 4): 849–861. [PMC free article] [PubMed]
  • Thanassi DG, Hultgren SJ (2000) Multiple pathways allow protein secretion across the bacterial outer membrane. Curr Opin Cell Biol 12: 420–430. [PubMed]
  • Turk DC (1984) The pathogenicity of Haemophilus influenzae. J Med Microbiol 18: 1–16. [PubMed]
  • Veiga E, Sugawara E, Nikaido H, de Lorenzo V, Fernandez LA (2002) Export of autotransported proteins proceeds through an oligomeric ring shaped by C-terminal domains. EMBO J 21: 2122–2131. [PMC free article] [PubMed]

Articles from The EMBO Journal are provided here courtesy of The European Molecular Biology Organization

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...