Transmembrane Helical Domains Research Articles

Structural Insights into the Active Site of Human Sodium Dependent Glucose Co-Transporter 2: Homology Modelling, Molecular Docking, and 3D - QSAR Studies

Human sodium dependent glucose co-transporter 2 (hSGLT2) is a target for diabetes mellitus type 2 (T2DM). The 3D (three dimensional) homology model of hSGLT2 comprising 14 transmembrane helical domains was constructed and molecular docking of the inhibitors, C-aryl glucoside analogues, into the active site was studied. The 3D-QSAR (quantitative structure activity relationship) analysis was carried out on 43 C-aryl glucoside analogues as a training set. The molecular field analysis (MFA) with G/PLS (genetic partial least-squares) method was used to generate statistically significant 3D-QSAR (r2 = 0.857) based on a molecular field generated using electrostatic and steric probes. The QSAR model was validated using leave-one-out cross-validation, bootstrapping, and randomisation methods, and finally with an external test set comprising 10 inhibitors. The molecular docking studies provide structural insights into the active site and key interactions involved in the binding of inhibitors to hSGLT2 and these results corroborate with the 3D-QSAR analysis that provide the active conformation of inhibitors and the nature of interactive fields important for activity.

Bacterial proteins with cleaved or uncleaved signal peptides of the general secretory pathway

Correct protein compartmentalization is a key step for molecular function and cell viability, and this is especially true for membrane and externalized proteins of bacteria. Recent proteomic reports of Bacillus subtilis have shown that many proteins with Sec-like signal peptides and absence of a transmembrane helix domain are still observed in membrane-enriched fractions, but further evidence about signal peptide cleavage or soluble protein contamination is still needed. Here we report a proteomic screening of identified peptides in culture filtrate, membrane fraction and whole cell lysate of Mycobacterium tuberculosis. We were able to detect peptide sequencing evidence that shows that the predicted signal peptide was kept uncleaved for several types of proteins such as mammalian cell entry (Mce) proteins and PE or PE-PGRS proteins. Label-free quantitation of all proteins identified in each fraction showed that the majority of these proteins with uncleaved signal peptides are, indeed, enriched in the Triton X-114 lipid phase. Some of these proteins are likely to be located in the inner membrane while others may be outer membrane proteins.

A new in-silico method for determination of helical transmembrane domains based on the PepLook scan: application to IL-2Rβ and IL-2Rγc receptor chains

BackgroundModeling of transmembrane domains (TMDs) requires correct prediction of interfacial residues for in-silico modeling and membrane insertion studies. This implies the defining of a target sequence long enough to contain interfacial residues. However, too long sequences induce artifactual polymorphism: within tested modeling methods, the longer the target sequence, the more variable the secondary structure, as though the procedure were stopped before the end of the calculation (which may in fact be unreachable). Moreover, delimitation of these TMDs can produce variable results with sequence based two-dimensional prediction methods, especially for sequences showing polymorphism. To solve this problem, we developed a new modeling procedure using the PepLook method. We scanned the sequences by modeling peptides from the target sequence with a window of 19 residues.ResultsUsing sequences whose NMR-structures are already known (GpA, EphA1 and Erb2-HER2), we first determined that the hydrophobic to hydrophilic accessible surface area ratio (ASAr) was the best criterion for delimiting the TMD sequence. The length of the helical structure and the Impala method further supported the determination of the TMD limits. This method was applied to the IL-2Rβ and IL-2Rγ TMD sequences of Homo sapiens, Rattus norvegicus, Mus musculus and Bos taurus.ConclusionsWe succeeded in reducing the variation in the TMD limits to only 2 residues and in gaining structural information.

Investigation of Transmembrane Helix Hetero-Dimerization by Combinatorial Peptide Libraries and High-Throughput Measurement of FÖRSTER Resonance Energy Transfer in Liposomes

Receptor tyrosine kinases (RTKs) are membrane proteins containing an N-terminal extracellular ligand-binding domain, a single helical transmembrane (TM) domain, and an intracellular catalytic kinase domain. Lateral dimerization driven in part by the TM domain is proposed to be a crucial intermediate step in signal transduction across the plasma membrane. Defects in this process such as over-dimerization are closely linked to unregulated signaling and disease. Therefore, dimerization inhibitors developed using the chemical-physical basis of RTK TM domain dimerization could be promising therapeutic agents. Here we present a first step towards this goal. We designed a rational combinatorial peptide library based on the TM domain of Neu, an RTK from the Erb-B/HER epidermal growth factor receptor family (EGFR) with a pathogenic V to E mutation in the TM domain. Förster Resonance Energy Transfer (FRET) in lipid vesicles was used as a method to probe the dimerization between the library members and mutant Neu sequence. To enable high-throughput screening in liposomes, we developed a novel multi-well FRET assay. FRET donor-labeled library members and acceptor-labeled Neu peptides were incorporated into lipid vesicles in a rapid high-throughput 96-well plate format. We are using this high throughput screen to identify inhibitors of Neu dimerization. Our findings will be discussed in a context of throughput and sensitivity along with a statistical analysis that takes into account the FRET that arises from random proximity of donors and acceptors in the bilayer.

Hydrophobicity profiles in G protein-coupled receptor transmembrane helical domains

The lack of a crystallographically derived structure for all but three G (TP [guanosine triphosphate]-binding) protein-coupled receptor (GPCRs) proteins necessitates the use of computationally derived methods to determine their structures. Computational methodologies allow a mechanistic glimpse into GPCR-ligand interactions at a molecular level to better understand the initial steps leading to a protein's biologic functions, ie, protecting the ligands that activate, deactivate, or inhibit the protein, stabilizing protein structure in the membrane's lipid bilayer, and ensuring that the hydrophilic environment within the GPCR-binding pocket is maintained. Described here is a formalism that quantifies the amphiphilic nature of a helix, by determining the effective hydrophobicity (or hydrophilicity) at specific positions around it. This formalism will enable computational protein modelers to position helices so that the functional aspects of GPCRs are adequately represented in the model. Hydro-Eff®, an online tool, allows users to calculate effective helical hydrophobicities.

Synthesis and analysis of the membrane proximal external region epitopes of HIV‐1

The membrane proximal external region (MPER) of gp41 abuts the viral membrane at the base of HIV-1 envelope glycoprotein spikes. The MPER is highly conserved and is rich in Trp and other lipophilic residues. The MPER is also required for the infection of host cells by HIV-1 and is the target of the broadly neutralizing antibodies, 4E10, 2F5, and Z13e1. These neutralizing antibodies are valuable tools for understanding relevant conformations of the MPER and for studying HIV-1 neutralization, but multiple approaches used to elicit MPER binding antibodies with similar neutralization properties have failed. Here we report our efforts to mimic the MPER using linear as well as constrained peptides. Unnatural amino acids were also introduced into the core epitope of 4E10 to probe requirements of antibody binding. Peptide analogs with C-terminal Api or Aib residues designed to be helical transmembrane (TM) domain surrogates exhibit enhanced binding to the 4E10 and Z13e1 antibodies. However, we find that placement of constrained amino acids at nonbinding sites within the core epitope significantly reduce binding. These results are relevant to an understanding of native MPER structure on HIV-1, and form a basis for a chemical synthesis approach to mimic MPER stricture and to construct an MPER-based vaccine.

Interactions between Intracellular Domains as Key Determinants of the Quaternary Structure and Function of Receptor Heteromers

G protein-coupled receptor (GPCR) heteromers are macromolecular complexes with unique functional properties different from those of its individual protomers. Little is known about what determines the quaternary structure of GPCR heteromers resulting in their unique functional properties. In this study, using resonance energy transfer techniques in experiments with mutated receptors, we provide for the first time clear evidence for a key role of intracellular domains in the determination of the quaternary structure of GPCR heteromers between adenosine A(2A), cannabinoid CB(1), and dopamine D(2) receptors. In these interactions, arginine-rich epitopes form salt bridges with phosphorylated serine or threonine residues from CK1/2 consensus sites. Each receptor (A(2A), CB(1), and D(2)) was found to include two evolutionarily conserved intracellular domains to establish selective electrostatic interactions with intracellular domains of the other two receptors, indicating that these particular electrostatic interactions constitute a general mechanism for receptor heteromerization. Mutation experiments indicated that the interactions of the intracellular domains of the CB(1) receptor with A(2A) and D(2) receptors are fundamental for the correct formation of the quaternary structure needed for the function (MAPK signaling) of the A(2A)-CB(1)-D(2) receptor heteromers. Analysis of MAPK signaling in striatal slices of CB(1) receptor KO mice and wild-type littermates supported the existence of A(1)-CB(1)-D(2) receptor heteromer in the brain. These findings allowed us to propose the first molecular model of the quaternary structure of a receptor heteromultimer.

Mutational analysis of residues important for ligand interaction with the human P2Y12 receptor

The P2Y12 receptor, a Gi protein-coupled receptor, plays a central role in platelet activation. In this study, we did a mutational analysis of residues possibly involved in the ligand interactions with the human P2Y12 receptor. Mutant receptors were stably expressed in CHO-K1 cells with an HA-tag at the N-terminus. Expression of wild-type and mutant receptors was confirmed by detecting the HA-tag on the cell membrane. Residues in transmembrane helical domains (TMs) 3, 5, 6, and 7, which are homologous to residues important for P2Y1 receptor activation and ligand recognition, were replaced by site-directed mutagenesis. ADP-induced inhibition of forskolin-stimulated cAMP levels in the presence or absence of antagonist AR-C69931MX were investigated for each of the mutant receptors. F104S and S288P significantly increased agonist-induced receptor function without affecting the antagonism by AR-C69931MX. Arg256 in TM6 and Arg 265 in extracellular loop 3 (EL3) are more important for antagonist recognition than effect on agonist-mediated receptor function. Compared to wild-type P2Y12 receptor, mutations in Arg 256 or/and Arg 265 significantly increased the sensitivity to antagonist AR-C69931MX. Our study shows that the cytosolic side of TM3 and the exofacial side of TM5 are critical for P2Y12 receptor function, which is different from P2Y1. Arg 256 in TM6 and Arg265 in EL3 appear to play a role in antagonist recognition rather than effects on agonist-induced receptor function.

Molecular Recognition at the Membrane−Water Interface: Controlling Integral Peptide Helices by Off-Membrane Nucleobase Pairing

The aggregation and organization of membrane proteins and transmembrane peptides is related to the interacting molecular species itself and strongly depends on the lipid environment. Because of the complexity and dynamics of these interactions, they are often hardly traceable and nearly impossible to predict. For this reason, peptide model systems are a valuable tool in studying membrane associated processes since they are synthetically accessible and can be readily modified. To control and study the aggregation of peptide transmembrane domains (TMDs) the interacting interfaces of the TMDs themselves can be altered. A second less extensively studied approach targets the TMD assembly by using interaction and recognition of domains at the membrane outside as frequently found in the membrane protein interplay and protein assembly. In the present study, double helical transmembrane domains were designed and synthesized on the basis of a recently reported d,l-alternating peptide pore motif derived from gramicidin A. The highly hydrophobic and aromatic transmembrane peptide was covalently functionalized with a short peptide nucleic acid (PNA) used as specific outer-membrane recognition unit. The PNA sequences were chosen with high polarity to ensure localization within the aqueous phase. To estimate the impact of the membrane adjacent recognition on the TMD assembly by Förster resonance energy transfer (FRET), fluorescence probes were covalently attached to the side chains of the membrane spanning peptide helices. Dimerization of the TMD-peptide/PNA conjugates within unilamellar lipid vesicles was observed. The dimer/monomer ratio of TMDs can be controlled by temperature variation.

Properties of the TRPML3 Channel Pore and Its Stable Expansion by the Varitint-Waddler-causing Mutation

TRPML3 is a H(+)-regulated Ca(2+) channel that shuttles between intracellular compartments and the plasma membrane. The A419P mutation causes the varitint-waddler phenotype as a result of gain-of-function (GOF). The mechanism by which A419P leads to GOF is not known. Here, we show that the TRPML3 pore is dynamic when conducting Ca(2+) to change its conductance and permeability, which appears to be mediated by trapping Ca(2+) within the pore. The pore properties can be restored by strong depolarization or by conducting Na(+) through the pore. The A419P mutation results in expanded channel pore with altered permeability that limits modulation of the pore by Ca(2+). This effect is specific for the A419P mutation and is not reproduced by other GOF mutations, including A419G, H283A, and proline mutations in the fifth transmembrane domain. These findings describe a novel mode of a transient receptor potential channel behavior and suggest that pore expansion by the A419P mutation may contribute to the varitint-waddler phenotype.

Sequence-Dependent Oligomerization of the Neu Transmembrane Domain Suggests Inhibition of “Conformational Switching” by an Oncogenic Mutant

Membrane-spanning epidermal growth factor receptor ErbB2 is of key importance in cell division, in which a dimeric complex of the protein is responsible for tyrosine kinase activation following ligand binding. The rat homologue of this receptor (Neu) is prone to a valine to glutamic acid mutation in the transmembrane domain (TM), resulting in permanent activation and oncogenesis. In this study, the TM domains of Neu and the corresponding oncogenic mutant Neu*, which contains a V to E mutation at position 664 in the TM domain, have been analyzed to improve our understanding of the structural effects of the oncogenic V(664)E mutation. Building on previous work, we have focused here on understanding the sequence dependence of TM helix-helix interactions and any differences in behavior upon introduction of the V(664)E mutation. Using a variety of biochemical and biophysical methods, we find that the rat Neu TM domain forms strong oligomers and, similar to previous observations for the human ErbB2 TM domain, the oncogenic mutation results in a reduced level of self-association. Our data also strongly indicate that the proto-oncogenic Neu TM domain can adopt multiple (at least two) oligomeric conformations in the membrane, possibly corresponding to the active and inactive forms of the receptor, and can "switch" between the two. Further, the oncogenic Neu* mutant appears to inhibit this "conformational switching" of TM dimers, as we observe that dimerization of the Neu* TM domain in the Escherichia coli inner membrane strongly favors a single conformation stabilized by an IXXXV motif (I(659)-XXX-V(663)) originally identified by site-specific infrared spectroscopic studies.

Cysteine Mutagenesis Reveals Transmembrane Residues Associated with Charge Translocation in Prestin

The solute carrier transmembrane protein prestin (SLC26A5) drives an active electromechanical transduction process in cochlear outer hair cells that increases hearing sensitivity and frequency discrimination in mammals. A large intramembraneous charge movement, the nonlinear capacitance (NLC), is the electrical signature of prestin function. The transmembrane domain (TMD) helices and residues involved in the intramembrane charge displacement remain unknown. We have performed cysteine-scanning mutagenesis with serine or valine replacement to investigate the importance of cysteine residues to prestin structure and function. The distribution of oligomeric states and membrane abundance of prestin was also probed to investigate whether cysteine residues participate in prestin oligomerization and/or NLC. Our results reveal that 1) Cys-196 (TMD 4) and Cys-415 (TMD 10) do not tolerate serine replacement, and thus maintaining hydrophobicity at these locations is important for the mechanism of charge movement; 2) Cys-260 (TMD 6) and Cys-381 (TMD 9) tolerate serine replacement and are probably water-exposed; and 3) if disulfide bonds are present, they do not serve a functional role as measured via NLC. These novel findings are consistent with a recent structural model, which proposes that prestin contains an occluded aqueous pore, and we posit that the orientations of transmembrane domain helices 4 and 10 are essential for proper prestin function.

Biological indications of a novel "short" µ opiate receptor in domestic chicken.

Previous work from our laboratory has established that cellular signaling processes of endogenous morphine are mediated by cognate G protein coupled receptor (GPCR) proteins, designated µ3 and µ4 opiate receptors. µ3 and µ4 opiate receptors are structurally unique “short” 6 transmembrane helical (TMH) domain GPCRs that are selectively responsive to endogenous morphine, not to families of endogenous opioid peptides, and are uniquely coupled to activation of constitutive nitric oxide synthase (cNOS). Based on high resolution predictive measures, it appears likely that domestic poultry express a µ opiate receptor mRNA encoding potentially two novel GPCRs with similar biochemical characteristics as described for µ3 and µ4 opiate receptors as well as traditional µ1 opioid receptors. The biological indications of these novel µ opiate receptors are discussed within the context of this short review.

Properties of the M3-M4 intracellular loop of the GABAA receptor revealed through site-directed mutagenesis

Event Abstract Back to Event Properties of the M3-M4 intracellular loop of the GABAA receptor revealed through site-directed mutagenesis Kate O'Toole1* and Andrew Jenkins1 1 Emory University, Graduate Program in Neuroscience, United States The γ-amino butyric acid receptor type A (GABAAR) functions as a chemical to voltage transducer in the central nervous system, converting inhibitory neurotransmitter signals to changes in postsynaptic membrane potential. GABAAR is a member of the cys-loop family of ligand-gated ion channels along with nAChR, GlyR, and 5-HT3R. GABAAR is functionally hetero-pentameric and this study focuses on the most common synaptic subunit arrangement of α1:β2alpha;1:β:2γ2s. Each subunit is comprised of a large extracellular domain, four alpha helical transmembrane domains (M1-M4), and a variable intracellular loop (IL) between M3 and M4. The current theory of ion permeation relies on rings of charged residues contributed by each subunit that line the central pore and interact with permeating anions. These residues are located in M2 and the extracellular vestibule. Traditionally, the IL is thought to control channel function via interactions with trafficking and scaffold proteins. However, a partial structure of nAChR [1] shows an intracellular vestibule which may also contribute a portion of the permeation pathway. Therefore, we performed a site-directed mutagenesis scan assaying individual α1 IL charge switch mutations for GABA concentration response properties, ion selectivity, and properties of the current-voltage relationship. Results show a definite role for IL charged residues in controlling anion permeation. Mutagenesis produced significant changes in GABA apparent affinity, current magnitude, ion selectivity, rectification, and hysteresis of channel activation. These properties are regionally localized, which suggests different critical roles for subdomains of the IL. Overall, these findings show that the intracellular loop of the α1 subunit of GABAAR does contribute to the functional properties of channel activity.

Valine-Induced Packing Deficiencies of Transmembrane Domains Promote Helix Flexibility and Membrane Fusion

The helical transmembrane domains of fusion proteins are known to be functionally important and display an overabundance of helix-destabilizing Ile and Val residues. In an effort to systematically study the relationship of helix flexibility and fusogenicity, synthetic LV-peptides were designed whose hydrophobic core consists of Leu and Val residues at different ratios and at different positions (Hofmann et al., 2004; Poschner et al., 2009). The ability of the LV-peptides to fuse membranes increases with the content of helix-destabilizing residues. Molecular dynamics simulations were performed in order to characterize the backbone dynamics of these peptides in membrane-mimicking 80% TFE solvent and to relate the hydrogen-bond dynamics to experimental deuterium/hydrogen exchange kinetics. The analysis revealed that (i) the backbone dynamics of the helices increases systematically with Val content, (ii) that the impact of Val is due to stereochemical constraints within the helical structure and (iii) that side-chain packing mainly determines exchange kinetics. As a consequence, VxxV and VVxVV motifs promote helix destabilization whose relevance for membrane fusogenicity will be discussed.Hofmann, M.W., K. Weise, J. Ollesch, A. Agrawal, H. Stalz, W. Stelzer, F. Hulsbergen, H. deGroot, K. Gerwert, J. Reed, and D. Langosch. 2004. De novo design of conformationally flexible transmembrane peptides driving membrane fusion. Proc. Natl. Acad. Sci. U S A 101:14776-14781.Poschner, B.C., S. Quint, M. Hofmann, and D. Langosch. 2009. Sequence-specific conformational dynamics of model transmembrane domains determines their membrane fusogenic function. J. Mol. Biol. 386:733-741.

Estimation of Phi-Values for the Am1 Domain of Neuromuscular Acetylcholine Receptors

The AChR forward ‘gating’ isomerization occurs as a conformational cascade that starts at the transmitter binding sites and propagates to the gate region. This pattern has been deduced from the progressive reduction in a experimental parameter called Φ. In two the α-subunit transmembrane domains (TMDs), Φ-values are consistent for the membrane-facing residues of M4 (Φ∼0.54) and M3 (Φ∼0.3), as expected from a rigid body gating motion. Most αM2 residues (17′-1′) have a Φ∼0.64, but there are low-Φ side chains near the gate region (12′-9′; Φ∼0.3) and high-Φ ones at the N-terminal ‘cap domain. (27′-18′; Φ∼0.9). Towards the completion of the map of Φ in the α subunit, we are mapping values for the αM1 domain. Here we present preliminary results on diliganded isomerization equilibrium constant (E2) consequent to mutations of αM1 residues (HEK, mouse (α1)2βδe subunits transfected, 23 °C, −100 mV, cell-attached single-channel recording, 20 mM Cho or 500 ∈μM ACh). Mutations (n=2-6) at each of the following positions altered E2 by 5-fold were at positions: F214, I215, V216, N217, V218, L224, F225, S226 and L228. Single-channel currents were observed for mutations at position P221 (to A, C, F, G, R, S, T, V, and Y) although the resulting kinetic behavior was complex. The largest effect in terms of fold-change in E2 observed so far was at position 228 (L-to-A, ∼300-fold, 3.4 kcal/mol). Overall, the energetic consequences of the mutations examined so far in αM1 are moderate compared to the other α subunit TMD helices.

Analysis of Transmembrane Domains 1 and 4 of the Human Angiotensin II AT1 Receptor by Cysteine-scanning Mutagenesis

The octapeptide hormone angiotensin II (AngII) exerts a wide variety of cardiovascular effects through the activation of the AT(1) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein-coupled receptors, the AT(1) receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. Here, we investigated the role of the first and fourth transmembrane domains (TMDs) in the formation of the binding pocket of the human AT(1) receptor using the substituted-cysteine accessibility method. Each residue within the Phe-28((1.32))-Ile-53((1.57)) fragment of TMD1 and Leu-143((4.40))-Phe-170((4.67)) fragment of TMD4 was mutated, one at a time, to a cysteine. The resulting mutant receptors were expressed in COS-7 cells, which were subsequently treated with the charged sulfhydryl-specific alkylating agent methanethiosulfonate ethylammonium (MTSEA). This treatment led to a significant reduction in the binding affinity of TMD1 mutants M30C((1.34))-AT(1) and T33C((1.37))-AT(1) and TMD4 mutant V169C((4.66))-AT(1). Although this reduction in binding of the TMD1 mutants was maintained when examined in a constitutively active receptor (N111G-AT(1)) background, we found that V169C((4.66))-AT(1) remained unaffected when treated with MTSEA compared with untreated in this context. Moreover, the complete loss of binding observed for R167C((4.64))-AT(1) was restored upon treatment with MTSEA. Our results suggest that the extracellular portion of TMD1, particularly residues Met-30((1.34)) and Thr-33((1.37)), as well as residues Arg-167((4.64)) and Val-169((4.66)) at the junction of TMD4 and the second extracellular loop, are important binding determinants within the AT(1) receptor binding pocket but that these TMDs undergo very little movement, if at all, during the activation process.

The Fifth Transmembrane Domain of Angiotensin II Type 1 Receptor Participates in the Formation of the Ligand-binding Pocket and Undergoes a Counterclockwise Rotation upon Receptor Activation

The octapeptide hormone angiotensin II exerts a wide variety of cardiovascular effects through the activation of the angiotensin II Type 1 (AT(1)) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein- coupled receptors, the AT(1) receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. The role of the fifth transmembrane domain (TMD5) was investigated using the substituted cysteine accessibility method. All of the residues within Thr-190 to Leu-217 region were mutated one at a time to cysteine, and after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of L197C-AT(1), N200C-AT(1), I201C-AT(1), G203C-AT(1), and F204C-AT(1) mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT(1) receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD5 reporter cysteines engineered in a constitutively active N111G-AT(1) receptor background. Indeed, mutant I201C-N111G-AT(1) became more sensitive to MTSEA, whereas mutant G203C-N111G-AT(1) lost some sensitivity. Our results suggest that constitutive activation of AT(1) receptor causes an apparent counterclockwise rotation of TMD5 as viewed from the extracellular side.

Identification of a polymorphic collagen-like protein in the crustacean bacteria Pasteuria ramosa

Pasteuria ramosa is a spore-forming bacterium that infects Daphnia species. Previous results demonstrated a high specificity of host clone/parasite genotype interactions. Surface proteins of bacteria often play an important role in attachment to host cells prior to infection. We analyzed surface proteins of P. ramosa spores by two-dimensional gel electrophoresis. For the first time, we prove that two isolates selected for their differences in infectivity reveal few but clear-cut differences in protein patterns. Using internal sequencing and LC/MS/MS, we identified a collagen-like protein named Pcl1a ( Pasteuria collagen-like protein 1a). This protein, reconstructed with the help of Pasteuria genome sequences, contains three domains: a 75-amino-acid amino-terminal domain with a potential transmembrane helix domain, a central collagen-like region (CLR) containing Gly-Xaa-Yaa (GXY) repeats, and a 7-amino-acid carboxy-terminal domain. The CLR region is polymorphic among the two isolates with amino-acid substitutions and a variable number of GXY triplets. Collagen-like proteins are rare in prokaryotes, although they have been described in several pathogenic bacteria, including Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis, closely related to Pasteuria species, in which they could be involved in the adherence of bacteria to host cells.

Juxtamembranous Region of the Amino Terminus of the Family B G Protein-coupled Calcitonin Receptor Plays a Critical Role in Small-molecule Agonist Action

The Family B G protein-coupled calcitonin receptor is an important drug target. The aim of this work was to elucidate the molecular mechanism of action of small-molecule agonist ligands acting at this receptor, comparing it with the action mechanism of the receptor's natural peptide ligand. cAMP responses to four non-peptidyl ligands and calcitonin were studied in COS-1 cells expressing wild-type and chimeric calcitonin-secretin receptors. All compounds were full agonists at the calcitonin receptor with no activity at the secretin receptor. Only chimeric constructs including the calcitonin receptor amino terminus exhibited responses to any of these ligands. We progressively truncated this domain and tested constructs for cAMP responses. Although calcitonin was able to activate the calcitonin receptor fully with the first 58 residues absent, its potency was 3 orders of magnitude lower than that at the wild-type receptor. After truncation of 114 residues, there was no response to calcitonin. In contrast, small-molecule ligands were fully active at receptors having up to 149 amino-terminal residues absent. Those compounds finally became inactive after truncation of 153 residues. Deletion and/or alanine replacement of the region of the calcitonin receptor between residues 150 and 153 resulted in marked reduction in cAMP responses to these compounds, with some compound-specific differences observed, supporting a critical role for this region. Binding studies further supported distinct sites of action of small molecules relative to that of calcitonin. These findings focus attention on the potential importance of the juxtamembranous region of the amino terminus of the Family B calcitonin receptor for agonist drug action.