scholarly journals Insights into substrate-mediated assembly of the chloroplast TAT receptor complex

2020 ◽  
Author(s):  
Qianqian Ma ◽  
Christopher Paul New ◽  
Carole Dabney-Smith
Keyword(s):  
Structural Model ◽  
Substrate Recognition ◽  
Transmembrane Helix ◽  
Complex Function ◽  
Transmembrane Helices ◽  
Translocation Event ◽  
Active Substrate ◽  
Tat System ◽  
Folded Proteins ◽  
Transport Complex

AbstractThe Twin Arginine Transport (TAT) system translocates fully folded proteins across the thylakoid membrane in the chloroplast (cp) and the cytoplasmic membrane of bacteria. In chloroplasts, cpTAT transport is achieved by three components: Tha4, Hcf106, and cpTatC. Hcf106 and cpTatC function as the substrate recognition/binding complex while Tha4 is thought to play a significant role in forming the translocation pore. Recent studies challenged this idea by suggesting that cpTatC-Hcf106-Tha4 function together in the active translocase. Here, we have mapped the inter-subunit contacts of cpTatC-Hcf106 during the resting state and built a cpTatC-Hcf106 structural model based on our crosslinking data. In addition, we have identified a substrate-mediated reorganization of cpTatC-Hcf106 contact sites during active substrate translocation. The proximity of Tha4 to the cpTatC-Hcf106 complex was also identified. Our data suggest a model for cpTAT function in which the transmembrane helices of Hcf106 and Tha4 may each contact the fifth transmembrane helix of cpTatC while the insertion of the substrate signal peptide may rearrange the cpTatC-Hcf106-Tha4 complex and initiate the translocation event.One sentence summaryProtein subunits of the thylakoidal twin arginine transport complex function together during substrate recognition and translocase assembly.

scholarly journals Substrate-triggered position-switching of TatA and TatB is an essential step in the Escherichia coli Tat protein export pathway

2017 ◽  
Author(s):  
Johann Habersetzer ◽  
Kristoffer Moore ◽  
Jon Cherry ◽  
Grant Buchanan ◽  
Phillip Stansfeld ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Sites ◽  
Protein Transport ◽  
Transmembrane Helix ◽  
Tat Protein ◽  
Disulfide Crosslinking ◽  
Additional Sequence ◽  
Tat System ◽  
Folded Proteins ◽  
Related Proteins

AbstractThe twin arginine protein transport (Tat) machinery mediates the translocation of folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. The Escherichia coli Tat system comprises TatC and two additional sequence-related proteins, TatA and TatB. Here we use disulfide crosslinking and molecular modelling to show there are two binding sites for TatA/B proteins on TatC. TatA and TatB are each able to occupy both sites if they are the only TatA/B protein present. However, under resting conditions the sites are differentially occupied with TatB occupying the ‘polar cluster’ site while TatA binds adjacently at the TatC transmembrane helix 6 binding site. When the Tat system is activated by the overproduction of a substrate, TatA and TatB switch their binding sites. We propose that this substrate-triggered positional exchange is a key step in the assembly of an active Tat translocase.

scholarly journals Assembling the Tat protein translocase

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Felicity Alcock ◽  
Phillip J Stansfeld ◽  
Hajra Basit ◽  
Johann Habersetzer ◽  
Matthew AB Baker ◽  
...  
Keyword(s):  
Structural Model ◽  
Protein Translocation ◽  
Transmembrane Helix ◽  
Tat Protein ◽  
Polar Cluster ◽  
Evolution Analysis ◽  
Protein Interfaces ◽  
Multiple Copies ◽  
Folded Proteins ◽  
Combine Sequence

The twin-arginine protein translocation system (Tat) transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membranes of plant chloroplasts. The Tat transporter is assembled from multiple copies of the membrane proteins TatA, TatB, and TatC. We combine sequence co-evolution analysis, molecular simulations, and experimentation to define the interactions between the Tat proteins of Escherichia coli at molecular-level resolution. In the TatBC receptor complex the transmembrane helix of each TatB molecule is sandwiched between two TatC molecules, with one of the inter-subunit interfaces incorporating a functionally important cluster of interacting polar residues. Unexpectedly, we find that TatA also associates with TatC at the polar cluster site. Our data provide a structural model for assembly of the active Tat translocase in which substrate binding triggers replacement of TatB by TatA at the polar cluster site. Our work demonstrates the power of co-evolution analysis to predict protein interfaces in multi-subunit complexes.

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.

scholarly journals Functional analysis of the polar amino acid in TatA transmembrane helix

2021 ◽  
Author(s):  
Binhan Hao ◽  
Wenjie Zhou ◽  
Steven M Theg
Keyword(s):  
Amino Acid ◽  
Potential Role ◽  
Transmembrane Helix ◽  
Amino Acid Side Chain ◽  
Polar Amino Acid ◽  
Twin Arginine Translocation ◽  
N Terminus ◽  
Biochemical Approach ◽  
Folded Proteins ◽  
Highly Correlated

The twin-arginine translocation (Tat) pathway utilizes the proton-motive force (PMF) to transport folded proteins across cytoplasmic membranes in bacteria and archaea, as well as across the thylakoid membrane in plants and the inner membrane in mitochondria. In most species, the minimal components required for Tat activity consist of three subunits, TatA, TatB, and TatC. Previous studies have shown that a polar amino acid is present at the N-terminus of the TatA transmembrane helix (TMH) across many different species. In order to systematically assess the functional importance of this polar amino acid in the TatA TMH in Escherichia coli, a complete set of 19-amino-acid substitutions was examined. Unexpectedly, although being preferred overall, our experiments suggest that the polar amino acid is not necessary for a functional TatA. Hydrophobicity and helix stabilizing properties of this polar amino acid were found to be highly correlated with the Tat activity. Specifically, change in charge status of the amino acid side chain due to pH resulted in a shift in hydrophobicity, which was demonstrated to impact the Tat transport activity. Furthermore, a four-residue motif at the N-terminus of the TatA TMH was identified by sequence alignment. Using a biochemical approach, the N-terminal motif was found to be functionally significant, with evidence indicating a potential role in the preference for utilizing different PMF components. Taken together, these findings yield new insights into the functionality of TatA and its potential role in the Tat transport mechanism.

Branching airway network models for analyzing high-frequency lung input impedance

1993 ◽  
Vol 75 (1) ◽  
pp. 217-227 ◽  
Author(s):  
A. C. Jackson ◽  
B. Suki ◽  
M. Ucar ◽  
R. Habib
Keyword(s):  
Input Impedance ◽  
Structural Model ◽  
Complex Function ◽  
Low Frequency ◽  
Network Models ◽  
Scaling Factor ◽  
Forced Oscillation Technique ◽  
Geometrical Factors ◽  
Airway Walls ◽  
Parameter Values

The input impedance of the lung (Zin) at high frequencies (> 100 Hz) is a complex function of the airway geometry and the mechanical properties of the airway walls. To investigate how the purely geometrical factors influence Zin, we measured Zin between 16 and 1,520 Hz in six dried dog lungs with the forced oscillation technique. In each of the lungs we found three resonances, at 36 +/- 5, 648 +/- 100, and 1,289 +/- 150 Hz, and at least two antiresonances (relative maxima in the real part of Zin), at 372 +/- 60 and 1,105 +/- 110 Hz. These data were fit with models featuring a detailed asymmetric branching network of the airways obtained from morphometric data published by Horsfield et al. (J. Appl. Physiol. 52: 21–26, 1982). On the basis of low-frequency (< 100 Hz) data alone, we first established a model of the acini, which was then attached to the end of the airway branching model. With a single scaling factor for the radius and length of the airways, the fit was unsatisfactory. Using sensitivity analysis techniques we determined which candidate variables of the structural model could influence Zin in a manner to improve the fit. We found that a two-parameter model accounting for separate central and peripheral airway diameter scaling provided a reasonable fit to Zin. On average the model required central diameter scaling close to unity (0.94 +/- 0.09), and the peripheral diameter scaling factor was 0.87 +/- 0.38. Over a range of parameter values that we believed were physiologically reasonable (i.e., scaling factors between 0.5 and 1.5), a single set of parameter values was found in all lungs. These results suggest that structurally based inverse models of Zin that include multiple antiresonances may provide information about airway geometry.

Substrate processing in intramembrane proteolysis by γ-secretase – the role of protein dynamics

2017 ◽  
Vol 398 (4) ◽  
pp. 441-453 ◽  
Author(s):  
Dieter Langosch ◽  
Harald Steiner
Keyword(s):  
Transmembrane Helix ◽  
Peptide Bond ◽  
Common Denominator ◽  
Transmembrane Helices ◽  
Intramembrane Proteases ◽  
Bond Scission ◽  
Catalytic Sites ◽  
Substrate Processing

Abstract Intramembrane proteases comprise a number of different membrane proteins with different types of catalytic sites. Their common denominator is cleavage within the plane of the membrane, which usually results in peptide bond scission within the transmembrane helices of their substrates. Despite recent progress in the determination of high-resolution structures, as illustrated here for the γ-secretase complex and its substrate C99, it is still unknown how these enzymes function and how they distinguish between substrates and non-substrates. In principle, substrate/non-substrate discrimination could occur at the level of substrate binding and/or cleavage. Focusing on the γ-secretase/C99 pair, we will discuss recent observations suggesting that global motions within a substrate transmembrane helix may be much more important for defining a substrate than local unraveling at cleavage sites.

scholarly journals A twin arginine signal peptide and the pH gradient trigger reversible assembly of the thylakoid ΔpH/Tat translocase

2002 ◽  
Vol 157 (2) ◽  
pp. 205-210 ◽  
Author(s):  
Hiroki Mori ◽  
Kenneth Cline
Keyword(s):  
Signal Peptide ◽  
Protein Translocation ◽  
Permeability Barrier ◽  
Synthetic Signal ◽  
Tat Pathway ◽  
Precursor Proteins ◽  
Varied Size ◽  
Tat System ◽  
Folded Proteins ◽  
Chemical Cross Linking

The thylakoid ΔpH-dependent/Tat pathway is a novel system with the remarkable ability to transport tightly folded precursor proteins using a transmembrane ΔpH as the sole energy source. Three known components of the transport machinery exist in two distinct subcomplexes. A cpTatC–Hcf106 complex serves as precursor receptor and a Tha4 complex is required after precursor recognition. Here we report that Tha4 assembles with cpTatC–Hcf106 during the translocation step. Interactions among components were examined by chemical cross-linking of intact thylakoids followed by immunoprecipitation and immunoblotting. cpTatC and Hcf106 were consistently associated under all conditions tested. In contrast, Tha4 was only associated with cpTatC and Hcf106 in the presence of a functional precursor and the ΔpH. Interestingly, a synthetic signal peptide could replace intact precursor in triggering assembly. The association of all three components was transient and dissipated upon the completion of protein translocation. Such an assembly–disassembly cycle could explain how the ΔpH/Tat system can assemble translocases to accommodate folded proteins of varied size. It also explains in part how the system can exist in the membrane without compromising its ion and proton permeability barrier.

scholarly journals The Lactococcin G Immunity Protein Recognizes Specific Regions in Both Peptides Constituting the Two-Peptide Bacteriocin Lactococcin G

2009 ◽  
Vol 76 (4) ◽  
pp. 1267-1273 ◽  
Author(s):  
Camilla Oppegård ◽  
Linda Emanuelsen ◽  
Lisbeth Thorbek ◽  
Gunnar Fimland ◽  
Jon Nissen-Meyer
Keyword(s):  
Structural Model ◽  
Transmembrane Helix ◽  
Cellular Component ◽  
Expression Vectors ◽  
Wild Type ◽  
Terminal Part ◽  
Gene Encoding ◽  
Immunity Protein ◽  
Hybrid Peptides ◽  
Helix Structure

ABSTRACT Lactococcin G and enterocin 1071 are two homologous two-peptide bacteriocins. Expression vectors containing the gene encoding the putative lactococcin G immunity protein (lagC) or the gene encoding the enterocin 1071 immunity protein (entI) were constructed and introduced into strains sensitive to one or both of the bacteriocins. Strains that were sensitive to lactococcin G became immune to lactococcin G when expressing the putative lactococcin G immunity protein, indicating that the lagC gene in fact encodes a protein involved in lactococcin G immunity. To determine which peptide or parts of the peptide(s) of each bacteriocin that are recognized by the cognate immunity protein, combinations of wild-type peptides and hybrid peptides from the two bacteriocins were assayed against strains expressing either of the two immunity proteins. The lactococcin G immunity protein rendered the enterococcus strain but not the lactococcus strains resistant to enterocin 1071, indicating that the functionality of the immunity protein depends on a cellular component. Moreover, regions important for recognition by the immunity protein were identified in both peptides (Lcn-α and Lcn-β) constituting lactococcin G. These regions include the N-terminal end of Lcn-α (residues 1 to 13) and the C-terminal part of Lcn-β (residues 14 to 24). According to a previously proposed structural model of lactococcin G, these regions will be positioned adjacent to each other in the transmembrane helix-helix structure, and the model thus accommodates the present results.

Sign in / Sign up

Export Citation Format

Share Document