scholarly journals Heptad repeat regions of coiled-coil domains of Mfn1 are crucial for Mfn1 mediated mitochondrial fusion

2018 ◽  
Author(s):  
Sansrity Sinha ◽  
Gopala Krishna Aradhyam
Keyword(s):  
Coiled Coil ◽  
Helical Structure ◽  
Heptad Repeat ◽  
Outer Mitochondrial Membrane ◽  
Mitochondrial Membranes ◽  
Gtpase Domain ◽  
Helical Bundles ◽  
Gtpase Effector Domain ◽  
Heptad Repeats

Mitofusin mediate fusion of outer mitochondrial membranes (OMM). Recent studies have explained the role of GTPase domain for Mfn1 dimerization and outer mitochondrial membrane (OMM) fusion. Coiled-coiled domains [namely, coiled-coil/Middle domains (CC1MD) and coiled-coil-2 GTPase effector domain (CC2/GED)] form helical bundles that mediate open-to-close conformations of Mfn1 upon GTP binding and have been previously reported to be important for OMM tethering and OMM fusion. To further decipher the significance of helical structure of MD, we functionally characterized the heptad repeat regions of MD. Consistent with previous studies, we show that MD consists of two heptad repeats (HR1, namely HR1a and HR1b) and both of these are crucial for Mfn1 mediated OMM fusion.

scholarly journals Extremely Thermostabilizing Core Mutations in Coiled-Coil Mimetic Proteins of HIV-1 gp41 Produce Diverse Effects on Target Binding but Do Not Affect Their Inhibitory Activity

Biomolecules ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 566
Author(s):  
Mario Cano-Muñoz ◽  
Samuele Cesaro ◽  
Bertrand Morel ◽  
Julie Lucas ◽  
Christiane Moog ◽  
...  
Keyword(s):  
Inhibitory Activity ◽  
Coiled Coil ◽  
Helical Structure ◽  
Heptad Repeat ◽  
Single Chain ◽  
Cell Infection ◽  
Polar Residues ◽  
Hiv 1

A promising strategy to neutralize HIV-1 is to target the gp41 spike subunit to block membrane fusion with the cell. We previously designed a series of single-chain proteins (named covNHR) that mimic the trimeric coiled-coil structure of the gp41 N-terminal heptad repeat (NHR) region and potently inhibit HIV-1 cell infection by avidly binding the complementary C-terminal heptad repeat (CHR) region. These proteins constitute excellent tools to understand the structural and thermodynamic features of this therapeutically important interaction. Gp41, as with many coiled-coil proteins, contains in core positions of the NHR trimer several highly conserved, buried polar residues, the role of which in gp41 structure and function is unclear. Here we produced three covNHR mutants by substituting each triad of polar residues for the canonical isoleucine. The mutants preserve their helical structure and show an extremely increased thermal stability. However, increased hydrophobicity enhances their self-association. Calorimetric analyses show a marked influence of mutations on the binding thermodynamics of CHR-derived peptides. The mutations do not affect however the in vitro HIV-1 inhibitory activity of the proteins. The results support a role of buried core polar residues in maintaining structural uniqueness and promoting an energetic coupling between conformational stability and NHR–CHR binding.

Guidelines for the assembly of novel coiled-coil structures: α-sheets and α-cylinders

2001 ◽  
Vol 68 ◽  
pp. 111-123 ◽  
Author(s):  
John Walshaw ◽  
Jennifer M. Shipway ◽  
Derek N. Woolfson
Keyword(s):  
Protein Design ◽  
Protein Interactions ◽  
Protein Structures ◽  
Coiled Coil ◽  
Data Bank ◽  
Coiled Coils ◽  
Heptad Repeat ◽  
Protein Protein Interactions ◽  
Helical Bundles ◽  
Heptad Repeats

The coiled coil is a ubiquitous motif that guides many different protein-protein interactions. The accepted hallmark of coiled coils is a seven-residue (heptad) sequence repeat. The positions of this repeat are labelled a-b-c-d-e-f-g, with residues at a and d tending to be hydrophobic. Such sequences form amphipathic α-helices, which assemble into helical bundles via knobs-into-holes interdigitation of residues from neighbouring helices. We wrote an algorithm, SOCKET, to identify this packing in protein structures, and used this to gather a database of coiled-coil structures from the Protein Data Bank. Surprisingly, in addition to commonly accepted structures with a single, contiguous heptad repeat, we identified sequences with multiple, offset heptad repeats. These 'new' sequence patterns help to explain oligomer-state specification in coiled coils. Here we focus on the structural consequences for sequences with two heptad repeats offset by two residues, i.e. a/f′-b/g′-c/a′-d/b′-e/c′-f/d′-g/e′. This sets up two hydrophobic seams on opposite sides of the helix formed. We describe how such helices may combine to bury these hydrophobic surfaces in two different ways and form two distinct structures: open 'α-sheets' and closed 'α-cylinders'. We highlight these with descriptions of natural structures and outline possibilities for protein design.

Expression in Escherichia coli of fragments of the coiled-coil rod domain of rabbit myosin: influence of different regions of the molecule on aggregation and paracrystal formation

1991 ◽  
Vol 99 (4) ◽  
pp. 823-836
Author(s):  
S.J. Atkinson ◽  
M. Stewart
Keyword(s):  
Escherichia Coli ◽  
Ionic Strength ◽  
Coiled Coil ◽  
Periodic Variation ◽  
Heptad Repeat ◽  
C Terminus ◽  
N Terminus ◽  
Low Ionic Strength ◽  
Myosin Rod

We have expressed in Escherichia coli a cDNA clone corresponding broadly to rabbit light meromyosin (LMM) together with a number of modified polypeptides and have used this material to investigate the role of different aspects of molecular structure on the solubility properties of LMM. The expressed material was characterized biochemically and structurally to ensure that it retained the coiled-coil conformation of the native molecule. Full-length recombinant LMM retained the general solubility properties of myosin and, although soluble at high ionic strength, precipitated when the ionic strength was reduced below 0.3 M. Constructs in which the ‘skip’ residues (that disrupt the coiled-coil heptad repeat) were deleted had solubility properties indistinguishable from the wild type, which indicated that the skip residues did not play a major role in determining the molecular interactions involved in assembly. Deletions from the N terminus of LMM did not alter the solubility properties of the expressed material, but deletion of 92 residues from the C terminus caused a large increase in solubility at low ionic strength, indicating that a determinant important for interaction between LMM molecules was located in this region. The failure of deletions from the molecule's N terminus to alter its solubility radically suggested that the periodic variation of charge along the myosin rod may not be as important as proposed for determining the strength of binding between molecules and thus the solubility of myosin.

scholarly journals Probing Protein Folding with Sequence-Reversed α-Helical Bundles

2021 ◽  
Vol 22 (4) ◽  
pp. 1955
Author(s):  
Aikaterini Kefala ◽  
Maria Amprazi ◽  
Efstratios Mylonas ◽  
Dina Kotsifaki ◽  
Mary Providaki ◽  
...  
Keyword(s):  
Protein Folding ◽  
Amino Acid ◽  
Amino Acid Sequences ◽  
Size Exclusion ◽  
Heptad Repeat ◽  
Amino Acid Residues ◽  
Helical Bundles ◽  
X Ray Scattering ◽  
Exclusion Chromatography ◽  
Heptad Repeats

Recurrent protein folding motifs include various types of helical bundles formed by α-helices that supercoil around each other. While specific patterns of amino acid residues (heptad repeats) characterize the highly versatile folding motif of four-α-helical bundles, the significance of the polypeptide chain directionality is not sufficiently understood, although it determines sequence patterns, helical dipoles, and other parameters for the folding and oligomerization processes of bundles. To investigate directionality aspects in sequence-structure relationships, we reversed the amino acid sequences of two well-characterized, highly regular four-α-helical bundle proteins and studied the folding, oligomerization, and structural properties of the retro-proteins, using Circular Dichroism Spectroscopy (CD), Size Exclusion Chromatography combined with Multi-Angle Laser Light Scattering (SEC-MALS), and Small Angle X-ray Scattering (SAXS). The comparison of the parent proteins with their retro-counterparts reveals that while the α-helical character of the parents is affected to varying degrees by sequence reversal, the folding states, oligomerization propensities, structural stabilities, and shapes of the new molecules strongly depend on the characteristics of the heptad repeat patterns. The highest similarities between parent and retro-proteins are associated with the presence of uninterrupted heptad patterns in helical bundles sequences.

scholarly journals Human Immunodeficiency Virus (HIV) gp41 Escape Mutants: Cross-Resistance to Peptide Inhibitors of HIV Fusion and Altered Receptor Activation of gp120

2005 ◽  
Vol 79 (8) ◽  
pp. 4774-4781 ◽  
Author(s):  
Emmanuel Desmezieres ◽  
Nidhi Gupta ◽  
Russell Vassell ◽  
Yong He ◽  
Keith Peden ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Coiled Coil ◽  
Receptor Activation ◽  
Peptide Inhibitors ◽  
Dominant Negative ◽  
Heptad Repeat ◽  
Cross Resistance ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Heptad Repeats

ABSTRACT Human immunodeficiency virus (HIV) infects cells by fusing with cellular membranes. Fusion occurs when the envelope glycoprotein (Env) undergoes conformational changes while binding to cellular receptors. Fusogenic changes involve assembly of two heptad repeats in the ectodomain of the gp41 transmembrane subunit to form a six-helix bundle (6HB), consisting of a trimeric N heptad repeat (N-HR) coiled-coil core with three antiparallel C heptad repeats (C-HRs) that pack in the coiled-coil grooves. Peptides corresponding to the N-and C-HRs (N and C peptides, respectively) interfere with formation of the 6HB in a dominant-negative manner and are emerging as a new class of antiretroviral therapeutics for treating HIV infection. We generated an escape mutant virus with resistance to an N peptide and show that early resistance involved two mutations, one each in the N- and C-HRs. The mutations conferred resistance not only to the selecting N peptide but also to C peptides, as well as other types of N-peptide inhibitors. Moreover, the N-HR mutation altered sensitivity to soluble CD4. Biophysical studies suggest that the 6HB with the resistance mutations is more stable than the wild-type 6HB and the 6HB formed by inhibitor binding to either wild-type or mutant C-HR. These findings provide new insights into potential mechanisms of resistance to HIV peptide fusion inhibitors and dominant-negative inhibitors in general. The results are discussed in the context of current models of Env-mediated membrane fusion.

scholarly journals The role of Fis1p–Mdv1p interactions in mitochondrial fission complex assembly

2005 ◽  
Vol 171 (2) ◽  
pp. 291-301 ◽  
Author(s):  
Mary Anne Karren ◽  
Emily M. Coonrod ◽  
Teresa K. Anderson ◽  
Janet M. Shaw
Keyword(s):  
Mitochondrial Membrane ◽  
Mitochondrial Fission ◽  
Coiled Coil ◽  
Cytoplasmic Domain ◽  
Tetratricopeptide Repeat ◽  
Outer Mitochondrial Membrane ◽  
Complex Assembly ◽  
Binding Surface ◽  
New Evidence

Mitochondrial division requires coordinated interactions among Fis1p, Mdv1p, and the Dnm1p GTPase, which assemble into fission complexes on the outer mitochondrial membrane. The integral outer membrane protein Fis1p contains a cytoplasmic domain consisting of a tetratricopeptide repeat (TPR)–like fold and a short NH2-terminal helix. Although it is known that the cytoplasmic domain is necessary for assembly of Mdv1p and Dnm1p into fission complexes, the molecular details of this assembly are not clear. In this study, we provide new evidence that the Fis1p–Mdv1p interaction is direct. Furthermore, we show that conditional mutations in the Fis1p TPR-like domain cause fission complex assembly defects that are suppressed by mutations in the Mdv1p-predicted coiled coil. We also define separable functions for the Fis1p NH2-terminal arm and TPR-like fold. These studies suggest that the concave binding surface of the Fis1p TPR-like fold interacts with Mdv1p during mitochondrial fission and that Mdv1p facilitates Dnm1p recruitment into functional fission complexes.

scholarly journals Examination of a Fusogenic Hexameric Core from Human Metapneumovirus and Identification of a Potent Synthetic Peptide Inhibitor from the Heptad Repeat 1 Region

2006 ◽  
Vol 81 (1) ◽  
pp. 141-149 ◽  
Author(s):  
Scott A. Miller ◽  
Sharon Tollefson ◽  
James E. Crowe ◽  
John V. Williams ◽  
David W. Wright
Keyword(s):  
Membrane Proteins ◽  
Coiled Coil ◽  
Helical Structure ◽  
Human Metapneumovirus ◽  
Integral Membrane Proteins ◽  
Heptad Repeat ◽  
Type I ◽  
Viral Fusion ◽  
Fusion Inhibitors ◽  
Major Player

ABSTRACT Paramyxoviruses are a leading cause of childhood illness worldwide. A recently discovered paramyxovirus, human metapneumovirus (hMPV), has been studied by our group in order to determine the structural relevance of its fusion (F) protein to other well-characterized viruses utilizing type I integral membrane proteins as fusion aids. Sequence analysis and homology models suggested the presence of requisite heptad repeat (HR) regions. Synthetic peptides from HR regions 1 and 2 (HR-1 and -2, respectively) were induced to form a thermostable (melting temperature, ∼90°C) helical structure consistent in mass with a hexameric coiled coil. Inhibitory studies of hMPV HR-1 and -2 indicated that the synthetic HR-1 peptide was a significant fusion inhibitor with a 50% inhibitory concentration and a 50% effective concentration of ∼50 nM. Many viral fusion proteins are type I integral membrane proteins utilizing the formation of a hexameric coiled coil of HR peptides as a major driving force for fusion. Our studies provide evidence that hMPV also uses a coiled-coil structure as a major player in the fusion process. Additionally, viral HR-1 peptide sequences may need further investigation as potent fusion inhibitors.

scholarly journals The trans-Golgi network GRIP-domain proteins form α-helical homodimers

2005 ◽  
Vol 388 (3) ◽  
pp. 835-841 ◽  
Author(s):  
Michael R. LUKE ◽  
Fiona HOUGHTON ◽  
Matthew A. PERUGINI ◽  
Paul A. GLEESON
Keyword(s):  
Protein Interactions ◽  
Coiled Coil ◽  
Helical Structure ◽  
Cd Spectroscopy ◽  
Protein Protein Interactions ◽  
Yeast Two Hybrid ◽  
Trans Golgi Network ◽  
Heptad Repeats ◽  
Golgi Network ◽  
Two Hybrid

A recently described family of TGN (trans-Golgi network) proteins, all of which contain a GRIP domain targeting sequence, has been proposed to play a role in membrane transport. On the basis of the high content of heptad repeats, GRIP domain proteins are predicted to contain extensive coiled-coil regions that have the potential to mediate protein–protein interactions. Four mammalian GRIP domain proteins have been identified which are targeted to the TGN through their GRIP domains, namely p230, golgin-97, GCC88 and GCC185. In the present study, we have investigated the ability of the four mammalian GRIP domain proteins to interact. Using a combination of immunoprecipitation experiments of epitope-tagged GRIP domain proteins, cross-linking experiments and yeast two-hybrid interactions, we have established that the GRIP proteins can self-associate to form homodimers exclusively. Two-hybrid analysis indicated that the N- and C-terminal fragments of GCC88 can interact with themselves but not with each other, suggesting that the GRIP domain proteins form parallel coiled-coil dimers. Analysis of purified recombinant golgin-97 by CD spectroscopy indicated a 67% α-helical structure, consistent with a high content of coiled-coil sequences. These results support a model for GRIP domain proteins as extended rod-like homodimeric molecules. The formation of homodimers, but not heterodimers, indicates that each of the four mammalian TGN golgins has the potential to function independently.

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