Emerging roles of low-molecular-weight thiols at the host–microbe interface

Low-molecular-weight (LMW) thiols are an abundant class of cysteine-derived small molecules found in all forms of life that maintain reducing conditions within cells. While their contributions to cellular redox homeostasis are well established, LMW thiols can also mediate other aspects of cellular physiology, including intercellular interactions between microbial and host cells. Here we discuss emerging roles for these redox-active metabolites at the host–microbe interface. We begin by providing an overview of chemical and computational approaches to LMW-thiol discovery. Next, we highlight mechanisms of virulence regulation by LMW thiols in infected cells. Finally, we describe how microbial metabolism of these compounds may influence host physiology.     

TcaR–ssDNA complex crystal structure reveals new DNA binding mechanism of the MarR family proteins

The teicoplanin-associated locus regulator (TcaR) regulates gene expression of proteins on the intercellular adhesion (ica) locus involved in staphylococci poly-N-acetylglucosamine biosynthesis. The absence of TcaR increases poly-N-acetylglucosamine production and promotes biofilm formation. Until recently, the mechanism of multiple antibiotic resistance regulator family protein members, such as TcaR, was restricted to binding double-stranded DNA. However, we recently found that TcaR strongly interacts with single-stranded DNA, which is a new role for this family of proteins. In this study, we report Staphylococcus epidermidis TcaR–single-stranded DNA complex structures. Our model suggests that TcaR and single-stranded DNA form a 6₁-symmetry polymer composed of TcaR dimers with single-stranded DNA that wraps outside the polymer and 12 nt per TcaR dimer. Single-stranded DNA binding to TcaR involves a large conformational change at the DNA binding lobe. Several point mutations involving the single-stranded DNA binding surface validate interactions between single-stranded DNA and TcaR. Our results extend the novel role of multiple antibiotic resistance regulator family proteins in staphylococci.     

The X-ray crystal structure of pyrrolidone–carboxylate peptidase from hyperthermophilic archaea Pyrococcus horikoshii

The crystal structure of pyrrolidone–carboxylate peptidase (PCP) from hyperthermophilic archaea Pyrococcus horikoshii (PhoPCP) has been determined at 1.6-A resolution by X-ray crystallography. PCP belongs to the C15 family of cysteine protease, and specifically removes the amino terminal pyroglutamate residue from a wide range of N-terminal-blocking peptides. The crystal structure is very similar to that of other hyperthermophiles, Pyrococcus friosus and Thermococcus litoralis, and even that from the mesophile, Bacillus amyloliquefacience. The inter-subunit disulfide bonds, which have been proposed as one of the thermostabilizing factors of the PCP from such hyperthermophiles, was not present in PhoPCP. The result suggests that the thermostability of PhoPCP may be obtained by the accumulation of many weak factors. Abbreviations: NCS – non-crystallographic symmetry; PCP – pyrrolidone-carboxylate peptidase; rmsd – root mean square deviation     

The crystal structure of the C₂A domain of otoferlin reveals an unconventional top loop region

Otoferlin (Otof), whose genetic mutations cause profound deafness in humans, is a protein composed of at least six C₂ domains, which are known as Ca²⁺-binding and phospholipid-binding regions. Mammalian ferlin proteins are proposed to act in membrane fusion events, with Otof being specifically required for exocytosis in auditory hair cells. Ferlin C₂ domains exhibit a rather low level of sequence similarity to those of synaptotagmins, protein kinase C isoforms, or phospholipases. Here, we report the crystal structure of the N-terminal C₂ domain of Otof (C₂A) at 1.95-Å resolution. In contrast to previous predictions, we found that this C₂ domain is complete with eight β-strands. Comparing the structure of Otof C₂A to those of other C₂ domains revealed one top loop in Otof to be significantly shorter. This results in a depression of the surface, which is positively charged for the Otof C₂A domain, and contrasts with the head-like protrusion surrounded by a negatively charged “neck” typically found in other C₂ domains. Isothermal titration calorimetry and circular dichroism spectroscopy studies confirmed that Otof C₂A is unable to bind Ca²⁺, while the synaptotagmin-1 C₂A domain exhibited Ca²⁺ binding under the same conditions. Furthermore, floatation assays revealed a failure of Otof C₂A to bind to phospholipid membranes. Accordingly, no positively charged β-groove-like surface structure, which is known to bind phosphatidylinositol-4,5-bisphosphate in other C₂ domains, was found at the respective position in Otof C₂A. Taken together, these data demonstrate that the Otof C₂A domain differs structurally and functionally from other C₂ domains.     

Analysis of maternal-zygotic ugdh mutants reveals divergent roles for HSPGs in vertebrate embryogenesis and provides new insight into the initiation of left–right asymmetry

Growth factors and morphogens regulate embryonic patterning, cell fate specification, cell migration, and morphogenesis. The activity and behavior of these signaling molecules are regulated in the extracellular space through interactions with proteoglycans (Bernfield et al., 1999, Perrimon and Bernfield, 2000, Lander and Selleck, 2000 and Selleck, 2000). Proteoglycans are high molecular-weight proteins consisting of a core protein with covalently linked glycosaminoglycan (GAG) side chains, which are thought to mediate ligand interaction. Drosophila mutant embryos deficient for UDP-glucose dehydrogenase activity (Ugdh, required for GAG synthesis) exhibit abnormal Fgf, Wnt and TGFß signaling and die during gastrulation, indicating a broad and critical role for proteoglycans during early embryonic development (Lin et al., 1999 and Lin and Perrimon, 2000) (Hacker et al., 1997). Mouse Ugdh mutants also die at gastrulation, however, only Fgf signaling appears disrupted ( Garcia-Garcia and Anderson, 2003). These findings suggested a possible divergence in the requirement for proteoglycans during Drosophila and mouse embryogenesis, and that mammals may have evolved alternative means of regulating Wnt and TGFß activity. To further examine the function of proteoglycans in vertebrate development, we have characterized zebrafish mutants devoid of both maternal and zygotic Ugdh/Jekyll activity (MZjekyll). We demonstrate that MZjekyll mutant embryos display abnormal Fgf, Shh, and Wnt signaling activities, with concomitant defects in central nervous system patterning, cardiac ventricular fate specification and axial morphogenesis. Furthermore, we uncover a novel role for proteoglycans in left–right pattern formation. Our findings resolve longstanding questions into the evolutionary conservation of Ugdh function and provide new mechanistic insights into the initiation of left–right asymmetry.     

The X-ray structure of human serum ceruloplasmin at 3.1 Å: nature of the copper centres

The X-ray structure of human serum ceruloplasmin has been solved at a resolution of 3.1?Å. The structure reveals that the molecule is comprised of six plastocyanin-type domains arranged in a triangular array. There are six copper atoms; three form a trinuclear cluster sited at the interface of domains 1 and 6, and there are three mononuclear sites in domains 2, 4 and 6. Each of the mononuclear coppers is coordinated to a cysteine and two histidine residues, and those in domains 4 and 6 also coordinate to a methionine residue; in domain 2, the methionine is replaced by a leucine residue which may form van der Waals type contacts with the copper. The trinuclear centre and the mononuclear copper in domain 6 form a cluster essentially the same as that found in ascorbate oxidase, strongly suggesting an oxidase role for ceruloplasmin in the plasma.     

The Italian verb–noun anthroponymic compounds at the Syntax / morphology interface

This paper offers an analysis of Italian anthroponymic verb–noun compounds. It is argued that the first element of these compounds historically is an imperative (2nd sg) form. Such a view not only accounts adequately for the evolutionary process at work in the original naming process; it also rightly accounts for the actual morphological make-up of these compounds. It is argued as well that anthroponymic compounds involving imperatives provided for a structural model which is still traceable in the morphological make-up of non-anthroponymic compounds, even though a reanalysis process has led to the reinterpretation of the verbal element of V–N compounds as a bare stem. Crucially, such a reanalysis will be said to have been favoured by the morphological unmarkedness of imperatives: as zero inflected stems, imperatives may serve as a base for paradigmatic restructuration. Italian Verb–noun compounds will be shown to offer an illustration of this basicness of the imperative, following a pattern of word formation which is available in other languages.     

The fungal–mineral interface: challenges and considerations of micro-analytical developments

Over recent years, the role of fungi, especially mycorrhizal fungi, in the weathering of rock-forming minerals has been increasingly recognised. Much of our understanding of the effects of fungi on mineral weathering is based on macroscopic studies. However, the ability of fungi to translocate materials, including organic acids and siderophores, to specific areas of a mineral surface leads to significant spatial heterogeneity in the weathering process. Thus, geomycologists are confronted with unique challenges of how to comprehend and quantify such a high degree of diversity and complicated arrays of interactions. Recent advances in experimental and analytical techniques have increased our ability to probe the fungal–mineral interface at the resolution necessary to decouple significant biogeochemical processes. Modern microscopy, spectroscopy, mass spectrometry, wet chemistry, and scattering techniques allow for the selective extraction of physical, chemical, and structural data at the micro- to nano-scale. These techniques offer exciting possibilities to study fungal–mineral interactions at the scale of individual hyphae. In this review, we give an overview of some of these techniques with their characteristics, advantages and limitations, and how they can be used to further our understanding of biotic mineral weathering.     

The mechanical effects of different levels of cement penetration at the cement–bone interface

The mechanical effects of varying the depth of cement penetration in the cement–bone interface were investigated using finite element analysis (FEA) and validated using companion experimental data. Two FEA models of the cement–bone interface were created from micro-computed tomography data and the penetration of cement into the bone was varied over six levels each. The FEA models, consisting of the interdigitated cement–bone constructs with friction between cement and bone, were loaded to failure in tension and in shear. The cement and bone elements had provision for crack formation due to excessive stress. The interfacial strength showed a strong relationship with the average interdigitation (r²=0.97 and r²=0.93 in tension and shear, respectively). Also, the interface strength was strongly related with the contact area (r²=0.98 and r²=0.95 in tension and shear, respectively). The FEA results compared favorably to the stiffness–strength relationships determined experimentally. Overall, the cement–bone interface was 2.5 times stronger in shear than in tension and 1.15 times stiffer in tension than in shear, independent of the average interdigitation. More cracks occurred in the cement than in the bone, independent of the average interdigitation, consistent with the experimental results. In addition, more cracks were generated in shear than in tension. In conclusion, achieving and maintaining maximal infiltration of cement into the bone to obtain large interdigitation and contact area is key to optimizing the interfacial strength.     

The dimeric structure of the Cpn60.2 chaperonin of Mycobacterium tuberculosis at 2.8 Å reveals possible modes of function

Mycobacterium tuberculosis expresses two proteins (Cpn60.1 and Cpn60.2) that belong to the chaperonin (Cpn) family of heat shock proteins. Studies have shown that the two proteins have different functional roles in the bacterial life cycle and that Cpn60.2 is essential for cell viability and may be involved in M. tuberculosis pathogenicity. Cpn60.2 does not form a tetradecameric double ring, which is typical of other Cpns. We have determined the crystal structure of recombinant Cpn60.2 to 2.8 Å resolution by molecular replacement; the asymmetric unit (AU) contains a dimer, which is consistent with size-exclusion high-performance liquid chromatography and dynamic light-scattering measurements of the soluble recombinant protein. However, we suggest that the actual Cpn60.2 dimer may be different from that identified within the AU on the basis of surface contact stability, solvation free-energy gain, and functional aspects. Unlike the dimer found in the AU, which is formed through apical domain interactions, the dimeric form we propose here provides a free apical domain that is required for normal chaperone activity and may be involved in M. tuberculosis association with macrophages and arthrosclerosis plaque formation. Here we describe in detail the structural aspects that lead to Cpn60.2 dimer formation and prevent the formation of heptameric rings and tetradecameric double rings.     

The crystal structure of human protein α1M reveals a chromophore-binding site and two putative protein–protein interfaces

Lipocalin α1-microglobulin (α1M) is a conserved glycoprotein present in plasma and in the interstitial fluids of all tissues. α1M is linked to a heterogeneous yellow–brown chromophore of unknown structure, and interacts with several target proteins, including α1-inhibitor-3, fibronectin, prothrombin and albumin. To date, there is little knowledge about the interaction sites between α1M and its partners. Here, we report the crystal structure of the human α1M. Due to the crystallization occurring in a low ionic strength solution, the unidentified chromophore with heavy electron density is observed at a hydrophobic inner tube of α1M. In addition, two conserved surface regions of α1M are proposed as putative protein–protein interface sites. Further study is needed to unravel the detailed information about the interaction between α1M and its partners.     

The crystal structure of human α(1)-microglobulin reveals a potential haem-binding site

We describe the 2.3 ? (1 ?=0.1?nm) X-ray structure of α1m (α1-microglobulin), an abundant protein in human blood plasma, which reveals the β-barrel fold typical for lipocalins with a deep pocket lined by four loops at its open rim. Loop #1 harbours the residue Cys34 which is responsible for covalent cross-linking with plasma IgA. A single disulfide bond between Cys72 and Cys169 connects the C-terminal segment to the β-barrel, as in many other lipocalins. The exposed imidazole side chains of His122 and His123 in loop #4 give rise to a double Ni2+-binding site together with a crystallographic neighbour. The closest structural relatives of α1m are the complement protein component C8γ, the L-prostaglandin D synthase and lipocalin 15, three other structurally characterized members of the lipocalin family in humans that have only distant sequence similarity. In contrast with these, α1m is initially expressed as a bifunctional fusion protein with the protease inhibitor bikunin. Neither the electron density nor ESI-MS (electrospray ionization MS) provide evidence for a chromophore bound to the recombinant α1m, also known as 'yellow/brown lipocalin'. However, the three side chains of Lys92, Lys118 and Lys130 that were reported to be involved in covalent chromophore binding appear to be freely accessible to ligands accommodated in the hydrophobic pocket. A structural feature similar to the well-known Cys-Pro haem-binding motif indicates the presence of a haem-binding site within the loop region of α1m, which explains previous biochemical findings and supports a physiological role in haem scavenging, as well as redox-mediated detoxification.     

Phylogeny of the Rhizobium–Allorhizobium–Agrobacterium clade supports the delineation of Neorhizobium gen. nov.

The genera Agrobacterium, Allorhizobium, and Rhizobium belong to the family Rhizobiaceae. However, the placement of a phytopathogenic group of bacteria, the genus Agrobacterium, among the nitrogen-fixing bacteria and the unclear position of Rhizobium galegae have caused controversy in previous taxonomic studies. To resolve uncertainties in the taxonomy and nomenclature within this family, the phylogenetic relationships of generic members of Rhizobiaceae were studied, but with particular emphasis on the taxa included in Agrobacterium and the “R. galegae complex” (R. galegae and related taxa), using multilocus sequence analysis (MLSA) of six protein-coding housekeeping genes among 114 rhizobial and agrobacterial taxa. The results showed that R. galegae, R. vignae, R. huautlense, and R. alkalisoli formed a separate clade that clearly represented a new genus, for which the name Neorhizobium is proposed. Agrobacterium was shown to represent a separate cluster of mainly pathogenic taxa of the family Rhizobiaceae. A. vitis grouped with Allorhizobium, distinct from Agrobacterium, and should be reclassified as Allorhizobium vitis, whereas Rhizobium rhizogenes was considered to be the proper name for former Agrobacterium rhizogenes. This phylogenetic study further indicated that the taxonomic status of several taxa could be resolved by the creation of more novel genera.     

Fluid–structure interactions in micro-interlocked regions of the cement–bone interface

Experimental tests and computational modelling were used to explore the fluid dynamics at the trabeculae–cement interlock regions found in the tibial component of total knee replacements. A cement–bone construct of the proximal tibia was created to simulate the immediate post-operative condition. Gap distributions along nine trabeculae–cement regions ranged from 0 to 50.4 μm (mean = 12 μm). Micro-motions ranged from 0.56 to 4.7 μm with a 1 MPa compressive load to the cement. Fluid–structure analysis between the trabeculae and the cement used idealised models with parametric evaluation of loading direction, gap closing fraction (GCF), gap thickness, loading frequency and fluid viscosity. The highest fluid shear stresses (926 Pa) along the trabecular surface were found for conditions with very thin and large GCFs, much larger than reported physiological levels (～1–5 Pa). A second fluid–structure model was created with a provision for bone resorption using a constitutive model with resorption velocity proportional to fluid shear rate. A lower cut-off was used, below which bone resorption would not occur (50 s^? 1). Results showed that there was initially high shear rates (>1000 s^? 1) that diminished after initial trabecular resorption. Resorption continued in high shear rate regions, resulting in a final shape with bone left deep in the cement layer, and is consistent with morphology found in post-mortem retrievals. Small gaps between the trabecular surface and the cement in the immediate post-operative state produce fluid flow conditions that appear to be supra-physiologic; these may cause fluid-induced lysis of trabeculae in the micro-interlock regions.     

The crystal structure of the α-neurexin-1 extracellular region reveals a hinge point for mediating synaptic adhesion and function

α- and β-neurexins (NRXNs) are transmembrane cell adhesion proteins that localize to presynaptic membranes in neurons and interact with the postsynaptic neuroligins (NLGNs). Their gene mutations are associated with the autism spectrum disorders. The extracellular region of α-NRXNs, containing nine independently folded domains, has structural complexity and unique functional characteristics, distinguishing it from the smaller β-NRXNs. We have solved the X-ray crystal structure of seven contiguous domains of the α-NRXN-1 extracellular region at 3.0 ? resolution. The structure reveals an arrangement where the N-terminal five domains adopt a more rigid linear conformation and the two C-terminal domains form a separate arm connected by a flexible hinge. In an extended conformation the molecule is suitably configured to accommodate a bound NLGN molecule, as supported by structural comparison and surface plasmon resonance. These studies provide the structural basis for a multifunctional synaptic adhesion complex mediated by α-NRXN-1.