The functional assay identified authentic interactions between CAPA peptides and the CAPA receptor isoforms in Bemisia tabaci (Gennadius).

CAPA neuropeptides regulate the diuresis/ antidiuresis process in insects by activating specific cognate receptor, CAPAr. In this study, we characterized the CAPAr gene (BtabCAPAr) in the whitefly, Bemisia tabaci Asia II 1. The two alternatively spliced isoforms of BtabCAPAr gene, BtabCAPAr-1 and BtabCAPAr-2, having six and five exons, respectively, were identified. The BtabCAPAr gene expression was highest in adult whitefly as compared to gene expression in egg, nymphal and pupal stages. Among the three putative CAPA peptides, CAPA-PVK1 and CAPA-PVK2 strongly activated the BtabCAPAr-1 with very low EC50 values of 0.067 nM and 0.053 nM, respectively, in heterologous calcium mobilization assays. None of the peptide activated the alternatively spliced isoform BtabCAPAr-2 that has lost the transmembrane segments 3 and 4. Significant levels of mortality were observed when whiteflies were fed with CAPA-PVK1 at 1.0 µM (50.0%), CAPA-PVK2 at 100.0 nM (43.8%) and CAPA-tryptoPK 1.0 µM (40.0%) at the 96 h after the treatment. This study provides valuable information to design biostable peptides to develop a class of insecticides.

Transmembrane topology and oligomeric nature of an astrocytic membrane protein, MLC1.

MLC1 is a membrane protein mainly expressed in astrocytes, and genetic mutations lead to the development of a leukodystrophy, megalencephalic leukoencephalopathy with subcortical cysts disease. Currently, the biochemical properties of the MLC1 protein are largely unknown. In this study, we aimed to characterize the transmembrane (TM) topology and oligomeric nature of the MLC1 protein. Systematic immunofluorescence staining data revealed that the MLC1 protein has eight TM domains and that both the N- and C-terminus face the cytoplasm. We found that MLC1 can be purified as an oligomer and could form a trimeric complex in both detergent micelles and reconstituted proteoliposomes. Additionally, a single-molecule photobleaching experiment showed that MLC1 protein complexes could consist of three MLC1 monomers in the reconstituted proteoliposomes. These results can provide a basis for both the high-resolution structural determination and functional characterization of the MLC1 protein.

A Multitask Deep-Learning Method for Predicting Membrane Associations and Secondary Structures of Proteins.

Prediction of residue-level structural attributes and protein-level structural classes helps model protein tertiary structures and understand protein functions. Existing methods are either specialized on only one class of proteins or developed to predict only a specific type of residue-level attribute. In this work, we develop a new deep-learning method, named Membrane Association and Secondary Structure Predictor (MASSP), for accurately predicting both residue-level structural attributes (secondary structure, location, orientation, and topology) and protein-level structural classes (bitopic, α-helical, ß-barrel, and soluble). MASSP integrates a multilayer two-dimensional convolutional neural network (2D-CNN) with a long short-term memory (LSTM) neural network into a multitasking framework. Our comparison shows that MASSP performs equally well or better than the state-of-the-art methods in predicting residue-level secondary structures, boundaries of transmembrane segments, and topology. Furthermore, it achieves outstanding accuracy in predicting protein-level structural classes. MASSP automatically distinguishes the structural classes of input sequences and identifies transmembrane segments and topologies if present, making it broadly applicable to different classes of proteins. In summary, MASSP's good performance and broad applicability make it well suited for annotating residue-level attributes and protein-level structural classes at the proteome scale.

Secondary structure and transmembrane topology analysis of the N-terminal domain of the inner membrane protein EccE1 from M. smegmatis using site-directed spin labeling EPR.

Protein EccE1 is an essential component of the mycobacterial ESX-1 secretion system, which plays a crucial part in the process of virulence factors secretion, especially for pathogenic mycobacteria such as Mycobacterium tuberculosis. While EccE1 was previously postulated to be the inner membrane pore-forming unit of a membrane complex through which substrates are transported, the structural properties of EccE1 remains to be explored. In the present study, systematic Site-Directed Spin Labeling (SDSL) and Electron Paramagnetic Resonance (EPR) spectroscopic studies was carried out to reveal the secondary structure and transmembrane topology of the N-terminal Domain of EccE1 protein (EccE1-NTD) from M. smegmatis in detergent micelles. EPR-based mobility and accessibility analysis of the R1 side chain for 64 residue positions of EccE1-NTD indicates that the transmembrane domain adopts two α-helices spanning Phe7-Cys30 and Leu36-Ile54. A tentative structural topology model of EccE1-NTD embedded in membrane is also suggested based on EPR spectroscopic data in this study, which will provide further insights into this protein and the ESX secretion systems of mycobacteria.

Verification of the Saccharina japonica Translocon Tic20 and its Localization in the Chloroplast Membrane in Diatoms.

Tic20 is an important translocon protein that plays a role in protein transport in the chloroplast. The sequence of Tic20 was determined in the lower brown alga Saccharina japonica. Structural analysis of SjTic20 revealed a noncanonical structure consisting of an N-terminal non-cyanobacterium-originated EF-hand domain (a helix-loop-helix structural domain) and a C-terminal cyanobacterium-originated Tic20 domain. Subcellular localization and transmembrane analysis indicated that SjTic20 featured an "M"-type Nin-Cin-terminal orientation, with four transmembrane domains in the innermost membrane of the chloroplast in the microalga Phaeodactylum tricornutum, and the EF-hand domain was entirely extruded into the chloroplast stroma. Our study provides information on the structure, localization, and topological features of SjTic20, and further functional analysis of SjTic20 in S. japonica is needed.

Evolution and adaptation of single-pass transmembrane proteins.

A comparative analysis of 6039 single-pass (bitopic) membrane proteins from six evolutionarily distant organisms was performed based on data from the Membranome database. The observed repertoire of bitopic proteins is significantly enlarged in eukaryotic cells and especially in multicellular organisms due to the diversification of enzymes, emergence of proteins involved in vesicular trafficking, and expansion of receptors, structural, and adhesion proteins. The majority of bitopic proteins in multicellular organisms are located in the plasma membrane (PM) and involved in cell communication. Bitopic proteins from different membranes significantly diverge in terms of their biological functions, size, topology, domain architecture, physical properties of transmembrane (TM) helices and propensity to form homodimers. Most proteins from eukaryotic PM and endoplasmic reticulum (ER) have the N-out topology. The predicted lengths of TM helices and hydrophobic thicknesses, stabilities and hydrophobicities of TM α-helices are the highest for proteins from eukaryotic PM, intermediate for proteins from prokaryotic cells, ER and Golgi apparatus, and lowest for proteins from mitochondria, chloroplasts, and peroxisomes. Tyr and Phe residues accumulate at the cytoplasmic leaflet of PM and at the outer leaflet of membranes of bacteria, Golgi apparatus, and nucleus. The propensity for dimerization increases from unicellular to multicellular eukaryotes, from enzymes to receptors, and from intracellular membrane proteins to PM proteins. More than half of PM proteins form homodimers with a 2:1 ratio of right-handed to left-handed helix packing arrangements. The inverse ratio (1:2) was observed for dimers from the ER, Golgi and vesicles.

Transmembrane topology of the arsenite permease Acr3 from Saccharomyces cerevisiae.

Acr3 is a plasma membrane transporter, a member of the bile/arsenite/riboflavin transporter (BART) superfamily, which confers high-level resistance to arsenicals in the yeast Saccharomyces cerevisiae. We have previously shown that the yeast Acr3 acts as a low affinity As(III)/H+ and Sb(III)/H+ antiporter. We have also identified several amino acid residues that are localized in putative transmembrane helices (TM) and appeared to be critical for the Acr3 activity. In the present study, the topology of Acr3 was investigated by insertion of glycosylation and factor Xa protease cleavage sites at predicted hydrophilic regions. The analysis of the glycosylation pattern and factor Xa cleavage products of resulting Acr3 fusion constructs provide evidence supporting a topological model of Acr3 with 10 TM segments and cytoplasmically oriented N- and C-terminal domains. Next, we investigated the role of the hydrophilic loop connecting TM8 and TM9, the large size of which is unique to members of the yeast Acr3 family of metalloid transporters. We found that a 28 amino acid deletion in this region does not affect Acr3 folding, trafficking substrate binding, or transport activity. Finally, we constructed a homology-based structural model of Acr3 using the crystal structure of the Yersinia frederiksenii homologue of the human bile acid sodium symporter ASBT.