Functional interactions between the MutL and Vsr proteins of Escherichia coli are dependent on the N-terminus of Vsr

The crystal structure of the Escherichia coli Vsr endonuclease bound to a C(T/G)AGG substrate revealed that the DNA is held by a pincer composed of a trio of aromatic residues which intercalate into the major groove, and an N-terminus alpha helix which lies across the minor groove. We have constructed an N-terminus truncation (Delta14) which removes most of the alpha helix. The mutant is still fairly proficient in mediating very short patch repair. However, its endonuclease activity is considerably reduced and, in contrast to that of the wild type protein, cannot be stimulated by MutL. We had shown previously that excess Vsr in vivo causes mutagenesis, probably by inhibiting the participation of MutL in mismatch repair. The Delta14 mutant has diminished mutagenicity. In contrast, four enzymatically inactive mutants, with intact N-termini, are as mutagenic as the wild type protein. On the basis of these results we suggest that MutL causes a conformational change in the N-terminus of Vsr which enhances Vsr activity, and that this functional interaction between Vsr and MutL decreases the ability of MutL to carry out mismatch repair.     

Crystal Structure of a Chimeric Receptor Binding Protein Constructed from Two Lactococcal Phages

Lactococcus lactis, a gram-positive bacterium widely used by the dairy industry to manufacture cheeses, is subject to infection by a diverse population of virulent phages. We have previously determined the structures of three receptor binding proteins (RBPs) from lactococcal phages TP901-1, p2, and bIL170, each of them having a distinct host range. Virulent phages p2 and bIL170 are classified within the 936 group, while the temperate phage TP901-1 is a member of the genetically distinct P335 polythetic group. These RBPs comprise three domains: the N-terminal domain, binding to the virion particle; a β-helical linker domain; and the C-terminal domain, bearing the receptor binding site used for host recognition. Here, we have designed, expressed, and determined the structure of an RBP chimera in which the N-terminal and linker RBP domains of phage TP901-1 (P335) are fused to the C-terminal RBP domain of phage p2 (936). This chimera exhibits a stable structure that closely resembles the parental structures, while a slight displacement of the linker made RBP domain adaptation efficient. The receptor binding site is structurally indistinguishable from that of native p2 RBP and binds glycerol with excellent affinity.A broad number of products are manufactured by large-scale bacterial fermentation, including the value-added fermented dairy products. Most bacterial fermentation industries have experienced problems with phage contamination. Phage outbreaks are costly and time-consuming because they can slow or arrest the fermentation process and adversely affect product quality (15). For decades, the dairy industry has relied on an array of strategies to control this natural phenomenon, including rotation of their bacterial cultures (11, 24, 25). However, in spite of these efforts, new virulent lactococcal phages keep emerging. A better understanding of the various mechanisms affecting the genetic diversity of the phage population is necessary for optimal phage control strategies (18).Lactococcal phages are among the most studied bacterial viruses because of the economic importance of their hosts. Hundreds of lactococcal phages have been isolated, and the vast majority of them have a long, contractile tail, thereby belonging to the Siphoviridae family (1). Lactococcus lactis phages are currently classified into 10 genetically distinct groups (10), but only members of 3 of them are highly adapted to multiply in milk, namely, the 936, c2, and P335 groups (11, 24, 25). The first step for such an effective viral infection is host recognition, which necessitates the interaction between the adsorption device located at the distal tail end of the phage and the cell surface receptor (32). Members of the 936 and P335 groups recognize their host through an interaction between their receptor binding protein (RBP) (13) and receptors, probably lipoteichoic acids, at the host cell surface (27, 29-31).We have previously determined the crystal structures of three RBPs, from the virulent lactococcal phages p2 (30, 31) and bIL170 (936 group) (27) and from the temperate phage TP901-1 (P335 group) (29). The RBPs of these phages have a similar architecture of three protomers related by a threefold axis. Each protomer comprises three domains: the N terminus (named shoulders in p2), the interlaced β-prism linker (the “neck” domain), and the jelly-roll domain (2) at the C terminus (the “head” domain). This last domain harbors a saccharide binding site likely involved in host recognition, as it binds with high affinity to phosphoglycerol, a component of teichoic acid (8, 19, 27, 29-31). We have previously shown that the shoulder and neck domains are highly conserved in the RBPs of 936-like phages (8, 19, 27, 29-31). The individuality of the RBP C-terminal domain sequence likely dictates phage specificity for the receptor, which may specifically recognize different substitutions (H, GlcNAc, or d-Ala) of the phosphoglycerol moieties of the L. lactis teichoic acid polymers. Recently, the complete genomic sequence of the reference virulent phage P335 was determined, and comparative analysis revealed that the C terminus of its RBP showed homology to the RBP of the virulent lactococcal phage P475 of the 936 group (17). Such homology between RBP head domains was surprising because the two lactococcal phage groups rarely shared common genes or domains. This observation suggested that modular shuffling of domains can occur between these otherwise genetically distinct phage groups.The overall fold of the N-terminal RBP domain is different in 936- and P335-like phages. In the P335 group, the N-terminal domain comprises a unique helix that fits into the rest of the phage baseplate (28, 29) (Fig. ​(Fig.1A),1A), while in the 936 group, this 140-residue domain is a large β-sandwich with an external α-helix (30) (Fig. ​(Fig.1B).1B). Nonetheless, the N-terminal domains of the two RBPs may still be, related because both appear to be built using a coiled coil, although the 936-like phages have an additional β-sandwich. The β-prism linkers (neck domain) of the two phage groups also differ in sequence and in radius, but they have a similar fold, the latter being also close to that of T4 phage short fiber (33). The linker domain of phage TP901-1 is wider than that of p2 and exhibits a repeated motif (G-X-Y-X-Y, where X is polar and Y nonpolar). Finally, the C-terminal domains of both species share the same fold, a jelly-roll motif (2) also found in adenovirus (5) and reovirus (3, 4, 6).Open in a separate windowFIG. 1.Structures and sequences of RBPs from lactococcal phages. (A) Three-dimensional structure of the RBP from phage TP901-1 (P335 group; blue). (B) Three-dimensional structure of the RBP from phage p2 (936 group; magenta). (C) View of a model associating domains of TP901-1 (N terminus and linker domain, below red line, blue) and p2 (head, above red line, magenta) RBPs. (D) Three-dimensional crystal structure of chimera form 1 (yellow) assembled according to the model in panel C. (E) Sequence alignment of the RBPs of p2 (part) and TP901-1. The secondary structure is described above the alignment. The binding residues are shown with blue dots. The hinge proline (Pro 162/63) is identified by a red arrow. The chimera is composed of the N-terminal domain (residues 17 to 33) and the linker domain residues (residues 34 to 63) from phage TP901-1 RBP and the C-terminal domain (residues 163 to 264) from phage p2 RBP.The question addressed here was whether exchange between the C-terminal domains of two phage groups would lead to a stable protein with conserved binding capacity. To answer this question, we have generated an RBP chimera comprising the N-terminal and linker domains of phage TP901-1 fused to the C-terminal domain of phage p2. We have produced this chimera and determined its crystal structure and its sugar binding capacity. These results indicate that straightforward domain exchange produced a stable chimera with a conserved binding capacity and a structure close to that of each of the parental parts.     

Structural Basis for Regulation of the Human Acetyl-CoA Thioesterase 12 and Interactions with the Steroidogenic Acute Regulatory Protein-related Lipid Transfer (START) Domain

Acetyl-CoA plays a fundamental role in cell signaling and metabolic pathways, with its cellular levels tightly controlled through reciprocal regulation of enzymes that mediate its synthesis and catabolism. ACOT12, the primary acetyl-CoA thioesterase in the liver of human, mouse, and rat, is responsible for cleavage of the thioester bond within acetyl-CoA, producing acetate and coenzyme A for a range of cellular processes. The enzyme is regulated by ADP and ATP, which is believed to be mediated through the ligand-induced oligomerization of the thioesterase domains, whereby ATP induces active dimers and tetramers, whereas apo- and ADP-bound ACOT12 are monomeric and inactive. Here, using a range of structural and biophysical techniques, it is demonstrated that ACOT12 is a trimer rather than a tetramer and that neither ADP nor ATP exert their regulatory effects by altering the oligomeric status of the enzyme. Rather, the binding site and mechanism of ADP regulation have been determined to occur through two novel regulatory regions, one involving a large loop that links the thioesterase domains (Phe¹⁵⁴-Thr¹⁷⁸), defined here as RegLoop1, and a second region involving the C terminus of thioesterase domain 2 (Gln³⁰⁴-Gly³²⁶), designated RegLoop2. Mutagenesis confirmed that Arg³¹² and Arg³¹³ are crucial for this mode of regulation, and novel interactions with the START domain are presented together with insights into domain swapping within eukaryotic thioesterases for substrate recognition. In summary, these experiments provide the first structural insights into the regulation of this enzyme family, revealing an alternate hypothesis likely to be conserved throughout evolution.     

Mammalian G protein-coupled receptor expression in Escherichia coli: II. Refolding and biophysical characterization of mouse cannabinoid receptor 1 and human parathyroid hormone receptor 1

G protein-coupled receptors (GPCRs) represent approximately 3% of the human proteome. They are involved in a large number of diverse processes and, therefore, are the most prominent class of pharmacological targets. Besides rhodopsin, X-ray structures of classical GPCRs have only recently been resolved, including the β1 and β2 adrenergic receptors and the A2A adenosine receptor. This lag in obtaining GPCR structures is due to several tedious steps that are required before beginning the first crystallization experiments: protein expression, detergent solubilization, purification, and stabilization. With the aim to obtain active membrane receptors for functional and crystallization studies, we recently reported a screen of expression conditions for approximately 100 GPCRs in Escherichia coli, providing large amounts of inclusion bodies, a prerequisite for the subsequent refolding step. Here, we report a novel artificial chaperone-assisted refolding procedure adapted for the GPCR inclusion body refolding, followed by protein purification and characterization. The refolding of two selected targets, the mouse cannabinoid receptor 1 (muCB1R) and the human parathyroid hormone receptor 1 (huPTH1R), was achieved from solubilized receptors using detergent and cyclodextrin as protein folding assistants. We could demonstrate excellent affinity of both refolded and purified receptors for their respective ligands. In conclusion, this study suggests that the procedure described here can be widely used to refold GPCRs expressed as inclusion bodies in E. coli.     

Novel Microbiological and Spatial Statistical Methods to Improve Strength of Epidemiological Evidence in a Community-Wide Waterborne Outbreak

Failures in the drinking water distribution system cause gastrointestinal outbreaks with multiple pathogens. A water distribution pipe breakage caused a community-wide waterborne outbreak in Vuorela, Finland, July 2012. We investigated this outbreak with advanced epidemiological and microbiological methods. A total of 473/2931 inhabitants (16%) responded to a web-based questionnaire. Water and patient samples were subjected to analysis of multiple microbial targets, molecular typing and microbial community analysis. Spatial analysis on the water distribution network was done and we applied a spatial logistic regression model. The course of the illness was mild. Drinking untreated tap water from the defined outbreak area was significantly associated with illness (RR 5.6, 95% CI 1.9–16.4) increasing in a dose response manner. The closer a person lived to the water distribution breakage point, the higher the risk of becoming ill. Sapovirus, enterovirus, single Campylobacter jejuni and EHEC O157:H7 findings as well as virulence genes for EPEC, EAEC and EHEC pathogroups were detected by molecular or culture methods from the faecal samples of the patients. EPEC, EAEC and EHEC virulence genes and faecal indicator bacteria were also detected in water samples. Microbial community sequencing of contaminated tap water revealed abundance of Arcobacter species. The polyphasic approach improved the understanding of the source of the infections, and aided to define the extent and magnitude of this outbreak.     

The emerging role of acyl-CoA thioesterases and acyltransferases in regulating peroxisomal lipid metabolism

The importance of peroxisomes in lipid metabolism is now well established and peroxisomes contain approximately 60 enzymes involved in these lipid metabolic pathways. Several acyl-CoA thioesterase enzymes (ACOTs) have been identified in peroxisomes that catalyze the hydrolysis of acyl-CoAs (short-, medium-, long- and very long-chain), bile acid-CoAs, and methyl branched-CoAs, to the free fatty acid and coenzyme A. A number of acyltransferase enzymes, which are structurally and functionally related to ACOTs, have also been identified in peroxisomes, which conjugate (or amidate) bile acid-CoAs and acyl-CoAs to amino acids, resulting in the production of amidated bile acids and fatty acids. The function of ACOTs is to act as auxiliary enzymes in the α- and β-oxidation of various lipids in peroxisomes. Human peroxisomes contain at least two ACOTs (ACOT4 and ACOT8) whereas mouse peroxisomes contain six ACOTs (ACOT3, 4, 5, 6, 8 and 12). Similarly, human peroxisomes contain one bile acid-CoA:amino acid N-acyltransferase (BAAT), whereas mouse peroxisomes contain three acyltransferases (BAAT and acyl-CoA:amino acid N-acyltransferases 1 and 2: ACNAT1 and ACNAT2). This review will focus on the human and mouse peroxisomal ACOT and acyltransferase enzymes identified to date and discuss their cellular localizations, emerging structural information and functions as auxiliary enzymes in peroxisomal metabolic pathways. This article is part of a Special Issue entitled: Metabolic Functions and Biogenesis of Peroxisomes in Health and Disease.     

Comparative structural analysis of lipid binding START domains

Background

Steroidogenic acute regulatory (StAR) protein related lipid transfer (START) domains are small globular modules that form a cavity where lipids and lipid hormones bind. These domains can transport ligands to facilitate lipid exchange between biological membranes, and they have been postulated to modulate the activity of other domains of the protein in response to ligand binding. More than a dozen human genes encode START domains, and several of them are implicated in a disease.

Principal Findings

We report crystal structures of the human STARD1, STARD5, STARD13 and STARD14 lipid transfer domains. These represent four of the six functional classes of START domains.

Significance

Sequence alignments based on these and previously reported crystal structures define the structural determinants of human START domains, both those related to structural framework and those involved in ligand specificity.

Enhanced version

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A topological model of the baseplate of lactococcal phage Tuc2009

Phages infecting Lactococcus lactis, a Gram-positive bacterium, are a recurrent problem in the dairy industry. Despite their economical importance, the knowledge on these phages, belonging mostly to Siphoviridae, lags behind that accumulated for members of Myoviridae. The three-dimensional structures of the receptor-binding proteins (RBP) of three lactococcal phages have been determined recently, illustrating their modular assembly and assigning the nature of their bacterial receptor. These RBPs are attached to the baseplate, a large phage organelle, located at the tip of the tail. Tuc2009 baseplate is formed by the products of 6 open read frames, including the RBP. Because phage binding to its receptor induces DNA release, it has been postulated that the baseplate might be the trigger for DNA injection. We embarked on a structural study of the lactococcal phages baseplate, ultimately to gain insight into the triggering mechanism following receptor binding. Structural features of the Tuc2009 baseplate were established using size exclusion chromatography coupled to on-line UV-visible absorbance, light scattering, and refractive index detection (MALS/UV/RI). Combining the results of this approach with literature data led us to propose a "low resolution" model of Tuc2009 baseplate. This model will serve as a knowledge base to submit relevant complexes to crystallization trials.     

Crystal Structure of ORF12 from Lactococcus lactis Phage p2 Identifies a Tape Measure Protein Chaperone

We report here the characterization of the nonstructural protein ORF12 of the virulent lactococcal phage p2, which belongs to the Siphoviridae family. ORF12 was produced as a soluble protein, which forms large oligomers (6- to 15-mers) in solution. Using anti-ORF12 antibodies, we have confirmed that ORF12 is not found in the virion structure but is detected in the second half of the lytic cycle, indicating that it is a late-expressed protein. The structure of ORF12, solved by single anomalous diffraction and refined at 2.9-Å resolution, revealed a previously unknown fold as well as the presence of a hydrophobic patch at its surface. Furthermore, crystal packing of ORF12 formed long spirals in which a hydrophobic, continuous crevice was identified. This crevice exhibited a repeated motif of aromatic residues, which coincided with the same repeated motif usually found in tape measure protein (TMP), predicted to form helices. A model of a complex between ORF12 and a repeated motif of the TMP of phage p2 (ORF14) was generated, in which the TMP helix fitted exquisitely in the crevice and the aromatic patches of ORF12. We suggest, therefore, that ORF12 might act as a chaperone for TMP hydrophobic repeats, maintaining TMP in solution during the tail assembly of the lactococcal siphophage p2.During industrial milk fermentation, Lactococcus lactis cells are added to transform milk into an array of fermented products such as cheese. However, this manufacturing process may be impaired by lytic phages present in the factory environment as well as in the milk itself (30). Due to the destructive effects of phage infections on bacterial fermentation, much effort has been undertaken to isolate and study the biodiversity of these bacteriophages. Lactococcal bacteriophages belong to at least 10 different genetically distinct species of double-stranded DNA viruses (9). Of them, three lactococcal phage species, all belonging to the Siphoviridae family, are the major source of problems in milk fermentation, namely, the 936, P335, and c2 species (7, 28, 29). Furthermore, members of the 936 species are by far responsible for the majority of infections (50 to 80%) (1, 24, 41). Numerous phages of the 936 species have been isolated, and several have been characterized at the genome level (25). However, little is known concerning their molecular mechanisms of infection, although we recently solved the structure of the receptor-binding protein (RBP) of our model 936-like phage, namely, the virulent phage p2 (38, 43), and of phages belonging to the P335 species (27, 34, 37, 38).As with all viruses, bacteriophage genomes are quite compact, leaving little room for noncoding sequences (4). In fact, phage genes are disposed in an operon-type organization (4), and the order of genes corresponds to the different phases of the infection cycle. Moreover, genes are often in clusters (referred to as modules), with gene products from adjacent genes generally found to interact with each other. Interestingly, phage genome organization, including individual gene order, is often conserved within a given species, particularly within the Siphoviridae family. In the case of L. lactis virulent phages belonging to the 936 or P335 species, this principle applies particularly to the morphogenesis gene module, which includes all the genes coding for the phage structural protein genes. For the tail assembly, a module comprises a set of genes between the portal protein, which is connecting the tail to the capsid, and the RBP, which is located at the tip of the tail and is involved in host recognition (39, 43).The characterization of tail assembly genes of lactococcal phages has been more extensive for temperate siphophages belonging to the P335 species (27, 34, 37, 38). Because of the similarities in genome organization, the findings in this phage species can, in some cases, be used as clues toward understanding the morphology of 936-like phages. For the temperate phage Tuc2009 (P335 species), all structural proteins required for tail and baseplate assembly have been identified (27, 34, 37, 38). Genes located between those coding for the tape measure protein (TMP) and BppL (RBP) were identified as corresponding to components of the baseplate structure, located at the tail distal end. Furthermore, a gene coding for the major tail protein (MTP) was also identified at a position upstream from tmp. Between the genes coding for the MTP and those coding for the TMP in Tuc2009 are two gene products identified as gpG and gpGT, which are not present in the phage particle. These two proteins were named based on their likely role analogous to the tail assembly proteins present in coliphage lambda, a model virus belonging to the Siphoviridae family (21, 27, 47). gpGT has an essential role in lambda tail assembly, acting prior to tail shaft assembly, while the role of gpG in tail assembly is not known (21). Both gpG and gpGT are also absent from mature lambda virions (21). It has been argued that they may act as assembly chaperones (47).A close examination of 936 genomes indicates the presence of two genes coding for gpG and gpGT-like proteins. Analysis of the phage p2 genome, closely related to that of lactococcal phage sk1 (6), revealed that the putative tail assembly proteins could correspond to gene products ORF12 and ORF13. These two genes are followed by the TMP gene corresponding to orf14, other genes coding for other structural proteins, and the RBP gene orf18. During our ongoing investigation of the structure of phage p2, we report here the cloning, expression, and crystal structure of ORF12 in order to decipher its role in the tail assembly process.