Identification of an “α-helix-extended segment-α-helix” conformation of the sixth transmembrane domain in DMT1

The chloroplastic outer envelope protein Toc34 is inserted into the membrane by a COOH-terminal membrane anchor domain in the orientation Ncyto-Cin. The insertion is independent of ATP and a cleavable transit sequence. The cytosolic domain of Toc34 does not influence the insertion process and can be replaced by a different hydrophilic reporter peptide. Inversion of the COOH-terminal, 45-residue segment, including the membrane anchor domain (Toc34Cinv), resulted in an inverted topology of the protein, i.e., Nin-Ccyto. A mutual exchange of the charged amino acid residues NH2- and COOH-proximal of the hydrophobic α-helix indicates that a double-positive charge at the cytosolic side of the transmembrane α-helix is the sole determinant for its topology. When the inverted COOH-terminal segment was fused to the chloroplastic precursor of the ribulose-1,5-bisphosphate carboxylase small subunit (pS34Cinv), it engaged the transit sequence–dependent import pathway. The inverted peptide domain of Toc34 functions as a stop transfer signal and is released out of the outer envelope protein translocation machinery into the lipid phase. Simultaneously, the NH2-terminal part of the hybrid precursor remained engaged in the inner envelope protein translocon, which could be reversed by the removal of ATP, demonstrating that only an energy-dependent force but no further ionic interactions kept the precursor in the import machinery.

Download Full-text

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

Nature Communications ◽

10.1038/ncomms8093 ◽

2015 ◽

Vol 6 (1) ◽

Cited By ~ 16

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.

Download Full-text

Identification of a Contact Site for Residue 19 of Parathyroid Hormone (PTH) and PTH-Related Protein Analogs in Transmembrane Domain Two of the Type 1 PTH Receptor

Molecular Endocrinology ◽

10.1210/me.2003-0275 ◽

2003 ◽

Vol 17 (12) ◽

pp. 2647-2658 ◽

Cited By ~ 41

Author(s):

Robert C. Gensure ◽

Naoto Shimizu ◽

Janet Tsang ◽

Thomas J. Gardella

Keyword(s):

Transmembrane Domain ◽

Molecular Model ◽

Cross Linking ◽

Conformationally Constrained ◽

Functional Studies ◽

Extracellular Loops ◽

Mapping Analysis ◽

J Domain ◽

Α Helix

Abstract Recent functional studies have suggested that position 19 in PTH interacts with the portion of the PTH-1 receptor (P1R) that contains the extracellular loops and seven transmembrance helices (TMs) (the J domain). We tested this hypothesis using the photoaffinity cross-linking approach. A PTHrP(1–36) analog and a conformationally constrained PTH(1–21) analog, each containing para-benzoyl-l-phenylalanine (Bpa) at position 19, each cross-linked efficiently to the P1R expressed in COS-7 cells, and digestive mapping analysis localized the cross-linked site to the interval (Leu232-Lys240) at the extracellular end of TM2. Point mutation analysis identified Ala234, Val235, and Lys240 as determinants of cross-linking efficiency, and the Lys240→Ala mutation selectively impaired the binding of PTH(1–21) and PTH(1–19) analogs, relative to that of PTH(1–15) analogs. The findings support the hypothesis that residue 19 of the receptor-bound ligand contacts, or is close to, the P1R J domain—specifically, Lys240 at the extracellular end of TM2. The findings also support a molecular model in which the 1–21 region of PTH binds to the extracellular face of the P1R J domain as an α-helix.

Download Full-text

The Vaccinia Virus 14-Kilodalton (A27L) Fusion Protein Forms a Triple Coiled-Coil Structure and Interacts with the 21-Kilodalton (A17L) Virus Membrane Protein through a C-Terminal α-Helix

Journal of Virology ◽

10.1128/jvi.72.12.10126-10137.1998 ◽

1998 ◽

Vol 72 (12) ◽

pp. 10126-10137 ◽

Cited By ~ 44

Author(s):

María-Isabel Vázquez ◽

German Rivas ◽

David Cregut ◽

Luis Serrano ◽

Mariano Esteban

Keyword(s):

Amino Acids ◽

Vaccinia Virus ◽

Structural Model ◽

Analytical Ultracentrifugation ◽

Transmembrane Domain ◽

Coiled Coil ◽

C Terminus ◽

Α Helix ◽

Mutant Forms ◽

Helical Region

ABSTRACT The vaccinia virus 14-kDa protein (encoded by the A27L gene) plays an important role in the biology of the virus, acting in virus-to-cell and cell-to-cell fusions. The protein is located on the surface of the intracellular mature virus form and is essential for both the release of extracellular enveloped virus from the cells and virus spread. Sequence analysis predicts the existence of four regions in this protein: a structureless region from amino acids 1 to 28, a helical region from residues 29 to 37, a triple coiled-coil helical region from residues 44 to 72, and a Leu zipper motif at the C terminus. Circular dichroism spectroscopy, analytical ultracentrifugation, and chemical cross-linking studies of the purified wild-type protein and several mutant forms, lacking one or more of the above regions or with point mutations, support the above-described structural division of the 14-kDa protein. The two contiguous cysteine residues at positions 71 and 72 are not responsible for the formation of 14-kDa protein trimers. The location of hydrophobic residues at the a and d positions on a helical wheel and of charged amino acids in adjacent positions, e and g, suggests that the hydrophobic and ionic interactions in the triple coiled-coil helical region are involved in oligomer formation. This conjecture was supported by the construction of a three-helix bundle model and molecular dynamics. Binding assays with purified proteins expressed in Escherichia coli and cytoplasmic extracts from cells infected with a virus that does not produce the 14-kDa protein during infection (VVindA27L) show that the 21-kDa protein (encoded by the A17L gene) is the specific viral binding partner and identify the putative Leu zipper, the predicted third α-helix on the C terminus of the 14-kDa protein, as the region involved in protein binding. These findings were confirmed in vivo, following transfection of animal cells with plasmid vectors expressing mutant forms of the 14-kDa protein and infected with VVindA27L. We find the structural organization of 14kDa to be similar to that of other fusion proteins, such as hemagglutinin of influenza virus and gp41 of human immunodeficiency virus, except for the presence of a protein-anchoring domain instead of a transmembrane domain. Based on our observations, we have established a structural model of the 14-kDa protein.

Download Full-text

Sec22b-dependent assembly of endoplasmic reticulum Q-SNARE proteins

Biochemical Journal ◽

10.1042/bj20071304 ◽

2008 ◽

Vol 410 (1) ◽

pp. 93-100 ◽

Cited By ~ 21

Author(s):

Takehiro Aoki ◽

Masaki Kojima ◽

Katsuko Tani ◽

Mitsuo Tagaya

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Fusion ◽

Transmembrane Domain ◽

Cd Spectroscopy ◽

Snare Proteins ◽

Peripheral Membrane Proteins ◽

Protein Receptor ◽

Snare Motif ◽

Α Helix ◽

Protein Attachment

SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) proteins involved in membrane fusion usually contain a conserved α-helix (SNARE motif) that is flanked by a C-terminal transmembrane domain. They can be classified into Q-SNARE and R-SNARE based on the structural property of their motifs. Assembly of four SNARE motifs (Qa, b, c and R) is supposed to trigger membrane fusion. We have previously shown that ER (endoplasmic reticulum)-localized syntaxin 18 (Qa) forms a complex with BNIP1 (Qb), p31/Use1 (Qc), Sec22b (R) and several peripheral membrane proteins. In the present study, we examined the interaction of syntaxin 18 with other SNAREs using pulldown assays and CD spectroscopy. We found that the association of syntaxin 18 with Sec22b induces an increase in α-helicity of their SNARE motifs, which results in the formation of high-affinity binding sites for BNIP1 and p31. This R-SNARE-dependent Q-SNARE assembly is quite different from the assembly mechanisms of SNAREs localized in organelles other than the ER. The implication of the mechanism of ER SNARE assembly is discussed in the context of the physiological roles of the syntaxin 18 complex.

Download Full-text

Anisotropic rotational motions of poly(L-glutamic acid) in the α-helix conformation

Biopolymers ◽

10.1002/bip.360240704 ◽

1985 ◽

Vol 24 (7) ◽

pp. 1147-1156 ◽

Cited By ~ 3

Author(s):

U. Hahn ◽

H. Hanssum ◽

H. Rüterjans

Keyword(s):

Glutamic Acid ◽

Helix Conformation ◽

Rotational Motions ◽

Α Helix

Download Full-text

The Antimicrobial Peptides P-113Du and P-113Tri Function against Candida albicans

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00699-16 ◽

2016 ◽

Vol 60 (10) ◽

pp. 6369-6373 ◽

Cited By ~ 7

Author(s):

Guan-Yu Lin ◽

Hsueh-Fen Chen ◽

Yao-Peng Xue ◽

Ying-Chieh Yeh ◽

Chia-Lu Chen ◽

...

Keyword(s):

Candida Albicans ◽

Antifungal Activity ◽

Antimicrobial Peptides ◽

Antifungal Agents ◽

Low Ph ◽

Planktonic Cells ◽

Content Type ◽

Peptide Derivatives ◽

Helix Conformation ◽

Α Helix

ABSTRACTTwo antimicrobial P-113 peptide derivatives, P-113Du and P-113Tri, were investigated in this study. Notably, P-113Du and P-113Tri contained significant fractions of α-helix conformation and were less sensitive to high salt and low pH than P-113. Moreover, compared to P-113, these peptides exhibited increased antifungal activity against planktonic cells, biofilm cells, and clinical isolates ofCandida albicansand non-albicans Candidaspp. These results suggest that P-113Du and P-113Tri are promising candidates for development as novel antifungal agents.

Download Full-text

The α-helix conformation of polypeptides in solution: A re-evaluation of the small-angle X-ray scattering evidence

Journal of Molecular Biology ◽

10.1016/s0022-2836(66)80137-7 ◽

1966 ◽

Vol 15 (2) ◽

pp. 681-682 ◽

Cited By ~ 11

Author(s):

P. Saludjian ◽

V. Luzzati

Keyword(s):

Small Angle ◽

X Ray ◽

X Ray Scattering ◽

Helix Conformation ◽

Α Helix ◽

Ray Scattering

Download Full-text

Human immunodeficiency virus-1 BF intersubtype recombinant viral protein U second alpha helix plays an important role in viral release and BST-2 degradation

Journal of General Virology ◽

10.1099/vir.0.047746-0 ◽

2013 ◽

Vol 94 (4) ◽

pp. 758-766 ◽

Cited By ~ 1

Author(s):

Cristian De Candia ◽

Constanza Espada ◽

Gabriel Duette ◽

Horacio Salomón ◽

Mauricio Carobene

Keyword(s):

Viral Protein ◽

Transmembrane Domain ◽

Alpha Helix ◽

Protein Variant ◽

Viral Release ◽

Subtype B ◽

Viral Protein U ◽

Intersubtype Recombination ◽

Α Helix ◽

The Impact

We previously reported a naturally occurring BF intersubtype recombinant viral protein U (Vpu) variant with an augmented capacity to enhance viral replication. Structural analysis of this variant revealed that its transmembrane domain and α-helix I in the cytoplasmic domain (CTD) corresponded to subtype B, whereas the α-helix II in the CTD corresponded to subtype F1. In this study, we aimed to evaluate the role of the Vpu cytoplasmic α-helix II domain in viral release enhancement and in the down-modulation of BST-2 and CD4 from the cell surface. In addition, as serine residues in Vpu amino acid positions 61 or 64 have been shown to regulate Vpu intracellular half-life, which in turn could influence the magnitude of viral release, we also studied the impact of these residues on the VpuBF functions, since S61 and S64 are infrequently found among BF recombinant Vpu variants. Our results showed that the exchange of Vpu α-helix II between subtypes (B→F) directly correlated with the enhancement of viral release and, to a lesser extent, with changes in the capacity of the resulting chimera to down-modulate BST-2 and CD4. No differences in viral release and BST-2 down-modulation were observed between VpuBF and VpuBF-E61S. On the other hand, VpuBF-A64S showed a slightly reduced capacity to enhance viral production, but was modestly more efficient than VpuBF in down-modulating BST-2. In summary, our observations clearly indicate that α-helix II is actively involved in Vpu viral-release-promoting activity and that intersubtype recombination between subtypes B and F1 created a protein variant with a higher potential to boost the spread of the recombinant strain that harbours it.

Download Full-text