Biophysical Studies of the Membrane-Embedded and Cytoplasmic Forms of the Glucose-Specific Enzyme II of the E. coli Phosphotransferase System (PTS)

The glucose Enzyme II transporter complex of the Escherichia coli phosphotransferase system (PTS) exists in at least two physically distinct forms: a membrane-integrated dimeric form, and a cytoplasmic monomeric form, but little is known about the physical states of these enzyme forms. Six approaches were used to evaluate protein-protein and protein-lipid interactions in this system. Fluorescence energy transfer (FRET) using MBP-IIGlc-YFP and MBP-IIGlc-CFP revealed that the homodimeric Enzyme II complex in cell membranes is stable (FRET-) but can be dissociated and reassociated to the heterodimer only in the presence of Triton X100 (FRET+). The monomeric species could form a heterodimeric species (FRET+) by incubation and purification without detergent exposure. Formaldehyde cross linking studies, conducted both in vivo and in vitro, revealed that the dimeric MBP-IIGlc activity decreased dramatically with increasing formaldehyde concentrations due to both aggregation and activity loss, but that the monomeric MBP-IIGlc retained activity more effectively in response to the same formaldehyde treatments, and little or no aggregation was observed. Electron microscopy of MBP-IIGlc indicated that the dimeric form is larger than the monomeric form. Dynamic light scattering confirmed this conclusion and provided quantitation. NMR analyses provided strong evidence that the dimeric form is present primarily in a lipid bilayer while the monomeric form is present as micelles. Finally, lipid analyses of the different fractions revealed that the three lipid species (PE, PG and CL) are present in all fractions, but the monomeric micellar structure contains a higher percentage of anionic lipids (PG & CL) while the dimeric bilayer form has a higher percentage of zwitterion lipids (PE). Additionally, evidence for a minor dimeric micellar species, possibly an intermediate between the monomeric micellar and the dimeric bilayer forms, is presented. These results provide convincing evidence for interconvertible physical forms of Enzyme-IIGlc

Defense Against Cannibalism: The SdpI Family of Bacterial Immunity/Signal Transduction Proteins

The SdpI family consists of putative bacterial toxin immunity and signal transduction proteins. One member of the family in Bacillus subtilis, SdpI, provides immunity to cells from cannibalism in times of nutrient limitation. SdpI family members are transmembrane proteins with 3, 4, 5, 6, 7, 8, or 12 putative transmembrane α-helical segments (TMSs). These varied topologies appear to be genuine rather than artifacts due to sequencing or annotation errors. The basic and most frequently occurring element of the SdpI family has 6 TMSs. Homologues of all topological types were aligned to determine the homologous TMSs and loop regions, and the positive-inside rule was used to determine sidedness. The two most conserved motifs were identified between TMSs 1 and 2 and TMSs 4 and 5 of the 6 TMS proteins. These showed significant sequence similarity, leading us to suggest that the primordial precursor of these proteins was a 3 TMS–encoding genetic element that underwent intragenic duplication. Various deletional and fusional events, as well as intragenic duplications and inversions, may have yielded SdpI homologues with topologies of varying numbers and positions of TMSs. We propose a specific evolutionary pathway that could have given rise to these distantly related bacterial immunity proteins. We further show that genes encoding SdpI homologues often appear in operons with genes for homologues of SdpR, SdpI’s autorepressor. Our analyses allow us to propose structure–function relationships that may be applicable to most family members

The Bacterial Intimins and Invasins: A Large and Novel Family of Secreted Proteins

Gram-negative bacteria have developed a limited repertoire of solutions for secreting proteins from the cytoplasmic compartment to the exterior of the cell. Amongst the spectrum of secreted proteins are the intimins and invasins (the Int/Inv family; TC# 1.B.54) which are characterized by an N-terminal β-barrel domain and a C-terminal surface localized passenger domain. Despite the important role played by members of this family in diseases mediated by several species of the Enterobacteriaceae, there has been little appreciation for the distribution and diversity of these proteins amongst Gram-negative bacteria. Furthermore, there is little understanding of the molecular events governing secretion of these proteins to the extracellular milieu.In silico approaches were used to analyze the domain organization and diversity of members of this secretion family. Proteins belonging to this family are predominantly associated with organisms from the γ-proteobacteria. Whilst proteins from the Chlamydia, γ-, β- and ε-proteobacteria possess β-barrel domains and passenger domains of various sizes, Int/Inv proteins from the α-proteobacteria, cyanobacteria and chlorobi possess only the predicted β-barrel domains. Phylogenetic analyses revealed that with few exceptions these proteins cluster according to organismal type, indicating that divergence occurred contemporaneously with speciation, and that horizontal transfer was limited. Clustering patterns of the β-barrel domains correlate well with those of the full-length proteins although the passenger domains do so with much less consistency. The modular subdomain design of the passenger domains suggests that subdomain duplication and deletion have occurred with high frequency over evolutionary time. However, all repeated subdomains are found in tandem, suggesting that subdomain shuffling occurred rarely if at all. Topological predictions for the β-barrel domains are presented.Based on our in silico analyses we present a model for the biogenesis of these proteins. This study is the first of its kind to describe this unusual family of bacterial adhesins

Domain-Based Identification and Analysis of Glutamate Receptor Ion Channels and Their Relatives in Prokaryotes

Voltage-gated and ligand-gated ion channels are used in eukaryotic organisms for the purpose of electrochemical signaling. There are prokaryotic homologues to major eukaryotic channels of these sorts, including voltage-gated sodium, potassium, and calcium channels, Ach-receptor and glutamate-receptor channels. The prokaryotic homologues have been less well characterized functionally than their eukaryotic counterparts. In this study we identify likely prokaryotic functional counterparts of eukaryotic glutamate receptor channels by comprehensive analysis of the prokaryotic sequences in the context of known functional domains present in the eukaryotic members of this family. In particular, we searched the nonredundant protein database for all proteins containing the following motif: the two sections of the extracellular glutamate binding domain flanking two transmembrane helices. We discovered 100 prokaryotic sequences containing this motif, with a wide variety of functional annotations. Two groups within this family have the same topology as eukaryotic glutamate receptor channels. Group 1 has a potassium-like selectivity filter. Group 2 is most closely related to eukaryotic glutamate receptor channels. We present analysis of the functional domain architecture for the group of 100, a putative phylogenetic tree, comparison of the protein phylogeny with the corresponding species phylogeny, consideration of the distribution of these proteins among classes of prokaryotes, and orthologous relationships between prokaryotic and human glutamate receptor channels. We introduce a construct called the Evolutionary Domain Network, which represents a putative pathway of domain rearrangements underlying the domain composition of present channels. We believe that scientists interested in ion channels in general, and ligand-gated ion channels in particular, will be interested in this work. The work should also be of interest to bioinformatics researchers who are interested in the use of functional domain-based analysis in evolutionary and functional discovery

Global Proteome Analysis of Leptospira interrogans

Comparative global proteome analyses were performed on Leptospira interrogans serovar Copenhageni grown under conventional in vitro conditions and those mimicking in vivo conditions (iron limitation and serum presence). Proteomic analyses were conducted using iTRAQ and LC-ESI-tandem mass spectrometry complemented with two-dimensional gel electrophoresis and MALDI-TOF mass spec-trometry. A total of 563 proteins were identified in this study. Altered expression of 65 proteins, including upregulation of the L. interrogans virulence factor Loa22 and 5 novel proteins with homology to virulence factors found in other pathogens, was observed between the comparative conditions. Immunoblot analyses confirmed upregulation of 5 of the known or putative virulence factors in L. interrogans exposed to the in vivo-like environmental conditions. Further, ELISA analyses using serum from patients with leptospirosis and immunofluorescence studies performed on liver sections derived from L. interrogans-infected hamsters verified expression of all but one of the identified proteins during infection. These studies, which represent the first documented comparative global proteome analysis of Leptospira, demonstrated proteome alterations under conditions that mimic in vivo infection and allowed for the identification of novel putative L. interrogans virulence factors

Genome of the Avirulent Human-Infective Trypanosome—Trypanosoma rangeli

Background: Trypanosoma rangeli is a hemoflagellate protozoan parasite infecting humans and other wild and domestic mammals across Central and South America. It does not cause human disease, but it can be mistaken for the etiologic agent of Chagas disease, Trypanosoma cruzi. We have sequenced the T. rangeli genome to provide new tools for elucidating the distinct and intriguing biology of this species and the key pathways related to interaction with its arthropod and mammalian hosts.  Methodology/Principal Findings: The T. rangeli haploid genome is ,24 Mb in length, and is the smallest and least repetitive trypanosomatid genome sequenced thus far. This parasite genome has shorter subtelomeric sequences compared to those of T. cruzi and T. brucei; displays intraspecific karyotype variability and lacks minichromosomes. Of the predicted 7,613 protein coding sequences, functional annotations could be determined for 2,415, while 5,043 are hypothetical proteins, some with evidence of protein expression. 7,101 genes (93%) are shared with other trypanosomatids that infect humans. An ortholog of the dcl2 gene involved in the T. brucei RNAi pathway was found in T. rangeli, but the RNAi machinery is non-functional since the other genes in this pathway are pseudogenized. T. rangeli is highly susceptible to oxidative stress, a phenotype that may be explained by a smaller number of anti-oxidant defense enzymes and heatshock proteins.  Conclusions/Significance: Phylogenetic comparison of nuclear and mitochondrial genes indicates that T. rangeli and T. cruzi are equidistant from T. brucei. In addition to revealing new aspects of trypanosome co-evolution within the vertebrate and invertebrate hosts, comparative genomic analysis with pathogenic trypanosomatids provides valuable new information that can be further explored with the aim of developing better diagnostic tools and/or therapeutic targets

Substrate binding and translocation of the serotonin transporter studied by docking and molecular dynamics simulations

The serotonin (5-HT) transporter (SERT) plays an important role in the termination of 5-HT-mediated neurotransmission by transporting 5-HT away from the synaptic cleft and into the presynaptic neuron. In addition, SERT is the main target for antidepressant drugs, including the selective serotonin reuptake inhibitors (SSRIs). The three-dimensional (3D) structure of SERT has not yet been determined, and little is known about the molecular mechanisms of substrate binding and transport, though such information is very important for the development of new antidepressant drugs. In this study, a homology model of SERT was constructed based on the 3D structure of a prokaryotic homologous leucine transporter (LeuT) (PDB id: 2A65). Eleven tryptamine derivates (including 5-HT) and the SSRI (S)-citalopram were docked into the putative substrate binding site, and two possible binding modes of the ligands were found. To study the conformational effect that ligand binding may have on SERT, two SERT–5-HT and two SERT–(S)-citalopram complexes, as well as the SERT apo structure, were embedded in POPC lipid bilayers and comparative molecular dynamics (MD) simulations were performed. Our results show that 5-HT in the SERT–5-HTB complex induced larger conformational changes in the cytoplasmic parts of the transmembrane helices of SERT than any of the other ligands. Based on these results, we suggest that the formation and breakage of ionic interactions with amino acids in transmembrane helices 6 and 8 and intracellular loop 1 may be of importance for substrate translocation

Incorporating Genomics and Bioinformatics across the Life Sciences Curriculum

Undergraduate life sciences education needs an overhaul, as clearly described in the National Research Council of the National Academies’ publication BIO 2010: Transforming Undergraduate Education for Future Research Biologists. Among BIO 2010’s top recommendations is the need to involve students in working with real data and tools that reflect the nature of life sciences research in the 21st century [1]. Education research studies support the importance of utilizing primary literature, designing and implementing experiments, and analyzing results in the context of a bona fide scientific question [1–12] in cultivating the analytical skills necessary to become a scientist. Incorporating these basic scientific methodologies in undergraduate education leads to increased undergraduate and post-graduate retention in the sciences [13–16]. Toward this end, many undergraduate teaching organizations offer training and suggestions for faculty to update and improve their teaching approaches to help students learn as scientists, through design and discovery (e.g., Council of Undergraduate Research [www.cur.org] and Project Kaleidoscope [ www.pkal.org])

Structural basis for Mep2 ammonium transceptor activation by phosphorylation

Mep2 proteins are fungal transceptors that play an important role as ammonium sensors in fungal development. Mep2 activity is tightly regulated by phosphorylation, but how this is achieved at the molecular level is not clear. Here we report X-ray crystal structures of the Mep2 orthologues from Saccharomyces cerevisiae and Candida albicans and show that under nitrogen-sufficient conditions the transporters are not phosphorylated and present in closed, inactive conformations. Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif. The phosphorylation site in the CTR is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. The crystal structure of phosphorylation-mimicking Mep2 variants from C. albicans show large conformational changes in a conserved and functionally important region of the CTR. The results allow us to propose a model for regulation of eukaryotic ammonium transport by phosphorylation