The First Structure of a Lantibiotic Immunity Protein,SpaI from Bacillus subtilis,Reveals a Novel Fold

Lantibiotics are peptide-derived antibiotics that inhibit the growth of Gram-positive bacteria via interactions with lipid II and lipid II-dependent pore formation in the bacterial membrane. Due to their general mode of action the Gram-positive producer strains need to express immunity proteins (LanI proteins) for protection against their own lantibiotics. Little is known about the immunity mechanism protecting the producer strain against its own lantibiotic on the molecular level. So far, no structures have been reported for any LanI protein. We solved the structure of SpaI, a LanI protein from the subtilin producing strain Bacillus subtilis ATCC 6633. SpaI is a 16.8-kDa lipoprotein that is attached to the outside of the cytoplasmic membrane via a covalent diacylglycerol anchor. SpaI together with the ABC transporter SpaFEG protects the B. subtilis membrane from subtilin insertion. The solution-NMR structure of a 15-kDa biologically active C-terminal fragment reveals a novel fold. We also demonstrate that the first 20 N-terminal amino acids not present in this C-terminal fragment are unstructured in solution and are required for interactions with lipid membranes. Additionally, growth tests reveal that these 20 N-terminal residues are important for the immunity mediated by SpaI but most likely are not part of a possible subtilin binding site. Our findings are the first step on the way of understanding the immunity mechanism of B. subtilis in particular and of other lantibiotic producing strains in general.     

Solution Structure of the Oncogenic MIEN1 Protein Reveals a Thioredoxin-Like Fold with a Redox-Active Motif

The novel tumor biomarker MIEN1, identified by representational difference analysis, is overexpressed in breast cancer and prostate cancer. MIEN1 is considered an oncogenic protein, because MIEN1 overexpression functionally enhances migration and invasion of tumor cells via modulating the activity of AKT. However, the structure and molecular function of MIEN1 is little understood. Here, we report the solution structure of MIEN1, which adopts a thioredoxin-like fold with a redox-active motif. Comparison of backbone chemical shifts showed that most of the residues for both oxidized and reduced MIEN1 possessed the same backbone conformation, with differences limited to the active motif and regions in proximity. The redox potential of this disulfide bond was measured as −225 mV, which compares well with that of disulfides for other thioredoxin-like proteins. Overall, our results suggest that MIEN1 may have an important regulatory role in phosphorylation of AKT with its redox potential.     

An Atlas of the Thioredoxin Fold Class Reveals the Complexity of Function-Enabling Adaptations

The group of proteins that contain a thioredoxin (Trx) fold is huge and diverse. Assessment of the variation in catalytic machinery of Trx fold proteins is essential in providing a foundation for understanding their functional diversity and predicting the function of the many uncharacterized members of the class. The proteins of the Trx fold class retain common features—including variations on a dithiol CxxC active site motif—that lead to delivery of function. We use protein similarity networks to guide an analysis of how structural and sequence motifs track with catalytic function and taxonomic categories for 4,082 representative sequences spanning the known superfamilies of the Trx fold. Domain structure in the fold class is varied and modular, with 2.8% of sequences containing more than one Trx fold domain. Most member proteins are bacterial. The fold class exhibits many modifications to the CxxC active site motif—only 56.8% of proteins have both cysteines, and no functional groupings have absolute conservation of the expected catalytic motif. Only a small fraction of Trx fold sequences have been functionally characterized. This work provides a global view of the complex distribution of domains and catalytic machinery throughout the fold class, showing that each superfamily contains remnants of the CxxC active site. The unifying context provided by this work can guide the comparison of members of different Trx fold superfamilies to gain insight about their structure-function relationships, illustrated here with the thioredoxins and peroxiredoxins.     

The Crystal Structure of Cancer Osaka Thyroid Kinase Reveals an Unexpected Kinase Domain Fold

Macrophages are important cellular effectors in innate immune responses and play a major role in autoimmune diseases such as rheumatoid arthritis. Cancer Osaka thyroid (COT) kinase, also known as mitogen-activated protein kinase kinase kinase 8 (MAP3K8) and tumor progression locus 2 (Tpl-2), is a serine-threonine (ST) kinase and is a key regulator in the production of pro-inflammatory cytokines in macrophages. Due to its pivotal role in immune biology, COT kinase has been identified as an attractive target for pharmaceutical research that is directed at the discovery of orally available, selective, and potent inhibitors for the treatment of autoimmune disorders and cancer. The production of monomeric, recombinant COT kinase has proven to be very difficult, and issues with solubility and stability of the enzyme have hampered the discovery and optimization of potent and selective inhibitors. We developed a protocol for the production of recombinant human COT kinase that yields pure and highly active enzyme in sufficient yields for biochemical and structural studies. The quality of the enzyme allowed us to establish a robust in vitro phosphorylation assay for the efficient biochemical characterization of COT kinase inhibitors and to determine the x-ray co-crystal structures of the COT kinase domain in complex with two ATP-binding site inhibitors. The structures presented in this study reveal two distinct ligand binding modes and a unique kinase domain architecture that has not been observed previously. The structurally versatile active site significantly impacts the design of potent, low molecular weight COT kinase inhibitors.     

Solution Structure of IseA,an Inhibitor Protein of dl-Endopeptidases from Bacillus subtilis,Reveals a Novel Fold with a Characteristic Inhibitory Loop

In Bacillus subtilis, LytE, LytF, CwlS, and CwlO are vegetative autolysins, dl-endopeptidases in the NlpC/P60 family, and play essential roles in cell growth and separation. IseA (YoeB) is a proteinaceous inhibitor against the dl-endopeptidases, peptidoglycan hydrolases. Overexpression of IseA caused significantly long chained cell morphology, because IseA inhibits the cell separation dl-endopeptidases post-translationally. Here, we report the first three-dimensional structure of IseA, determined by NMR spectroscopy. The structure includes a single domain consisting of three α-helices, one 3₁₀-helix, and eight β-strands, which is a novel fold like a “hacksaw.” Noteworthy is a dynamic loop between β4 and the 3₁₀-helix, which resembles a “blade.” The electrostatic potential distribution shows that most of the surface is positively charged, but the region around the loop is negatively charged. In contrast, the LytF active-site cleft is expected to be positively charged. NMR chemical shift perturbation of IseA interacting with LytF indicated that potential interaction sites are located around the loop. Furthermore, the IseA mutants D100K/D102K and G99P/G101P at the loop showed dramatic loss of inhibition activity against LytF, compared with wild-type IseA, indicating that the β4–3₁₀ loop plays an important role in inhibition. Moreover, we built a complex structure model of IseA-LytF by docking simulation, suggesting that the β4–3₁₀ loop of IseA gets stuck deep in the cleft of LytF, and the active site is occluded. These results suggest a novel inhibition mechanism of the hacksaw-like structure, which is different from known inhibitor proteins, through interactions around the characteristic loop regions with the active-site cleft of enzymes.     

The Class III Cyclobutane Pyrimidine Dimer Photolyase Structure Reveals a New Antenna Chromophore Binding Site and Alternative Photoreduction Pathways

Photolyases are proteins with an FAD chromophore that repair UV-induced pyrimidine dimers on the DNA in a light-dependent manner. The cyclobutane pyrimidine dimer class III photolyases are structurally unknown but closely related to plant cryptochromes, which serve as blue-light photoreceptors. Here we present the crystal structure of a class III photolyase termed photolyase-related protein A (PhrA) of Agrobacterium tumefaciens at 1.67-Å resolution. PhrA contains 5,10-methenyltetrahydrofolate (MTHF) as an antenna chromophore with a unique binding site and mode. Two Trp residues play pivotal roles for stabilizing MTHF by a double π-stacking sandwich. Plant cryptochrome I forms a pocket at the same site that could accommodate MTHF or a similar molecule. The PhrA structure and mutant studies showed that electrons flow during FAD photoreduction proceeds via two Trp triads. The structural studies on PhrA give a clearer picture on the evolutionary transition from photolyase to photoreceptor.     

The Structure of Mouse Cytomegalovirus m04 Protein Obtained from Sparse NMR Data Reveals a Conserved Fold of the m02-m06 Viral Immune Modulator Family

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Crystal Structure of ATVORF273, a New Fold for a Thermo- and Acido-Stable Protein from the Acidianus Two-Tailed Virus

Acidianus two-tailed virus (ATV) infects crenarchaea of the genus Acidianus living in terrestrial thermal springs at extremely high temperatures and low pH. ATV is a member of the Bicaudaviridae virus family and undergoes extra-cellular development of two tails, a process that is unique in the viral world. To understand this intriguing phenomenon, we have undertaken structural studies of ATV virion proteins and here we present the crystal structure of one of these proteins, ATV. ATV forms tetramers in solution and a molecular envelope is provided for the tetramer, computed from small-angle X-ray scattering (SAXS) data. The crystal structure has properties typical of hyperthermostable proteins, including a relatively high number of salt bridges. However, the protein also exhibits flexible loops and surface pockets. Remarkably, ATV displays a new protein fold, consistent with the absence of homologues of this protein in public sequence databases.     

The Caenorhabditis elegans bus-2 Mutant Reveals a New Class of O-Glycans Affecting Bacterial Resistance

Microbacterium nematophilum causes a deleterious infection of the C. elegans hindgut initiated by adhesion to rectal and anal cuticle. C. elegans bus-2 mutants, which are resistant to M. nematophilum and also to the formation of surface biofilms by Yersinia sp., carry genetic lesions in a putative glycosyltransferase containing conserved domains of core-1 β1,3-galactosyltransferases. bus-2 is predicted to act in the synthesis of core-1 type O-glycans. This observation implies that the infection requires the presence of host core-1 O-glycoconjugates and is therefore carbohydrate-dependent. Chemical analysis reported here reveals that bus-2 is indeed deficient in core-1 O-glycans. These mutants also exhibit a new subclass of O-glycans whose structures were determined by high performance tandem mass spectrometry; these are highly fucosylated and have a novel core that contains internally linked GlcA. Lectin studies showed that core-1 glycans and this novel class of O-glycans are both expressed in the tissue that is infected in the wild type worms. In worms having the bus-2 genetic background, core-1 glycans are decreased, whereas the novel fucosyl O-glycans are increased in abundance in this region. Expression analysis using a red fluorescent protein marker showed that bus-2 is expressed in the posterior gut, cuticle seam cells, and spermatheca, the first two of which are likely to be involved in secreting the carbohydrate-rich surface coat of the cuticle. Therefore, in the bus-2 background of reduced core-1 O-glycans, the novel fucosyl glycans likely replace or mask remaining core-1 ligands, leading to the resistance phenotype. There are more than 35 Microbacterium species, some of which are pathogenic in man. This study is the first to analyze the biochemistry of adhesion to a host tissue by a Microbacterium species.     

The Structure of the Poxvirus A33 Protein Reveals a Dimer of Unique C-Type Lectin-Like Domains

The current vaccine against smallpox is an infectious form of vaccinia virus that has significant side effects. Alternative vaccine approaches using recombinant viral proteins are being developed. A target of subunit vaccine strategies is the poxvirus protein A33, a conserved protein in the Chordopoxvirinae subfamily of Poxviridae that is expressed on the outer viral envelope. Here we have determined the structure of the A33 ectodomain of vaccinia virus. The structure revealed C-type lectin-like domains (CTLDs) that occur as dimers in A33 crystals with five different crystal lattices. Comparison of the A33 dimer models shows that the A33 monomers have a degree of flexibility in position within the dimer. Structural comparisons show that the A33 monomer is a close match to the Link module class of CTLDs but that the A33 dimer is most similar to the natural killer (NK)-cell receptor class of CTLDs. Structural data on Link modules and NK-cell receptor-ligand complexes suggest a surface of A33 that could interact with viral or host ligands. The dimer interface is well conserved in all known A33 sequences, indicating an important role for the A33 dimer. The structure indicates how previously described A33 mutations disrupt protein folding and locates the positions of N-linked glycosylations and the epitope of a protective antibody.Poxviruses are large DNA viruses that infect a wide range of hosts. The smallpox virus devastated human populations until its eradication 3 decades ago. Other poxviruses are emerging, such as monkeypox virus, which also infects humans and causes disease (61). The smallpox vaccine is a model of vaccine efficacy, but how the vaccine induces protection is not well understood. Knowledge of how the vaccine produces protection will also likely be important for efforts to produce vaccines that are effective against other pathogens. Though highly successful in the population overall, the smallpox vaccine uses an infectious strain of vaccinia virus and has a significant level of serious side effects (6). One focus of current research is to develop vaccines using recombinant poxvirus proteins that are as protective as the live virus vaccine but produce fewer complications. A more detailed structural characterization of the protein antigens that are important for conferring protection will improve our knowledge of how the smallpox vaccine works and lead to a better understanding of poxvirus biology.There are two morphologically distinct forms of poxviruses: the mature virion (MV) and the enveloped virion (EV) (53). The MV, also known as the intracellular mature virion (IMV), is found inside the infected cell (62, 65). Enveloped virions are formed from MVs that have been wrapped by modified Golgi or endosomal membranes (53). MVs are thought to be responsible for host-to-host proliferation of the virus, while the EVs mediate virus spread within a host (40, 49). EVs that are attached to the cell surface, also termed cell-associated enveloped virions (CEV), are thought to propagate viral infection to neighboring cells (65). EVs that are released from the cell surface, also termed extracellular enveloped virus (EEV), mediate longer-range dissemination in the host (65). The outer membranes of the MV and EV forms each have a distinct assemblage of proteins. Candidate subunit vaccines have been shown to require proteins from both virus forms to be most effective (17, 27, 28).A33 is a type II integral membrane glycoprotein found on the surface of the EV form of the virus and is also expressed on the host cell membrane (62). A33 is a disulfide-bonded homodimer with both N- and O-linked glycosylation (57, 62). Deletion of the A33R gene in vaccinia virus results in a small-plaque phenotype, defects in actin tail formation, and inefficient cell-to-cell spread in cell culture (63). Evidence implicates A33 in the spreading of virus from cell to cell by a mechanism that is antibody resistant (40). Antibodies against A33 in cell culture prevent the formation of comet-shaped viral plaques, which are assayed in an overlay of plaques with liquid and are indicative of cell-to-cell spreading of the EV (2, 17). A33 has been shown to interact through its cytoplasmic and transmembrane regions with the EV proteins A36 (18, 64, 72, 76) and B5 (58, 64). Vaccination with A33 is protective in a number of animal models of poxvirus infection as a component of protein subunit vaccination (16, 17, 19, 77), DNA vaccination (19, 27-29), or a combination of the two methods (24). Despite the inclusion of A33 in vaccination studies, the functions of A33 in the virus are unclear.Members of the C-type lectin-like domain (CTLD) superfamily of proteins are found in organisms ranging from bacteria to humans (13, 78). The first crystal structures of carbohydrate-binding domains with this fold gave rise to the name “lectin” for the family, but many family members do not bind carbohydrates. The classification of a domain as being C type lectin-like is currently based on similarities in protein sequence and fold (74). CTLDs have been shown to bind noncarbohydrate small molecules, lipids, proteins, and other structures, such as ice (13, 78). One group of type II transmembrane proteins that contain CTLDs is comprised of the natural killer (NK)-cell receptors of the innate immune system (78). The NK-cell receptors are composed of dimers of CTLDs, which are the only dimers out of the several hundred C-type lectin-like structures that are known. Protein binding by NK-cell receptors occurs on a surface formed by the “long loop” and nearby residues that are on the opposite side of the dimer from the N and C termini (60). A second group of proteins contains a monomeric CTLD, termed a “Link module” CTLD, that binds glycosaminoglycans but lacks the “long loop” region that is present in almost all other CTLDs (78).To gain a better understanding of the structure and possible functions of A33 and to further its development in vaccines, we determined the X-ray crystal structure of the A33 ectodomain from vaccinia virus. Based on the structure and sequence of A33, the carbohydrate-binding site of the canonical CTLD is not present. The structure revealed A33 to have dimers of CTLDs. Comparison of A33 with other CTLDs, including dimers from NK-cell receptors and monomers from Link modules, indicates that A33 contains an unusual CTLD that likely binds ligands of host or virus origins.     

Structure of an Unfolding Intermediate of an RRM Domain of ETR-3 Reveals Its Native-like Fold

Prevalence of one or more partially folded intermediates during protein unfolding with different secondary and ternary conformations has been identified as an integral character of protein unfolding. These transition-state species need to be characterized structurally for elucidation of their folding pathways. We have determined the three-dimensional structure of an intermediate state with increased conformational space sampling under urea-denaturing condition. The protein unfolds completely at 10 M urea but retains residual secondary structural propensities with restricted motion. Here, we describe the native state, observable intermediate state, and unfolded state for ETR-3 RRM-3, which has canonical RRM fold. These observations can shed more light on unfolding events for RRM-containing proteins.     

Structural Analysis of Semi-specific Oligosaccharide Recognition by a Cellulose-binding Protein of Thermotoga maritima Reveals Adaptations for Functional Diversification of the Oligopeptide Periplasmic Binding Protein Fold

Periplasmic binding proteins (PBPs) constitute a protein superfamily that binds a wide variety of ligands. In prokaryotes, PBPs function as receptors for ATP-binding cassette or tripartite ATP-independent transporters and chemotaxis systems. In many instances, PBPs bind their cognate ligands with exquisite specificity, distinguishing, for example, between sugar epimers or structurally similar anions. By contrast, oligopeptide-binding proteins bind their ligands through interactions with the peptide backbone but do not distinguish between different side chains. The extremophile Thermotoga maritima possesses a remarkable array of carbohydrate-processing metabolic systems, including the hydrolysis of cellulosic polymers. Here, we present the crystal structure of a T. maritima cellobiose-binding protein (tm0031) that is homologous to oligopeptide-binding proteins. T. maritima cellobiose-binding protein binds a variety of lengths of β(1→4)-linked glucose oligomers, ranging from two rings (cellobiose) to five (cellopentaose). The structure reveals that binding is semi-specific. The disaccharide at the nonreducing end binds specifically; the other rings are located in a large solvent-filled groove, where the reducing end makes several contacts with the protein, thereby imposing an upper limit of the oligosaccharides that are recognized. Semi-specific recognition, in which a molecular class rather than individual species is selected, provides an efficient solution for the uptake of complex mixtures.     

Random Field Model Reveals Structure of the Protein Recombinational Landscape

We are interested in how intragenic recombination contributes to the evolution of proteins and how this mechanism complements and enhances the diversity generated by random mutation. Experiments have revealed that proteins are highly tolerant to recombination with homologous sequences (mutation by recombination is conservative); more surprisingly, they have also shown that homologous sequence fragments make largely additive contributions to biophysical properties such as stability. Here, we develop a random field model to describe the statistical features of the subset of protein space accessible by recombination, which we refer to as the recombinational landscape. This model shows quantitative agreement with experimental results compiled from eight libraries of proteins that were generated by recombining gene fragments from homologous proteins. The model reveals a recombinational landscape that is highly enriched in functional sequences, with properties dominated by a large-scale additive structure. It also quantifies the relative contributions of parent sequence identity, crossover locations, and protein fold to the tolerance of proteins to recombination. Intragenic recombination explores a unique subset of sequence space that promotes rapid molecular diversification and functional adaptation.     

Crystal Structure of a Mucus-binding Protein Repeat Reveals an Unexpected Functional Immunoglobulin Binding Activity

Lactobacillus reuteri mucus-binding protein (MUB) is a cell-surface protein that is involved in bacterial interaction with mucus and colonization of the digestive tract. The 353-kDa mature protein is representative of a broadly important class of adhesins that have remained relatively poorly characterized due to their large size and highly modular nature. MUB contains two different types of repeats (Mub1 and Mub2) present in six and eight copies, respectively, and shown to be responsible for the adherence to intestinal mucus. Here we report the 1.8-Å resolution crystal structure of a type 2 Mub repeat (184 amino acids) comprising two structurally related domains resembling the functional repeat found in a family of immunoglobulin (Ig)-binding proteins. The N-terminal domain bears striking structural similarity to the repeat unit of Protein L (PpL) from Peptostreptococcus magnus, suggesting binding in a non-immune Fab-dependent manner. A distorted PpL-like fold is also seen in the C-terminal domain. As with PpL, Mub repeats were able to interact in vitro with a large repertoire of mammalian Igs, including secretory IgA. This hitherto undetected activity is consistent with the current model that antibody responses against commensal flora are of broad specificity and low affinity.     

Crystal Structure of a Mammalian CTP: Phosphocholine Cytidylyltransferase Catalytic Domain Reveals Novel Active Site Residues within a Highly Conserved Nucleotidyltransferase Fold

CTP:phosphocholine cytidylyltransferase (CCT) is the key regulatory enzyme in the synthesis of phosphatidylcholine, the most abundant phospholipid in eukaryotic cell membranes. The CCT-catalyzed transfer of a cytidylyl group from CTP to phosphocholine to form CDP-choline is regulated by a membrane lipid-dependent mechanism imparted by its C-terminal membrane binding domain. We present the first analysis of a crystal structure of a eukaryotic CCT. A deletion construct of rat CCTα spanning residues 1–236 (CCT236) lacks the regulatory domain and as a result displays constitutive activity. The 2.2-Å structure reveals a CCT236 homodimer in complex with the reaction product, CDP-choline. Each chain is composed of a complete catalytic domain with an intimately associated N-terminal extension, which together with the catalytic domain contributes to the dimer interface. Although the CCT236 structure reveals elements involved in binding cytidine that are conserved with other members of the cytidylyltransferase superfamily, it also features nonconserved active site residues, His-168 and Tyr-173, that make key interactions with the β-phosphate of CDP-choline. Mutagenesis and kinetic analyses confirmed their role in phosphocholine binding and catalysis. These results demonstrate structural and mechanistic differences in a broadly conserved protein fold across the cytidylyltransferase family. Comparison of the CCT236 structure with those of other nucleotidyltransferases provides evidence for substrate-induced active site loop movements and a disorder-to-order transition of a loop element in the catalytic mechanism.