The type III secretion system needle tip complex mediates host cell sensing and translocon insertion

Type III secretion systems (T3SSs) are essential virulence determinants of many Gram-negative bacterial pathogens. The Shigella T3SS consists of a cytoplasmic bulb, a transmembrane region and a hollow 'needle' protruding from the bacterial surface. Physical contact with host cells initiates secretion and leads to assembly of a pore, formed by IpaB and IpaC, in the host cell membrane, through which proteins that facilitate host cell invasion are translocated. As the needle is implicated in host cell sensing and secretion regulation, its tip should contain components that initiate host cell contact. Through biochemical and immunological studies of wild-type and mutant Shigella T3SS needles, we reveal tip complexes of differing compositions and functional states, which appear to represent the molecular events surrounding host cell sensing and pore formation. Our studies indicate that the interaction between IpaB and IpaD at needle tips is key to host cell sensing, orchestration of IpaC secretion and its subsequent assembly at needle tips. This allows insertion into the host cell membrane of a translocation pore that is continuous with the needle.     

The type III secretion system needle,tip, and translocon

Many Gram‐negative bacteria pathogenic to plants and animals deploy the type III secretion system (T3SS) to inject virulence factors into their hosts. All bacteria that rely on the T3SS to cause infectious diseases in humans have developed antibiotic resistance. The T3SS is an attractive target for developing new antibiotics because it is essential in virulence, and part of its structural component is exposed on the bacterial surface. The structural component of the T3SS is the needle apparatus, which is assembled from over 20 different proteins and consists of a base, an extracellular needle, a tip, and a translocon. This review summarizes the current knowledge on the structure and assembly of the needle, tip, and translocon.     

Self-chaperoning of the type III secretion system needle tip proteins IpaD and BipD

Bacteria expressing type III secretion systems (T3SS) have been responsible for the deaths of millions worldwide, acting as key virulence elements in diseases ranging from plague to typhoid fever. The T3SS is composed of a basal body, which traverses both bacterial membranes, and an external needle through which effector proteins are secreted. We report multiple crystal structures of two proteins that sit at the tip of the needle and are essential for virulence: IpaD from Shigella flexneri and BipD from Burkholderia pseudomallei. The structures reveal that the N-terminal domains of the molecules are intramolecular chaperones that prevent premature oligomerization, as well as sharing structural homology with proteins involved in eukaryotic actin rearrangement. Crystal packing has allowed us to construct a model for the tip complex that is supported by mutations designed using the structure.     

Shigella IpaD has a dual role: signal transduction from the type III secretion system needle tip and intracellular secretion regulation

Type III secretion systems (T3SSs) are protein injection devices essential for the interaction of many Gram‐negative bacteria with eukaryotic cells. While Shigella assembles its T3SS when the environmental conditions are appropriate for invasion, secretion is only activated after physical contact with a host cell. First, the translocators are secreted to form a pore in the host cell membrane, followed by effectors which manipulate the host cell. Secretion activation is tightly controlled by conserved T3SS components: the needle tip proteins IpaD and IpaB, the needle itself and the intracellular gatekeeper protein MxiC. To further characterize the role of IpaD during activation, we combined random mutagenesis with a genetic screen to identify ipaD mutant strains unable to respond to host cell contact. Class II mutants have an overall defect in secretion induction. They map to IpaD's C‐terminal helix and likely affect activation signal generation or transmission. The Class I mutant secretes translocators prematurely and is specifically defective in IpaD secretion upon activation. A phenotypically equivalent mutant was found in mxiC. We show that IpaD and MxiC act in the same intracellular pathway. In summary, we demonstrate that IpaD has a dual role and acts at two distinct locations during secretion activation.     

Functional insights into the Shigella type III needle tip IpaD in secretion control and cell contact

Type III secretion apparatus (T3SA) are complex nanomachines that insert a translocation pore into the host cell membrane through which effector proteins are injected into the cytosol. In Shigella, the pore is inserted by a needle tip complex that also controls secretion. IpaD is the key protein that rules the composition of the tip complex before and upon cell contact or Congo red (CR) induction. However, how IpaD is involved in secretion control and translocon insertion remains not fully understood. Here, we report the phenotypic analysis of 20 10‐amino acids deletion variants all along the coiled‐coil and the central domains of IpaD (residues 131–332). Our results highlight three classes of T3S phenotype; (i) wild‐type secretion, (ii) constitutive secretion of all classes of effectors, and (iii) constitutive secretion of translocators and early effectors, but not of late effectors. Our data also suggest that the composition of the tip complex defines both the T3SA inducibility state and late effectors secretion. Finally, we shed light on a new aspect regarding the contact of the needle tip with cell membrane by uncoupling the Shigella abilities to escape macrophage vacuole, and to insert the translocation pore or to invade non‐phagocytic cells.     

Crystal structure of an enzyme-substrate complex provides insight into the interaction between human arylsulfatase A and its substrates during catalysis

Arylsulfatase A (ASA) belongs to the sulfatase family whose members carry a C(alpha)-formylglycine that is post-translationally generated by oxidation of a conserved cysteine or serine residue. The crystal structures of two arylsulfatases, ASA and ASB, and kinetic studies on ASA mutants led to different proposals for the catalytic mechanism in the hydrolysis of sulfate esters.The structures of two ASA mutants that lack the functional C(alpha)-formylglycine residue 69, in complex with a synthetic substrate, have been determined in order to unravel the reaction mechanism. The crystal structure of the inactive mutant C69A-ASA in complex with p-nitrocatechol sulfate (pNCS) mimics a reaction intermediate during sulfate ester hydrolysis by the active enzyme, without the covalent bond to the key side-chain FGly69. The structure shows that the side-chains of lysine 123, lysine 302, serine 150, histidine 229, the main-chain of the key residue 69 and the divalent cation in the active center are involved in sulfate binding. It is proposed that histidine 229 protonates the leaving alcoholate after hydrolysis.C69S-ASA is able to bind covalently to the substrate and hydrolyze it, but is unable to release the resulting sulfate. Nevertheless, the resulting sulfation is low. The structure of C69S-ASA shows the serine side-chain in a single conformation, turned away from the position a substrate occupies in the complex. This suggests that the double conformation observed in the structure of wild-type ASA is more likely to correspond to a formylglycine hydrate than to a twofold disordered aldehyde oxo group, and accounts for the relative inertness of the C69S-ASA mutant. In the C69S-ASA-pNCS complex, the substrate occupies the same position as in the C69A-ASA-pNCS complex, which corresponds to the non-covalently bonded substrate. Based on the structural data, a detailed mechanism for sulfate ester cleavage is proposed, involving an aldehyde hydrate as the functional group.     

Helical structure of the needle of the type III secretion system of Shigella flexneri

Gram-negative bacteria commonly interact with animal and plant hosts using type III secretion systems (TTSSs) for translocation of proteins into eukaryotic cells during infection. 10 of the 25 TTSS-encoding genes are homologous to components of the bacterial flagellar basal body, which the TTSS needle complex morphologically resembles. This indicates a common ancestry, although no TTSS sequence homologues for the genes encoding the flagellum are found. We here present an approximately 16-A structure of the central component, the needle, of the TTSS. Although the needle subunit is significantly smaller and shares no sequence homology with the flagellar hook and filament, it shares a common helical architecture ( approximately 5.6 subunits/turn, 24-A helical pitch). This common architecture implies that there will be further mechanistic analogies in the functioning of these two bacterial systems.     

Crystal structure and mutational study of RecOR provide insight into its mode of DNA binding

The crystal structure of the complex formed between Deinococcus radiodurans RecR and RecO (drRecOR) has been determined. In accordance with previous biochemical characterisation, the drRecOR complex displays a RecR:RecO molecular ratio of 2:1. The biologically relevant drRecOR entity consists of a heterohexamer in the form of two drRecO molecules positioned on either side of the tetrameric ring of drRecR, with their OB (oligonucleotide/oligosaccharide-binding) domains pointing towards the interior of the ring. Mutagenesis studies validated the protein-protein interactions observed in the crystal structure and allowed mapping of the residues in the drRecOR complex required for DNA binding. Furthermore, the preferred DNA substrate of drRecOR was identified as being 3'-overhanging DNA, as encountered at ssDNA-dsDNA junctions. Together these results suggest a possible mechanism for drRecOR recognition of stalled replication forks.     

Crystal structure of Legionella DotD: insights into the relationship between type IVB and type II/III secretion systems

The Dot/Icm type IVB secretion system (T4BSS) is a pivotal determinant of Legionella pneumophila pathogenesis. L. pneumophila translocate more than 100 effector proteins into host cytoplasm using Dot/Icm T4BSS, modulating host cellular functions to establish a replicative niche within host cells. The T4BSS core complex spanning the inner and outer membranes is thought to be made up of at least five proteins: DotC, DotD, DotF, DotG and DotH. DotH is the outer membrane protein; its targeting depends on lipoproteins DotC and DotD. However, the core complex structure and assembly mechanism are still unknown. Here, we report the crystal structure of DotD at 2.0 Å resolution. The structure of DotD is distinct from that of VirB7, the outer membrane lipoprotein of the type IVA secretion system. In contrast, the C-terminal domain of DotD is remarkably similar to the N-terminal subdomain of secretins, the integral outer membrane proteins that form substrate conduits for the type II and the type III secretion systems (T2SS and T3SS). A short β-segment in the otherwise disordered N-terminal region, located on the hydrophobic cleft of the C-terminal domain, is essential for outer membrane targeting of DotH and Dot/Icm T4BSS core complex formation. These findings uncover an intriguing link between T4BSS and T2SS/T3SS.     

Crystal structure of a molybdopterin synthase-precursor Z complex: insight into its sulfur transfer mechanism and its role in molybdenum cofactor deficiency

In almost all biological life forms, molybdenum and tungsten are coordinated by molybdopterin (MPT), a tricyclic pyranopterin containing a cis-dithiolene group. Together, the metal and the pterin moiety form the redox reactive molybdenum cofactor (Moco). Mutations in patients with deficiencies in Moco biosynthesis usually occur in the enzymes catalyzing the first and second steps of biosynthesis, leading to the formation of precursor Z and MPT, respectively. The second step is catalyzed by the heterotetrameric MPT synthase protein consisting of two large (MoaE) and two small (MoaD) subunits with the MoaD subunits located at opposite ends of a central MoaE dimer. Previous studies have determined that the conversion of the sulfur- and metal-free precursor Z to MPT by MPT synthase involves the transfer of sulfur atoms from a C-terminal MoaD thiocarboxylate to the C-1' and C-2' positions of precursor Z. Here, we present the crystal structures of non-thiocarboxylated MPT synthase from Staphylococcus aureus in its apo form and in complex with precursor Z. A comparison of the two structures reveals conformational changes in a loop that participates in interactions with precursor Z. In the complex, precursor Z is bound by strictly conserved residues in a pocket at the MoaE dimer interface in close proximity of the C-terminal glycine of MoaD. Biochemical evidence indicates that the first dithiolene sulfur is added at the C-2' position.     

Mutations in the Yersinia pseudotuberculosis type III secretion system needle protein, YscF, that specifically abrogate effector translocation into host cells

The trafficking of effectors, termed Yops, from Yersinia spp. into host cells is a multistep process that requires the type III secretion system (TTSS). The TTSS has three main structural parts: a base, a needle, and a translocon, which work together to ensure the polarized movement of Yops directly from the bacterial cytosol into the host cell cytosol. To understand the interactions that take place at the interface between the tip of the TTSS needle and the translocon, we developed a screen to identify mutations in the needle protein YscF that separated its function in secretion from its role in translocation. We identified 25 translocation-defective (TD) yscF mutants, which fall into five phenotypic classes. Some classes exhibit aberrant needle structure and/or reduced levels of Yop secretion, consistent with known functions for YscF. Strikingly, two yscF TD classes formed needles and secreted Yops normally but displayed distinct translocation defects. Class I yscF TD mutants showed diminished pore formation, suggesting incomplete pore insertion and/or assembly. Class II yscF TD mutants formed pores but showed nonpolar translocation, suggesting unstable needle-translocon interactions. These results indicate that YscF functions in Yop secretion and translocation can be genetically separated. Furthermore, the identification of YscF residues that are required for the assembly of the translocon and/or productive interactions with the translocon has allowed us to initiate the mapping of the needle-translocon interface.     

Crystal structure of D-hydantoinase from Bacillus stearothermophilus: insight into the stereochemistry of enantioselectivity

Industrial production of antibiotics, such as semisynthetic penicillins and cephalosporins, requires optically pure D-p-hydroxylphenylglycine and its derivatives as important side-chain precursors. To produce optically pure D-amino acids, microbial D-hydantoinase (E.C. 3.5.2.2) is used for stereospecific hydrolysis of chemically synthesized cyclic hydantoins. We report the apo-crystal structure of D-hydantoinase from B. stearothermophilus SD1 at 3.0 A resolution. The structure has a classic TIM barrel fold. Despite an undetectable similarity in sequence, D-hydantoinase shares a striking structural similarity with the recently solved structure of dihydroorotase. A structural comparison of hydantoinase with dihydroorotase revealed that the catalytic chemistry is conserved, while the substrate recognition is not. This structure provides insight into the stereochemistry of enantioselectivity in hydrolysis and illustrates how the enzyme recognizes stereospecific exocyclic substituents and hydrolyzes hydantoins. It should also provide a rationale for further directed evolution of this enzyme for hydrolysis of new hydantoins with novel exocyclic substituents.     

Crystal structure of Bacillus stearothermophilus UvrA provides insight into ATP-modulated dimerization, UvrB interaction, and DNA binding

The nucleotide excision repair pathway corrects many structurally unrelated DNA lesions. Damage recognition in bacteria is performed by UvrA, a member of the ABC ATPase superfamily whose functional form is a dimer with four nucleotide-binding domains (NBDs), two per protomer. In the 3.2 A structure of UvrA from Bacillus stearothermophilus, we observe that the nucleotide-binding sites are formed in an intramolecular fashion and are not at the dimer interface as is typically found in other ABC ATPases. UvrA also harbors two unique domains; we show that one of these is required for interaction with UvrB, its partner in lesion recognition. In addition, UvrA contains three zinc modules, the number and ligand sphere of which differ from previously published models. Structural analysis, biochemical experiments, surface electrostatics, and sequence conservation form the basis for models of ATP-modulated dimerization, UvrA-UvrB interaction, and DNA binding during the search for lesions.     

Crystal structure of the hexameric traffic ATPase of the Helicobacter pylori type IV secretion system

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.     

Crystal structure of the human N-Myc downstream-regulated gene 2 protein provides insight into its role as a tumor suppressor

Considerable attention has recently been paid to the N-Myc downstream-regulated gene (NDRG) family because of its potential as a tumor suppressor in many human cancers. Primary amino acid sequence information suggests that the NDRG family proteins may belong to the α/β-hydrolase (ABH) superfamily; however, their functional role has not yet been determined. Here, we present the crystal structures of the human and mouse NDRG2 proteins determined at 2.0 and 1.7 Å resolution, respectively. Both NDRG2 proteins show remarkable structural similarity to the ABH superfamily, despite limited sequence similarity. Structural analysis suggests that NDRG2 is a nonenzymatic member of the ABH superfamily, because it lacks the catalytic signature residues and has an occluded substrate-binding site. Several conserved structural features suggest NDRG may be involved in molecular interactions. Mutagenesis data based on the structural analysis support a crucial role for helix α6 in the suppression of TCF/β-catenin signaling in the tumorigenesis of human colorectal cancer, via a molecular interaction.     

Crystal structure and mutagenesis of the metallochaperone MeaB: insight into the causes of methylmalonic aciduria

MeaB is an auxiliary protein that plays a crucial role in the protection and assembly of the B(12)-dependent enzyme methylmalonyl-CoA mutase. Impairments in the human homologue of MeaB, MMAA, lead to methylmalonic aciduria, an inborn error of metabolism. To explore the role of this metallochaperone, its structure was solved in the nucleotide-free form, as well as in the presence of product, GDP. MeaB is a homodimer, with each subunit containing a central alpha/beta-core G domain that is typical of the GTPase family, as well as alpha-helical extensions at the N and C termini that are not found in other metalloenzyme chaperone GTPases. The C-terminal extension appears to be essential for nucleotide-independent dimerization, and the N-terminal region is implicated in protein-protein interaction with its partner protein, methylmalonyl-CoA mutase. The structure of MeaB confirms that it is a member of the G3E family of P-loop GTPases, which contains other putative metallochaperones HypB, CooC, and UreG. Interestingly, the so-called switch regions, responsible for signal transduction following GTP hydrolysis, are found at the dimer interface of MeaB instead of being positioned at the surface of the protein where its partner protein methylmalonyl-CoA mutase should bind. This observation suggests a large conformation change of MeaB must occur between the GDP- and GTP-bound forms of this protein. Because of their high sequence homology, the missense mutations in MMAA that result in methylmalonic aciduria have been mapped onto MeaB and, in conjunction with mutagenesis data, provide possible explanations for the pathology of this disease.     

Crystal structure of DnaK protein complexed with nucleotide exchange factor GrpE in DnaK chaperone system: insight into intermolecular communication

The conserved, ATP-dependent bacterial DnaK chaperones process client substrates with the aid of the co-chaperones DnaJ and GrpE. However, in the absence of structural information, how these proteins communicate with each other cannot be fully delineated. For the study reported here, we solved the crystal structure of a full-length Geobacillus kaustophilus HTA426 GrpE homodimer in complex with a nearly full-length G. kaustophilus HTA426 DnaK that contains the interdomain linker (acting as a pseudo-substrate), and the N-terminal nucleotide-binding and C-terminal substrate-binding domains at 4.1-Å resolution. Each complex contains two DnaKs and two GrpEs, which is a stoichiometry that has not been found before. The long N-terminal GrpE α-helices stabilize the linker of DnaK in the complex. Furthermore, interactions between the DnaK substrate-binding domain and the N-terminal disordered region of GrpE may accelerate substrate release from DnaK. These findings provide molecular mechanisms for substrate binding, processing, and release during the Hsp70 chaperone cycle.     

Crystal structure of the retinoblastoma protein N domain provides insight into tumor suppression, ligand interaction, and holoprotein architecture

The retinoblastoma susceptibility protein, Rb, has a key role in regulating cell-cycle progression via interactions involving the central "pocket" and C-terminal regions. While the N-terminal domain of Rb is dispensable for this function, it is nonetheless strongly conserved and harbors missense mutations found in hereditary retinoblastoma, indicating that disruption of its function is oncogenic. The crystal structure of the Rb N-terminal domain (RbN), reveals a globular entity formed by two rigidly connected cyclin-like folds. The similarity of RbN to the A and B boxes of the Rb pocket domain suggests that Rb evolved through domain duplication. Structural and functional analysis provides insight into oncogenicity of mutations in RbN and identifies a unique phosphorylation-regulated site of protein interaction. Additionally, this analysis suggests a coherent conformation for the Rb holoprotein in which RbN and pocket domains directly interact, and which can be modulated through ligand binding and possibly Rb phosphorylation.     

Crystal structure of the Yersinia type III secretion protein YscE

The plague-causing bacterium Yersinia pestis utilizes a contact-dependent (type III) secretion system (T3SS) to transport virulence factors from the bacterial cytosol directly into the interior of mammalian cells where they interfere with signal transduction pathways that mediate phagocytosis and the inflammatory response. The type III secretion apparatus is composed of 20-25 different Yersinia secretion (Ysc) proteins. We report here the structure of YscE, the smallest Ysc protein, which is a dimer in solution. The probable mode of oligomerization is discussed.     

Crystal structure and solution NMR dynamics of a D (type II) peroxiredoxin glutaredoxin and thioredoxin dependent: a new insight into the peroxiredoxin oligomerism

Peroxiredoxins (Prxs) constitute a family of thiol peroxidases that reduce hydrogen peroxide, peroxinitrite, and hydroperoxides using a strictly conserved cysteine. Very abundant in all organisms, Prxs are produced as diverse isoforms characterized by different catalytic mechanisms and various thiol-containing reducing agents. The oligomeric state of Prxs and the link with their functionality is a subject of intensive research. We present here a combined X-ray and nuclear magnetic resonance (NMR) study of a plant Prx that belongs to the D-Prx (type II) subfamily. The Populus trichocarpa Prx is the first Prx shown to be regenerated in vitro by both the glutaredoxin and thioredoxin systems. The crystal structure and solution NMR provide evidence that the reduced protein is a specific noncovalent homodimer both in the crystal and in solution. The dimer interface is roughly perpendicular to the plane of the central beta sheet and differs from the interface of A- and B-Prx dimers, where proteins associate in the plane parallel to the beta sheet. The homodimer interface involves residues strongly conserved in the D (type II) Prxs, suggesting that all Prxs of this family can homodimerize. The study provides a new insight into the Prx oligomerism and the basis for protein-protein and enzyme-substrate interaction studies by NMR.