scholarly journals Sampling the conformational space of the catalytic subunit of human γ-secretase

2015 ◽  
Author(s):  
Xiao-chen Bai ◽  
Eeson Rajendra ◽  
Guanghui Yang ◽  
Yigong Shi ◽  
Sjors Scheres
Keyword(s):  
Secondary Structure ◽  
Transmembrane Domain ◽  
Transmembrane Helix ◽  
Butyl Ester ◽  
Catalytic Subunit ◽  
Conformational Space ◽  
Transmembrane Helices ◽  
Classification Procedure ◽  
Previous Sample ◽  
Molecular Plasticity

Human γ-secretase is an intra-membrane protease that cleaves many different substrates. Aberrant cleavage of Notch is implicated in cancer, while abnormalities in cutting amyloid precursor protein lead to Alzheimer's disease. Our previous cryo-EM structure of γ-secretase revealed considerable disorder in its catalytic subunit presenilin. Here, we introduce an image classification procedure that characterizes molecular plasticity at the secondary structure level, and apply this method to identify three distinct conformations in our previous sample. In one of these conformations, an additional transmembrane helix is visible that cannot be attributed to the known components of γ-secretase. In addition, we present a γ-secretase structure in complex with the dipeptidic inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). Our results reveal how conformational mobility in the second and sixth transmembrane helices of presenilin is greatly reduced upon binding of DAPT or the additional helix, and form the basis for a new model of how substrate enters the transmembrane domain.

scholarly journals Sampling the conformational space of the catalytic subunit of human γ-secretase

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Xiao-chen Bai ◽  
Eeson Rajendra ◽  
Guanghui Yang ◽  
Yigong Shi ◽  
Sjors HW Scheres
Keyword(s):  
Secondary Structure ◽  
Transmembrane Domain ◽  
Transmembrane Helix ◽  
Butyl Ester ◽  
Catalytic Subunit ◽  
Conformational Space ◽  
Transmembrane Helices ◽  
Classification Procedure ◽  
Previous Sample ◽  
Molecular Plasticity

Human γ-secretase is an intra-membrane protease that cleaves many different substrates. Aberrant cleavage of Notch is implicated in cancer, while abnormalities in cutting amyloid precursor protein lead to Alzheimer's disease. Our previous cryo-EM structure of γ-secretase revealed considerable disorder in its catalytic subunit presenilin. Here, we describe an image classification procedure that characterizes molecular plasticity at the secondary structure level, and apply this method to identify three distinct conformations in our previous sample. In one of these conformations, an additional transmembrane helix is visible that cannot be attributed to the known components of γ-secretase. In addition, we present a γ-secretase structure in complex with the dipeptidic inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). Our results reveal how conformational mobility in the second and sixth transmembrane helices of presenilin is greatly reduced upon binding of DAPT or the additional helix, and form the basis for a new model of how substrate enters the transmembrane domain.

scholarly journals Small Residues Inhibit Homo-Dimerization of the Human Carbonic Anhydrase XII Transmembrane Domain

Membranes ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 512
Author(s):  
Florian Cymer ◽  
Dirk Schneider
Keyword(s):  
Amino Acids ◽  
Transmembrane Domain ◽  
Biological Membrane ◽  
Transmembrane Helix ◽  
Accessible Surface Area ◽  
Side Chains ◽  
Carbonic Anhydrases ◽  
Transmembrane Helices ◽  
Accessible Surface ◽  
Small Side

Amino acids with small side chains and motifs of small residues in a distance of four are rather abundant in human single-span transmembrane helices. While interaction of such helices appears to be common, the role of the small residues in mediating and/or stabilizing transmembrane helix oligomers remains mostly elusive. Yet, the mere existence of (small)xxx(small) motifs in transmembrane helices is frequently used to model dimeric TM helix structures. The single transmembrane helix of the human carbonic anhydrases XII contains a large number of amino acids with small side chains, and critical involvement of these small amino acids in dimerization of the transmembrane domain has been suggested. Using the GALLEX assay, we show here that the transmembrane domain indeed forms a strong transmembrane helix oligomer within a biological membrane. However, single or multiple mutations of small residue(s) to isoleucine almost always increased, rather than decreased, the interaction propensities. Reduction of helix flexibility and of protein–lipid contacts caused by a reduced lipid accessible surface area likely results in stabilization of helix–helix interactions within the membrane.

scholarly journals Secondary structure and biophysical activity of synthetic analogues of the pulmonary surfactant polypeptide SP-C

1995 ◽  
Vol 307 (2) ◽  
pp. 535-541 ◽  
Author(s):  
J Johansson ◽  
G Nilsson ◽  
R Strömberg ◽  
B Robertson ◽  
H Jörnvall ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Pulmonary Surfactant ◽  
Transmembrane Helix ◽  
Helical Structure ◽  
Helical Conformation ◽  
Synthetic Analogues ◽  
Charged Amino Acids ◽  
Phospholipid Micelles ◽  
Helical Content ◽  
Biophysical Activity

Native pulmonary-surfactant-associated lipopolypeptide SP-C, its chemically depalmitoylated form and several synthetic analogues lacking the palmitoylcysteine residues were analysed for secondary structure in phospholipid micelles and for biophysical activity in 1,2-dipalmitoyl-sn-glycero-3- phosphocholine/phosphatidylglycerol/palmitic acid (68:22:9, by wt.). Compared with the native molecule, with the entire poly-valyl part in a known alpha-helical conformation, depalmitoylated SP-C was found to be still mainly alpha-helical, but with an approx. 20% decrease in the helical content. A synthetic hybrid polypeptide where the entire poly-valyl alpha-helical part of native SP-C had been replaced with the amino acid sequence of a transmembrane helix of bacteriorhodopsin is also predominantly alpha-helical. In contrast, synthetic SP-C analogues lacking only the palmitoyl groups, by replacement of the palmitoylcysteine residues with cysteine, phenylalanine or serine, or lacking the positively charged amino acids by replacement with alanine, are considerably less alpha-helical than both native and depalmitoylated SP-C. The data indicate that the SP-C palmitoyl groups are important for maintenance of the alpha-helical conformation in parts of the polypeptide, and that the poly-valyl alpha-helical conformation is not fully formed in synthetic SP-C polypeptides. Furthermore, the helical structure of both native and depalmitoylated SP-C in dodecylphosphocholine micelles is very resistant to thermal denaturation, exhibiting ordered structure at 90 degrees C. The alpha-helical content grossly parallels the peptide-induced acceleration of the spreading of phospholipids at an air/water interface and the increase of surface pressure. The data suggest that the alpha-helical conformation itself, rather than just the covalent structure, is of prime importance for the biological function of synthetic pulmonary-surfactant peptides.

scholarly journals Residues within the Transmembrane Domain of the Glucagon-Like Peptide-1 Receptor Involved in Ligand Binding and Receptor Activation: Modelling the Ligand-Bound Receptor

2011 ◽  
Vol 25 (10) ◽  
pp. 1804-1818 ◽  
Author(s):  
K. Coopman ◽  
R. Wallis ◽  
G. Robb ◽  
A. J. H. Brown ◽  
G. F. Wilkinson ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Structural Model ◽  
Transmembrane Domain ◽  
Transmembrane Helix ◽  
Receptor Activation ◽  
Agonist Affinity ◽  
Camp Generation ◽  
N Terminus ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1

The C-terminal regions of glucagon-like peptide-1 (GLP-1) bind to the N terminus of the GLP-1 receptor (GLP-1R), facilitating interaction of the ligand N terminus with the receptor transmembrane domain. In contrast, the agonist exendin-4 relies less on the transmembrane domain, and truncated antagonist analogs (e.g. exendin 9–39) may interact solely with the receptor N terminus. Here we used mutagenesis to explore the role of residues highly conserved in the predicted transmembrane helices of mammalian GLP-1Rs and conserved in family B G protein coupled receptors in ligand binding and GLP-1R activation. By iteration using information from the mutagenesis, along with the available crystal structure of the receptor N terminus and a model of the active opsin transmembrane domain, we developed a structural receptor model with GLP-1 bound and used this to better understand consequences of mutations. Mutation at Y152 [transmembrane helix (TM) 1], R190 (TM2), Y235 (TM3), H363 (TM6), and E364 (TM6) produced similar reductions in affinity for GLP-1 and exendin 9–39. In contrast, other mutations either preferentially [K197 (TM2), Q234 (TM3), and W284 (extracellular loop 2)] or solely [D198 (TM2) and R310 (TM5)] reduced GLP-1 affinity. Reduced agonist affinity was always associated with reduced potency. However, reductions in potency exceeded reductions in agonist affinity for K197A, W284A, and R310A, while H363A was uncoupled from cAMP generation, highlighting critical roles of these residues in translating binding to activation. Data show important roles in ligand binding and receptor activation of conserved residues within the transmembrane domain of the GLP-1R. The receptor structural model provides insight into the roles of these residues.

scholarly journals Point Mutations in Transmembrane Helices 2 and 3 of ExbB and TolQ Affect Their Activities in Escherichia coli K-12

2004 ◽  
Vol 186 (13) ◽  
pp. 4402-4406 ◽  
Author(s):  
Volkmar Braun ◽  
Christina Herrmann
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Transmembrane Helix ◽  
Point Mutations ◽  
Transmembrane Helices ◽  
The Third ◽  
Form Part ◽  
K 12

ABSTRACT Replacement of glutamate 176, the only charged amino acid in the third transmembrane helix of ExbB, with alanine (E176A) abolished ExbB activity in all determined ExbB-dependent functions of Escherichia coli. Combination of the mutations T148A in the second transmembrane helix and T181A in the third transmembrane helix, proposed to form part of a proton pathway through ExbB, also resulted in inactive ExbB. E176 and T148 are strictly conserved in ExbB and TolQ proteins, and T181 is almost strictly conserved in ExbB, TolQ, and MotA.

scholarly journals FOLDING ELASTIC TRANSMEMBRANE HELICES TO FIT IN A LOW-RESOLUTION IMAGE BY ELECTRON MICROSCOPY

2011 ◽  
Vol 09 (supp01) ◽  
pp. 37-50 ◽  
Author(s):  
YUTAKA UENO ◽  
KAZUNORI KAWASAKI ◽  
OSAMU SAITO ◽  
MASAFUMI ARAI ◽  
MAKIKO SUWA
Keyword(s):  
Membrane Proteins ◽  
Structure Prediction ◽  
Molecular Model ◽  
Imaging Techniques ◽  
Transmembrane Helix ◽  
Prediction Method ◽  
Molecular Shape ◽  
Coarse Grained ◽  
Transmembrane Helices ◽  
Low Resolution

Structure prediction of membrane proteins could be constrained and thereby improved by introducing data of the observed molecular shape. We studied a coarse-grained molecular model that relied on residue-based dummy atoms to fold the transmembrane helices of a protein in the observed molecular shape. Based on the inter-residue potential, the α-helices were folded to contact each other in a simulated annealing protocol to search optimized conformation. Fitting the model into a three-dimensional volume was tested for proteins with known structures and resulted in a fairly reasonable arrangement of helices. In addition, the constraint to the packing transmembrane helix with the two-dimensional region was tested and found to work as a very similar folding guide. The obtained models nicely represented α-helices with the desired slight bend. Our structure prediction method for membrane proteins well demonstrated reasonable folding results using a low-resolution structural constraint introduced from recent cell-surface imaging techniques.

Implications of an improved model of the TSH receptor transmembrane domain (TSHR-TMD -TRIO)

Endocrinology ◽  
2021 ◽  
Author(s):  
Mihaly Mezei ◽  
Rauf Latif ◽  
Bhaskar Das ◽  
Terry F Davies
Keyword(s):  
Small Molecules ◽  
Transmembrane Domain ◽  
Water Layer ◽  
Tsh Receptor ◽  
Transmembrane Helices ◽  
Transmembrane Region ◽  
Significant Preference ◽  
Group A ◽  
Improved Model ◽  
Intracellular Loops

Abstract The TSH receptor is a GPCR Group A family member with seven transmembrane helices. We generated three new models of its entire transmembrane region using a 600 ns molecular simulation. The simulation started from our previously published model which we have now revised by also modeling the intracellular loops and the C-terminal tail, adding internal waters and embedding it into a lipid bilayer with a water layer and with ions added to complete the system. We have named this model TSHR-TMD – TRIO since three representative dominant structures were then extracted from the simulation trajectory and compared with the original model. These structures each showed small but significant changes in the relative positions of the helices. The three models were also used as targets to dock a set of small molecules that are known active compounds including a new TSHR antagonist (BT362), which confirmed the appropriateness of the model with some small molecules showing significant preference for one or other of the structures.

scholarly journals The role of prolines and glycine in the transmembrane domain of LAT

2020 ◽  
Author(s):  
Daniela Glatzová ◽  
Harsha Mavila ◽  
Maria Chiara Saija ◽  
Tomáš Chum ◽  
Lukasz Cwiklik ◽  
...  
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Transmembrane Domain ◽  
Transmembrane Helix ◽  
Helical Structure ◽  
Surface Expression ◽  
Transmembrane Segment ◽  
Proline Residue ◽  
And Function ◽  
The Impact

ABSTRACTLAT is a critical regulator of T cell development and function. It organises signalling events at the plasma membrane. However, the mechanism, which controls LAT localisation at the plasma membrane is not fully understood. Here, we studied the impact of helix-breaking amino acids, two prolines and one glycine, in the transmembrane segment on localisation and function of LAT. Using in silico analysis, confocal and superresolution imaging and flow cytometry we demonstrate that central proline residue destabilises transmembrane helix by inducing a kink. The helical structure and dynamics is further regulated by glycine and another proline residue in the luminal part of LAT transmembrane domain. Replacement of these residues with aliphatic amino acids reduces LAT dependence on palmitoylation for sorting to the plasma membrane. However, surface expression of these mutants is not sufficient to recover function of non-palmitoylated LAT in stimulated T cells. These data indicate that geometry and dynamics of LAT transmembrane segment regulate its localisation and function in immune cells.

scholarly journals Yil102c-A is a Functional Homologue of the DPMII Subunit of Dolichyl Phosphate Mannose Synthase in Saccharomyces cerevisiae

2020 ◽  
Vol 21 (23) ◽  
pp. 8938
Author(s):  
Sebastian Piłsyk ◽  
Urszula Perlinska-Lenart ◽  
Anna Janik ◽  
Elżbieta Gryz ◽  
Marta Ajchler-Adamska ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Functional Analysis ◽  
Trichoderma Reesei ◽  
Transmembrane Domain ◽  
Human Cells ◽  
Catalytic Subunit ◽  
Yeast Saccharomyces Cerevisiae ◽  
Dolichyl Phosphate ◽  
Functional Homolog ◽  
Wide Range

In a wide range of organisms, dolichyl phosphate mannose (DPM) synthase is a complex of tree proteins Dpm1, Dpm2, and Dpm3. However, in the yeast Saccharomyces cerevisiae, it is believed to be a single Dpm1 protein. The function of Dpm3 is performed in S. cerevisiae by the C-terminal transmembrane domain of the catalytic subunit Dpm1. Until present, the regulatory Dpm2 protein has not been found in S. cerevisiae. In this study, we show that, in fact, the Yil102c-A protein interacts directly with Dpm1 in S. cerevisiae and influences its DPM synthase activity. Deletion of the YIL102c-A gene is lethal, and this phenotype is reversed by the dpm2 gene from Trichoderma reesei. Functional analysis of Yil102c-A revealed that it also interacts with glucosylphosphatidylinositol-N-acetylglucosaminyl transferase (GPI-GnT), similar to DPM2 in human cells. Taken together, these results show that Yil102c-A is a functional homolog of DPMII from T. reesei and DPM2 from humans.

Analysis of SARS-CoV-2 ORF3a structure reveals chloride binding sites

2020 ◽  
Author(s):  
Valeria Marquez-Miranda ◽  
Maximiliano Rojas ◽  
Yorley Duarte ◽  
Ignacio Diaz-Franulic ◽  
Miguel Holmgren ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Lung Epithelial Cells ◽  
Transmembrane Helices ◽  
Cellular Processes ◽  
Charged Amino Acids ◽  
Coordination Sites ◽  
Lung Epithelial ◽  
Dynamics Simulations ◽  
Positively Charged

AbstractSARS-CoV-2 ORF3a is believed to form ion channels, which may be involved in the modulation of virus release, and has been implicated in various cellular processes like the up-regulation of fibrinogen expression in lung epithelial cells, downregulation of type 1 interferon receptor, caspase-dependent apoptosis, and increasing IFNAR1 ubiquitination. ORF3a assemblies as homotetramers, which are stabilized by residue C133. A recent cryoEM structure of a homodimeric complex of ORF3a has been released. A lower-resolution cryoEM map of the tetramer suggests two dimers form it, arranged side by side. The dimer’s cryoEM structure revealed that each protomer contains three transmembrane helices arranged in a clockwise configuration forming a six helices transmembrane domain. This domain’s potential permeation pathway has six constrictions narrowing to about 1 Å in radius, suggesting the structure solved is in a closed or inactivated state. At the cytosol end, the permeation pathway encounters a large and polar cavity formed by multiple beta strands from both protomers, which opens to the cytosolic milieu. We modeled the tetramer following the arrangement suggested by the low-resolution tetramer cryoEM map. Molecular dynamics simulations of the tetramer embedded in a membrane and solvated with 0.5 M of KCl were performed. Our simulations show the cytosolic cavity is quickly populated by both K+ and Cl-, yet with different dynamics. K+ ions moved relatively free inside the cavity without forming proper coordination sites. In contrast, Cl- ions enter the cavity, and three of them can become stably coordinated near the intracellular entrance of the potential permeation pathway by an inter-subunit network of positively charged amino acids. Consequently, the central cavity’s electrostatic potential changed from being entirely positive at the beginning of the simulation to more electronegative at the end.

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