Hye-Jeong Yeo

Crystal Structure of the Hexameric Traffic ATPase of the Helicobacter pylori Type IV Secretion System

Abstract The type IV secretion system of Helicobacter pylori consists of 10–15 proteins responsible for transport of the transforming protein CagA into target epithelial cells. Secretion of CagA crucially depends on the hexameric ATPase, HP0525, a member of the VirB11-PulE family. We present the crystal structure of a binary complex of HP0525 bound to ADP. Each monomer consists of two domains formed by the N- and C-terminal halves of the sequence. ADP is bound at the interface between the two domains. In the hexamer, the N- and C-terminal domains form two rings, which together form a chamber open on one side and closed on the other. A model is proposed in which HP0525 functions as an inner membrane pore, the closure and opening of which is regulated by ATP binding and ADP release.

VirB11 ATPases are dynamic hexameric assemblies: New insights into bacterial type IV secretion

The coupling of ATP binding/hydrolysis to macromolecular secretion systems is crucial to the pathogenicity of Gram‐negative bacteria. We reported previously the structure of the ADP‐bound form of the hexameric traffic VirB11 ATPase of the Helicobacter pylori type IV secretion system (named HP0525), and proposed that it functions as a gating molecule at the inner membrane, cycling through closed and open forms regulated by ATP binding/hydrolysis. Here, we combine crystal structures with analytical ultracentrifugation experiments to show that VirB11 ATPases indeed function as dynamic hexameric assemblies. In the absence of nucleotide, the N‐terminal domains exhibit a collection of rigid‐body conformations. Nucleotide binding ‘locks’ the hexamer into a symmetric and compact structure. We propose that VirB11s use the mechanical leverage generated by such nucleotide‐dependent conformational changes to facilitate the export of substrates or the assembly of the type IV secretion apparatus. Bio chemical characterization of mutant forms of HP0525 coupled with electron microscopy and in vivo assays support such hypothesis, and establish the relevance of VirB11s ATPases as drug targets against pathogenic bacteria.

Structural basis for host recognition by the Haemophilus influenzae Hia autotransporter.

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.

Structural and functional characterization of the VirB5 protein from the type IV secretion system encoded by the conjugative plasmid pKM101

Type IV secretion systems mediate intercellular transfer of macro-molecules via a mechanism ancestrally related to that of bacterial conjugation machineries. TraC of the IncN plasmid pKM101 belongs to the VirB5 family of proteins, an essential component of most type IV secretion systems. Here, we present the structure of TraC. VirB5/TraC is a single domain protein, which consists of a three helix bundle and a loose globular appendage. Structure-based site-directed mutagenesis followed by functional studies indicates that VirB5 proteins participate in protein-protein interactions important for pilus assembly and function.

Unveiling Molecular Scaffolds of the Type IV Secretion System

In gram-negative bacteria, secretion of macromolecules across the two membranes takes place using diverse macromolecular transport assemblies, from simple one-component systems to complex multicomponent machineries. The type IV secretion system (T4SS) is one of the five major secretion systems that

Architecture and Adhesive Activity of the Haemophilus influenzae Hsf Adhesin

Haemophilus influenzae type b is an important cause of meningitis and other serious invasive diseases and initiates infection by colonizing the upper respiratory tract. Among the major adhesins in H. influenzae type b is a nonpilus protein called Hsf, a large protein that forms fiber-like structures on the bacterial surface and shares significant sequence similarity with the nontypeable H. influenzae Hia autotransporter. In the present study, we characterized the structure and adhesive activity of Hsf. Analysis of the predicted amino acid sequence of Hsf revealed three regions with high-level homology to the HiaBD1 and HiaBD2 binding domains in Hia. Based on examination of glutathione S-transferase fusion proteins corresponding to these regions, two of the three had adhesive activity and one was nonadhesive in assays with cultured epithelial cells. Structural modeling demonstrated that only the two regions with adhesive activity harbored an acidic binding pocket like the binding pocket identified in the crystal structure of HiaBD1. Consistent with these results, disruption of the acidic binding pockets in the adhesive regions eliminated adhesive activity. These studies advance our understanding of the architecture of Hsf and the family of trimeric autotransporters and provide insight into the structural determinants of H. influenzae type b adherence.

Enterococcus faecalis PcfC, a Spatially Localized Substrate Receptor for Type IV Secretion of the pCF10 Transfer Intermediate

Upon sensing of peptide pheromone, Enterococcus faecalis efficiently transfers plasmid pCF10 through a type IV secretion (T4S) system to recipient cells. The PcfF accessory factor and PcfG relaxase initiate transfer by catalyzing strand-specific nicking at the pCF10 origin of transfer sequence (oriT). Here, we present evidence that PcfF and PcfG spatially coordinate docking of the pCF10 transfer intermediate with PcfC, a membrane-bound putative ATPase related to the coupling proteins of gram-negative T4S machines. PcfC and PcfG fractionated with the membrane and PcfF with the cytoplasm, yet all three proteins formed several punctate foci at the peripheries of pheromone-induced cells as monitored by immunofluorescence microscopy. A PcfC Walker A nucleoside triphosphate (NTP) binding site mutant (K156T) fractionated with the E. faecalis membrane and also formed foci, whereas PcfC deleted of its N-terminal putative transmembrane domain (PcfCDelta N103) distributed uniformly throughout the cytoplasm. Native PcfC and mutant proteins PcfCK156T and PcfCDelta N103 bound pCF10 but not pcfG or Delta oriT mutant plasmids as shown by transfer DNA immunoprecipitation, indicating that PcfC binds only the processed form of pCF10 in vivo. Finally, purified PcfCDelta N103 bound DNA substrates and interacted with purified PcfF and PcfG in vitro. Our findings support a model in which (i) PcfF recruits PcfG to oriT to catalyze T-strand nicking, (ii) PcfF and PcfG spatially position the relaxosome at the cell membrane to stimulate substrate docking with PcfC, and (iii) PcfC initiates substrate transfer through the pCF10 T4S channel by an NTP-dependent mechanism.

The Actinobacillus pleuropneumoniae HMW1C-like glycosyltransferase mediates N-linked glycosylation of the Haemophilus influenzae HMW1 adhesin.

The Haemophilus influenzae HMW1 adhesin is an important virulence exoprotein that is secreted via the two-partner secretion pathway and is glycosylated at multiple asparagine residues in consensus N-linked sequons. Unlike the heavily branched glycans found in eukaryotic N-linked glycoproteins, the modifying glycan structures in HMW1 are mono-hexoses or di-hexoses. Recent work demonstrated that the H. influenzae HMW1C protein is the glycosyltransferase responsible for transferring glucose and galactose to the acceptor sites of HMW1. An Actinobacillus pleuropneumoniae protein designated ApHMW1C shares high-level homology with HMW1C and has been assigned to the GT41 family, which otherwise contains only O-glycosyltransferases. In this study, we demonstrated that ApHMW1C has N-glycosyltransferase activity and is able to transfer glucose and galactose to known asparagine sites in HMW1. In addition, we found that ApHMW1C is able to complement a deficiency of HMW1C and mediate HMW1 glycosylation and adhesive activity in whole bacteria. Initial structure-function studies suggested that ApHMW1C consists of two domains, including a 15-kDa N-terminal domain and a 55-kDa C-terminal domain harboring glycosyltransferase activity. These findings suggest a new subfamily of HMW1C-like glycosyltransferases distinct from other GT41 family O-glycosyltransferases.

A prototype two-partner secretion pathway: the Haemophilus influenzae HMW1 and HMW2 adhesin systems

Nontypable Haemophilus influenzae is a common cause of human disease and initiates infection by colonizing the upper respiratory tract. Adherence to respiratory epithelium is an important step in the process of colonization and is influenced by adhesive proteins called adhesins. In approximately 80% of nontypable H. influenzae isolates, the major adhesins are related proteins called HMW1 and HMW2. Here, we summarize recent advances in our understanding of HMW1 and HMW2 as prototype members of the bacterial two-partner secretion pathway and examples of the expanding number of bacterial glycoproteins, highlighting experimental approaches that might be useful in studies of other secreted proteins and glycoproteins.

The structure of the Haemophilus influenzae HMW1 pro-piece reveals a structural domain essential for bacterial two-partner secretion.

In pathogenic Gram-negative bacteria, many virulence factors are secreted via the two-partner secretion pathway, which consists of an exoprotein called TpsA and a cognate outer membrane translocator called TpsB. The HMW1 and HMW2 adhesins are major virulence factors in nontypeable Haemophilus influenzae and are prototype two-partner secretion pathway exoproteins. A key step in the delivery of HMW1 and HMW2 to the bacterial surface involves targeting to the HMW1B and HMW2B outer membrane translocators by an N-terminal region called the secretion domain. Here we present the crystal structure at 1.92Å of the HMW1 pro-piece (HMW1-PP), a region that contains the HMW1 secretion domain and is cleaved and released during HMW1 secretion. Structural analysis of HMW1-PP revealed a right-handed β-helix fold containing 12 complete parallel coils and one large extra-helical domain. Comparison of HMW1-PP and the Bordetella pertussis FHA secretion domain (Fha30) reveals limited amino acid homology but shared structural features, suggesting that diverse TpsA proteins have a common structural domain required for targeting to cognate TpsB proteins. Further comparison of HMW1-PP and Fha30 structures may provide insights into the keen specificity of TpsA-TpsB interactions.