scholarly journals Efficient integration of transmembrane domains depends on the folding properties of the upstream sequences

2021 ◽  
Vol 118 (33) ◽  
pp. e2102675118
Author(s):  
Marco Janoschke ◽  
Mirjam Zimmermann ◽  
Anna Brunauer ◽  
Raffael Humbel ◽  
Tina Junne ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Transmembrane Domains ◽  
Transmembrane Segment ◽  
Native State ◽  
Protein Fragments ◽  
Hydrophobic Residues ◽  
Intrinsically Disordered ◽  
Efficient Integration ◽  
Chaperone Binding ◽  
Integration Efficiency

The topology of most membrane proteins is defined by the successive integration of α-helical transmembrane domains at the Sec61 translocon. The translocon provides a pore for the transfer of polypeptide segments across the membrane while giving them lateral access to the lipid. For each polypeptide segment of ∼20 residues, the combined hydrophobicities of its constituent amino acids were previously shown to define the extent of membrane integration. Here, we discovered that different sequences preceding a potential transmembrane domain substantially affect its hydrophobicity requirement for integration. Rapidly folding domains, sequences that are intrinsically disordered or very short or capable of binding chaperones with high affinity, allow for efficient transmembrane integration with low-hydrophobicity thresholds for both orientations in the membrane. In contrast, long protein fragments, folding-deficient mutant domains, and artificial sequences not binding chaperones interfered with membrane integration, requiring higher hydrophobicity. We propose that the latter sequences, as they compact on their hydrophobic residues, partially folded but unable to reach a native state, expose hydrophobic surfaces that compete with the translocon for the emerging transmembrane segment, reducing integration efficiency. The results suggest that rapid folding or strong chaperone binding is required for efficient transmembrane integration.

scholarly journals Cryo-EM structure of amyloid fibrils formed by the entire low complexity domain of TDP-43

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Qiuye Li ◽  
W. Michael Babinchak ◽  
Witold K. Surewicz
Keyword(s):  
Amyloid Fibrils ◽  
Low Complexity ◽  
Structural Features ◽  
Protein Fragments ◽  
Hydrophobic Residues ◽  
Backbone Conformation ◽  
Hydrophobic Amino Acids ◽  
Insight Into ◽  
Lateral Sclerosis

AbstractAmyotrophic lateral sclerosis and several other neurodegenerative diseases are associated with brain deposits of amyloid-like aggregates formed by the C-terminal fragments of TDP-43 that contain the low complexity domain of the protein. Here, we report the cryo-EM structure of amyloid formed from the entire TDP-43 low complexity domain in vitro at pH 4. This structure reveals single protofilament fibrils containing a large (139-residue), tightly packed core. While the C-terminal part of this core region is largely planar and characterized by a small proportion of hydrophobic amino acids, the N-terminal region contains numerous hydrophobic residues and has a non-planar backbone conformation, resulting in rugged surfaces of fibril ends. The structural features found in these fibrils differ from those previously found for fibrils generated from short protein fragments. The present atomic model for TDP-43 LCD fibrils provides insight into potential structural perturbations caused by phosphorylation and disease-related mutations.

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.

Roles of the cytoplasmic and transmembrane domains of syntaxins in intracellular localization and trafficking

2001 ◽  
Vol 114 (17) ◽  
pp. 3115-3124 ◽  
Author(s):  
Kazuo Kasai ◽  
Kimio Akagawa
Keyword(s):  
Plasma Membrane ◽  
Transmembrane Domain ◽  
Intracellular Localization ◽  
Transmembrane Domains ◽  
Cytoplasmic Domains ◽  
Immunofluorescence Analysis ◽  
Attachment Protein ◽  
Transport Pathways ◽  
Sensitive Factor ◽  
Protein Receptors

Syntaxins are target-soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (t-SNAREs) involved in docking and fusion of vesicles in exocytosis and endocytosis. Many syntaxin isoforms have been isolated, and each one displays a distinct intracellular localization pattern. However, the signals that drive the specific intracellular localization of syntaxins are poorly understood. In this study, we used indirect immunofluorescence analysis to examine the localization of syntaxin chimeras, each containing a syntaxin transmembrane domain fused to a cytoplasmic domain derived from a different syntaxin. We show that the cytoplasmic domains of syntaxins 5, 6, 7 and 8 have important effects on intracellular localization. We also demonstrate that the transmembrane domain of syntaxin 5 is sufficient to localize the chimera to the compartment expected for wild-type syntaxin 5. Additionally, we find that syntaxins 6, 7 and 8, but not syntaxin 5, are present at the plasma membrane, and that these syntaxins cycle through the plasma membrane by virtue of their cytoplasmic domains. Finally, we find that di-leucine-based motifs in the cytoplasmic domains of syntaxins 7 and 8 are necessary for their intracellular localization and trafficking via distinct transport pathways. Combined, these results suggest that both the cytoplasmic and the transmembrane domains play important roles in intracellular localization and trafficking of syntaxins.

Potential aggregation hot spots in recombinant human keratinocyte growth factor; a computational study

2020 ◽  
Author(s):  
Mansoureh Shahbazi Dastjerdeh ◽  
Mohammad Ali Shokrgozar ◽  
Hamzeh Rahimi ◽  
Majid Golkar
Keyword(s):  
Growth Factor ◽  
Tertiary Structure ◽  
Computational Study ◽  
Keratinocyte Growth Factor ◽  
Native State ◽  
Hydrophobic Residues ◽  
Aggregation Propensity ◽  
State Aggregation ◽  
Human Keratinocyte ◽  
Aggregation Tendency

Abstract Background: Recombinant human keratinocyte growth factor is a highly aggregation-prone therapeutic protein. The high aggregation liability of rhKGF is manifested by loss of the monomeric form of the protein and accumulation of the aggregated species even at moderate temperatures. Here, we analyzed rhKGF for its vulnerability towards aggregation by detection of aggregation-prone regions (APRs) using several sequence-based computational tools including TANGO, SolubiS, ZipperDB, AGGRESCAN, Zyggregator, Camsol, PASTA, SALSA, WALTZ, SODA, Amylpred, AMYPDB, and structure-based tools including Aggrescan3D and molecular dynamics-based spatial aggregation propensity (SAP) algorithm. Results: The sequence-based prediction of APRs in rhKGF indicated that they are mainly located at positions 10-30, 40-60, 61-66, 88-120, and 130-140 which are rich in β-branched aliphatic, hydrophobic, aromatic and Glutamine/Aspargine (Q/N) residues. Mapping on the rhKGF tertiary structure revealed that most of these residues including F16-R25, I43, E45, R47-I56, F61, Y62, N66, L88-E91, E108-F110, A112, N114, T131, and H133-T140 are surface-exposed in the natively folded protein which can promote aggregation without major unfolding event or the conformational change may occur in the oligomers composed of natively folded monomers. The other regions are buried in the native state and their contribution to non-native aggregation is mediated by a preceding unfolding event in the monomeric state of the protein. The structure-based prediction of APRs using SAP tool limited the number of identified APRs to the dynamically-exposed hydrophobic residues including V12, A50, V51, L88, I89, L90, I118, L135, and I139 mediating the native-state aggregation. Conclusion: Our analysis of APRs in rhKGF identified the regions determining the intrinsic aggregation propensity in both folded (native) and unfolded state of the protein. These regions are the candidate positions for engineering the rhKGF sequence to reduce its aggregation tendency.

Transmembrane domain length determines intracellular membrane compartment localization of syntaxins 3, 4, and 5

2001 ◽  
Vol 281 (1) ◽  
pp. C215-C223 ◽  
Author(s):  
Robert T. Watson ◽  
Jeffrey E. Pessin
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Amino Acid ◽  
Glucose Transporter ◽  
Transmembrane Domain ◽  
Transmembrane Domains ◽  
Snare Protein ◽  
Glut 4 ◽  
Golgi Localization ◽  
Syntaxin 3

Insulin recruits glucose transporter 4 (GLUT-4) vesicles from intracellular stores to the plasma membrane in muscle and adipose tissue by specific interactions between the vesicle membrane-soluble N-ethylmaleimide-sensitive factor attachment protein target receptor (SNARE) protein VAMP-2 and the target membrane SNARE protein syntaxin 4. Although GLUT-4 vesicle trafficking has been intensely studied, few have focused on the mechanism by which the SNAREs themselves localize to specific membrane compartments. We therefore set out to identify the molecular determinants for localizing several syntaxin isoforms, including syntaxins 3, 4, and 5, to their respective intracellular compartments (plasma membrane for syntaxins 3 and 4; cis-Golgi for syntaxin 5). Analysis of a series of deletion and chimeric syntaxin constructs revealed that the 17-amino acid transmembrane domain of syntaxin 5 was sufficient to direct the cis-Golgi localization of several heterologous reporter constructs. In contrast, the longer 25-amino acid transmembrane domain of syntaxin 3 was sufficient to localize reporter constructs to the plasma membrane. Furthermore, truncation of the syntaxin 3 transmembrane domain to 17 amino acids resulted in a complete conversion to cis-Golgi compartmentalization that was indistinguishable from syntaxin 5. These data support a model wherein short transmembrane domains (≤17 amino acids) direct the cis-Golgi localization of syntaxins, whereas long transmembrane domains (≥23 amino acids) direct plasma membrane localization.

scholarly journals Role of a DEF/Y motif in histone H2A-H2B recognition and nucleosome editing

2020 ◽  
Vol 117 (7) ◽  
pp. 3543-3550 ◽  
Author(s):  
Yan Huang ◽  
Lu Sun ◽  
Leonidas Pierrakeas ◽  
Linchang Dai ◽  
Lu Pan ◽  
...  
Keyword(s):  
Dna Interaction ◽  
General Strategy ◽  
Histone H2a ◽  
Hydrophobic Residues ◽  
Intrinsically Disordered ◽  
Chromatin Regulators ◽  
A Subunit

The SWR complex edits the histone composition of nucleosomes at promoters to facilitate transcription by replacing the two nucleosomal H2A-H2B (A-B) dimers with H2A.Z-H2B (Z-B) dimers. Swc5, a subunit of SWR, binds to A-B dimers, but its role in the histone replacement reaction was unclear. In this study, we showed that Swc5 uses a tandem DEF/Y motif within an intrinsically disordered region to engage the A-B dimer. A 2.37-Å X-ray crystal structure of the histone binding domain of Swc5 in complex with an A-B dimer showed that consecutive acidic residues and flanking hydrophobic residues of Swc5 form a cap over the histones, excluding histone–DNA interaction. Mutations in Swc5 DEF/Y inhibited the nucleosome editing function of SWR in vitro. Swc5 DEF/Y interacts with histones in vivo, and the extent of this interaction is dependent on the remodeling ATPase of SWR, supporting a model in which Swc5 acts as a wedge to promote A-B dimer eviction. Given that DEF/Y motifs are found in other evolutionary unrelated chromatin regulators, this work provides the molecular basis for a general strategy used repeatedly during eukaryotic evolution to mobilize histones in various genomic functions.

scholarly journals Conformational rearrangements in the transmembrane domain of CNGA1 channels revealed by single-molecule force spectroscopy

2015 ◽  
Vol 6 (1) ◽  
Author(s):  
Sourav Maity ◽  
Monica Mazzolini ◽  
Manuel Arcangeletti ◽  
Alejandro Valbuena ◽  
Paolo Fabris ◽  
...  
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Transmembrane Domain ◽  
Force Spectroscopy ◽  
Dynamic Structure ◽  
Transmembrane Domains ◽  
Structural Studies ◽  
Single Molecule Force Spectroscopy ◽  
Conformational Rearrangements ◽  
Α Helix

Abstract Cyclic nucleotide-gated (CNG) channels are activated by binding of cyclic nucleotides. Although structural studies have identified the channel pore and selectivity filter, conformation changes associated with gating remain poorly understood. Here we combine single-molecule force spectroscopy (SMFS) with mutagenesis, bioinformatics and electrophysiology to study conformational changes associated with gating. By expressing functional channels with SMFS fingerprints in Xenopus laevis oocytes, we were able to investigate gating of CNGA1 in a physiological-like membrane. Force spectra determined that the S4 transmembrane domain is mechanically coupled to S5 in the open state, but S3 in the closed state. We also show there are multiple pathways for the unfolding of the transmembrane domains, probably caused by a different degree of α-helix folding. This approach demonstrates that CNG transmembrane domains have dynamic structure and establishes SMFS as a tool for probing conformational change in ion channels.

scholarly journals Mapping the functional anatomy of Orai1 transmembrane domains for CRAC channel gating

2018 ◽  
Vol 115 (22) ◽  
pp. E5193-E5202 ◽  
Author(s):  
Priscilla S.-W. Yeung ◽  
Megumi Yamashita ◽  
Christopher E. Ing ◽  
Régis Pomès ◽  
Douglas M. Freymann ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Mutational Analysis ◽  
Channel Gating ◽  
Functional Anatomy ◽  
Transmembrane Domains ◽  
Hydrophobic Residues ◽  
Binding Domains ◽  
Allosteric Interactions ◽  
Crac Channel ◽  
Dynamics Simulations

Store-operated Orai1 channels are activated through a unique inside-out mechanism involving binding of the endoplasmic reticulum Ca2+ sensor STIM1 to cytoplasmic sites on Orai1. Although atomic-level details of Orai structure, including the pore and putative ligand binding domains, are resolved, how the gating signal is communicated to the pore and opens the gate is unknown. To address this issue, we used scanning mutagenesis to identify 15 residues in transmembrane domains (TMs) 1–4 whose perturbation activates Orai1 channels independently of STIM1. Cysteine accessibility analysis and molecular-dynamics simulations indicated that constitutive activation of the most robust variant, H134S, arises from a pore conformational change that opens a hydrophobic gate to augment pore hydration, similar to gating evoked by STIM1. Mutational analysis of this locus suggests that H134 acts as steric brake to stabilize the closed state of the channel. In addition, atomic packing analysis revealed distinct functional contacts between the TM1 pore helix and the surrounding TM2/3 helices, including one set mediated by a cluster of interdigitating hydrophobic residues and another by alternative ridges of polar and hydrophobic residues. Perturbing these contacts via mutagenesis destabilizes STIM1-mediated Orai1 channel gating, indicating that these bridges between TM1 and the surrounding TM2/3 ring are critical for conveying the gating signal to the pore. These findings help develop a framework for understanding the global conformational changes and allosteric interactions between topologically distinct domains that are essential for activation of Orai1 channels.

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