Abstract P-14: Molecular Modeling of the Transmembrane Domain of the SARS Cov-2 S-Protein and its Interaction with the Membrane

Background: The spike glycoprotein of SARS-coronavirus mediates the early events leading to infection of cells, including fusion of the viral and cellular membranes. The spike is a type I membrane glycoprotein that possesses a conserved transmembrane anchor and an unusual cysteine-rich domain that bridges the putative junction of the anchor and the cytoplasmic tail. In this study, we examined the role of these carboxyl-terminal domains in S-protein interaction with membrane. Methods: Structural model of the trimeric TM domain and adjacent fragments of ecto- and endo domains (residues 1157-1256) of the S-protein was built by homology basing on the solution structure of the SARS-coronavirus S-protein HR2 domain (pdb-code 2fxp), the structure of the transmembrane domain of HIV-1 gp41 in bicelle (5jyn), and assumption of generally coiled-coil fold of the considered domain. C-terminus of the domain was left unstructured but fully palmitoylated. Molecular dynamics simulation in heterogeneous lipid bilayer was prepared with CHARMM-GUI and performed with Gromacs during 100 ns. Results: 1. Ectodomain fragment (residues 1157-1212) demonstrates a tilt by the angle of 40-60 degrees from the axis of the TM domain (residues 1213-1237). This tilt is facilitated by glycine residues in position 1204. 2. Cholesterol molecules of the bottom layer tend to localize around protein due to interaction with palmitoyl tails while lipids in the upper layer do not show such tendency. Conclusion: Performed molecular simulations show that both palmitoylation and a large cluster of aromatic residues provide high stability of the S-protein TM domain.

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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.

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Genetic Analysis of Determinants for Spike Glycoprotein Assembly into Murine Coronavirus Virions: Distinct Roles for Charge-Rich and Cysteine-Rich Regions of the Endodomain

Journal of Virology ◽

10.1128/jvi.78.18.9904-9917.2004 ◽

2004 ◽

Vol 78 (18) ◽

pp. 9904-9917 ◽

Cited By ~ 42

Author(s):

Rong Ye ◽

Cynthia Montalto-Morrison ◽

Paul S. Masters

Keyword(s):

Transmembrane Domain ◽

Mutational Analysis ◽

Hepatitis Virus ◽

Transmembrane Protein ◽

Terminal Segment ◽

Type I ◽

Viral Fusion ◽

S Protein ◽

Infected Cells ◽

Carboxy Terminal

ABSTRACT The coronavirus spike protein (S) forms the distinctive virion surface structures that are characteristic of this viral family, appearing in negatively stained electron microscopy as stems capped with spherical bulbs. These structures are essential for the initiation of infection through attachment of the virus to cellular receptors followed by fusion to host cell membranes. The S protein can also mediate the formation of syncytia in infected cells. The S protein is a type I transmembrane protein that is very large compared to other viral fusion proteins, and all except a short carboxy-terminal segment of the S molecule constitutes the ectodomain. For the prototype coronavirus mouse hepatitis virus (MHV), it has previously been established that S protein assembly into virions is specified by the carboxy-terminal segment, which comprises the transmembrane domain and the endodomain. We have genetically dissected these domains in the MHV S protein to localize the determinants of S incorporation into virions. Our results establish that assembly competence maps to the endodomain of S, which was shown to be sufficient to target a heterologous integral membrane protein for incorporation into MHV virions. In particular, mutational analysis indicated a major role for the charge-rich carboxy-terminal region of the endodomain. Additionally, we found that the adjacent cysteine-rich region of the endodomain is critical for fusion of infected cells, confirming results previously obtained with S protein expression systems.

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A trimeric hydrophobic zipper mediates the intramembrane assembly of SARS-CoV-2 spike

10.1101/2021.04.09.439203 ◽

2021 ◽

Author(s):

Qingshan Fu ◽

James J Chou

Keyword(s):

Transmembrane Domain ◽

Structural Information ◽

Heptad Repeat ◽

Host Immune System ◽

Type I ◽

Fold Axis ◽

Viral Fusion ◽

S Protein ◽

Viral Fusion Protein ◽

Exact Function

The S protein of the SARS-CoV-2 is a Type I membrane protein that mediates membrane fusion and viral entry. A vast amount of structural information is available for the ectodomain of S, a primary target by the host immune system, but much less is known regarding its transmembrane domain (TMD) and its membrane-proximal regions. Here, we determined the nuclear magnetic resonance (NMR) structure of the S protein TMD in bicelles that closely mimic a lipid bilayer. The TMD structure is a transmembrane α-helix (TMH) trimer that assembles spontaneously in membrane. The trimer structure shows an extensive hydrophobic core along the 3-fold axis that resembles that of a trimeric leucine/isoleucine zipper, but with tetrad, not heptad, repeat. The trimeric core is strong in bicelles, resisting hydrogen-deuterium exchange for weeks. Although highly stable, structural guided mutagenesis identified single mutations that can completely dissociate the TMD trimer. Multiple studies have shown that the membrane anchor of viral fusion protein can form highly specific oligomers, but the exact function of these oligomers remain unclear. Our findings should guide future experiments to address the above question for SARS coronaviruses.

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GxxxG Motif of Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Transmembrane Domain Is Not Involved in Trimerization and Is Not Important for Entry

Journal of Virology ◽

10.1128/jvi.00014-07 ◽

2007 ◽

Vol 81 (15) ◽

pp. 8352-8355 ◽

Cited By ~ 3

Author(s):

Jeroen Corver ◽

Rene Broer ◽

Puck van Kasteren ◽

Willy Spaan

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Transmembrane Domain ◽

Vital Role ◽

Sars Coronavirus ◽

Spike Glycoprotein ◽

S Protein ◽

Mutant Proteins ◽

Gxxxg Motif

ABSTRACT Recently, a paper was published in which it was proposed that the GxxxG motif of the severe acute respiratory syndrome (SARS) coronavirus spike (S) protein transmembrane domain plays a vital role in oligomerization of the protein (E. Arbely, Z. Granot, I. Kass, J. Orly, and I. T. Arkin, Biochemistry 45:11349-11356, 2006). Here, we show that the GxxxG motif is not involved in SARS S oligomerization by trimerization analysis of S GxxxG mutant proteins. In addition, the capability of S to mediate entry of SARS S-pseudotyped particles overall was affected moderately in the mutant proteins, also arguing for a nonvital role for the GxxxG motif in SARS coronavirus entry.

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A Conserved Transmembrane Domain Interface Regulates Integrin Function

Blood ◽

10.1182/blood.v112.11.2858.2858 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2858-2858 ◽

Cited By ~ 1

Author(s):

Bryan W Berger ◽

Lisa M. Span ◽

Daniel W Kulp ◽

Paul C. Billings ◽

William F. DeGrado ◽

...

Keyword(s):

Type I Collagen ◽

Transmembrane Domain ◽

Dominant Negative ◽

The Other ◽

Type I ◽

Wild Type ◽

Suspension Cells ◽

Α Subunit ◽

Tm Domain ◽

Domain Separation

Abstract Integrins are a superfamily of transmembrane (TM) α/β heterodimers that mediate fundamental cellular adhesive functions. Platelet integrins, for example, mediate stable platelet adhesion to collagen and fibronectin and the formation of stable platelet aggregates. Integrins reside on cell surfaces in an equilibrium between inactive and active conformations. An essential feature of this equilibrium is interaction of the integrin α and β subunit TM domains. Thus, when integrins are inactive, the α and β TM domains are in proximity, but they separate when integrins assume an active conformation. Moreover, inducing TM domain separation alone is sufficient to cause integrin activation. Previously, we reported that the TM domains of the platelet integrin αIIbβ3 interact both heteromerically and homomerically and that the strength of their heteromeric interaction is necessarily weak to allow regulated TM domain separation. To address whether these observations can be extended to the other members of the integrin superfamily, we focused initially on αvβ3, α2β1 and α5β1, integrins present in platelets, using a dominant-negative ToxR-based assay. ToxR is a single-pass TM transcriptional factor from V. cholera that activates the cholera toxin (ctx) promoter when it dimerizes in the inner membrane of E. coli. By co-expressing wild-type ToxR with either wild-type ToxR or an R96K ToxR mutant that can dimerize but is unable to activate the ctx promoter, we can measure the homomeric and heteromeric interaction of each integrin TM domain. Using alanine and leucine scanning mutagenesis, we found that like αIIb, homo-oligomerization of other integrin α subunit TM domains is preferred over hetero-oligomerization, and that the relative strength of homo-oligomerization correlates with the presence of a canonical small residue-xxx-small residue motif followed one turn of the TM helix by a leucine (G, A, S-xxx-G-xxx-L). This motif also mediates the hetero-oligomerization of these TM domains with either β3 or β1. By contrast, a different motif (V-xxx-I-xxx-G) mediates the heteromeric interaction of both β3 and β1 with their complementary α subunits. Mutations that disrupt either the αIIb or β3 interaction motif induce constitutive αIIbβ3 activation. To determine if this is also the case for β1-containing integrins, we introduced disruptive interfacial mutations into the full-length integrins and expressed the mutants in either the β1-deficient Jurkat A1 cells or in HEK293 suspension cells. We found that the β1 mutations V716A, I720A and G724L caused a substantial increase in the static adhesion of A1 cells to laminin, fibronectin, the α4β1-specific peptide H1, as well as type I, II and type IV collagen, whereas mutation of the canonical G-xxx-G motif did not. On the other hand, an increase in binding to type I collagen and fibronectin was observed for mutations of the interfacial α2 residues S1009, G1013, and L1017 and the interfacial α5 residues A964, G968, and L972, respectively. Thus, our studies indicate that β1 and β3 integrins employ a novel, specific, and conserved reciprocating ‘large-small’ TM packing interface that interacts less strongly than the canonical small-residue-xxx-small residue motif. It is also noteworthy that this interface is present in all integrins except β4 and is overrepresented in databases of TM helix-helix interaction as well. Accordingly, it is likely that this type of interface evolved to mediate TM domain interactions that are capable of regulation.

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Charged Residues Flanking the Transmembrane Domain of Two Related Toxin–Antitoxin System Toxins Affect Host Response

Toxins ◽

10.3390/toxins13050329 ◽

2021 ◽

Vol 13 (5) ◽

pp. 329

Author(s):

Andrew Holmes ◽

Jessie Sadlon ◽

Keith Weaver

Keyword(s):

Amino Acid ◽

Enterococcus Faecalis ◽

Host Response ◽

Cytoplasmic Membrane ◽

Transmembrane Domain ◽

The Other ◽

Type I ◽

Transcriptomic Response ◽

Specific Amino Acid ◽

Transporter Protein

A majority of toxins produced by type I toxin–antitoxin (TA-1) systems are small membrane-localized proteins that were initially proposed to kill cells by forming non-specific pores in the cytoplasmic membrane. The examination of the effects of numerous TA-1 systems indicates that this is not the mechanism of action of many of these proteins. Enterococcus faecalis produces two toxins of the Fst/Ldr family, one encoded on pheromone-responsive conjugative plasmids (FstpAD1) and the other on the chromosome, FstEF0409. Previous results demonstrated that overexpression of the toxins produced a differential transcriptomic response in E. faecalis cells. In this report, we identify the specific amino acid differences between the two toxins responsible for the differential response of a gene highly induced by FstpAD1 but not FstEF0409. In addition, we demonstrate that a transporter protein that is genetically linked to the chromosomal version of the TA-1 system functions to limit the toxicity of the protein.

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Structural Basis for the C-Terminal Domain of Mycobacterium tuberculosis Ribosome Maturation Factor RimM to Bind Ribosomal Protein S19

Biomolecules ◽

10.3390/biom11040597 ◽

2021 ◽

Vol 11 (4) ◽

pp. 597

Author(s):

Haoran Zhang ◽

Qiuxiang Zhou ◽

Chenyun Guo ◽

Liubin Feng ◽

Huilin Wang ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Ribosomal Protein ◽

Solution Structure ◽

Molecular Mechanisms ◽

Structural Model ◽

Ribosomal Subunit ◽

Structural Basis ◽

Hydrophobic Core ◽

Terminal Domain ◽

Ribosomal Protein S19

Multidrug-resistant tuberculosis (TB) is a serious threat to public health, calling for the development of new anti-TB drugs. Chaperon protein RimM, involved in the assembly of ribosomal protein S19 into 30S ribosomal subunit during ribosome maturation, is a potential drug target for TB treatment. The C-terminal domain (CTD) of RimM is primarily responsible for binding S19. However, both the CTD structure of RimM from Mycobacterium tuberculosis (MtbRimMCTD) and the molecular mechanisms underlying MtbRimMCTD binding S19 remain elusive. Here, we report the solution structure, dynamics features of MtbRimMCTD, and its interaction with S19. MtbRimMCTD has a rigid hydrophobic core comprised of a relatively conservative six-strand β-barrel, tailed with a short α-helix and interspersed with flexible loops. Using several biophysical techniques including surface plasmon resonance (SPR) affinity assays, nuclear magnetic resonance (NMR) assays, and molecular docking, we established a structural model of the MtbRimMCTD–S19 complex and indicated that the β4-β5 loop and two nonconserved key residues (D105 and H129) significantly contributed to the unique pattern of MtbRimMCTD binding S19, which might be implicated in a form of orthogonality for species-dependent RimM–S19 interaction. Our study provides the structural basis for MtbRimMCTD binding S19 and is beneficial to the further exploration of MtbRimM as a potential target for the development of new anti-TB drugs.

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Fibers Effects on Contract Turbulence Using a Coupling Euler Model

Applied Sciences ◽

10.3390/app11157126 ◽

2021 ◽

Vol 11 (15) ◽

pp. 7126

Author(s):

Wei Yang ◽

Pei Hu

Keyword(s):

Boundary Layer ◽

Velocity Profile ◽

Fiber Orientation ◽

Bottom Layer ◽

Orientation Distribution ◽

Industrial Applications ◽

Dynamics Simulation ◽

Fiber Concentration ◽

Fibers Orientation ◽

Process Strategy

Fiber additive will induce the rheological behavior of suspension, resulting in variation in velocity profile and fiber orientation especially for the non-dilute case. Based on the fluid-solid coupling dynamics simulation, it shows that the fiber orientation aligns along the streamline more and more quickly in the central turbulent region as the fiber concentration increases, especially contract ratio Cx > 4. However, fibers tend to maintain the original uniform orientation and are rarely affected by the contract ratio in the boundary layer. The fibers orientation in the near semi-dilute phase is lower than that in the dilute phase near the outlet, which may be the result of the hydrodynamic contact lubrication between fibers. The orientation distribution and concentration of the fibers change the viscous flow mechanism of the suspension microscopically, which makes a velocity profile vary with the phase concentration. The velocity profile of the approaching semi-dilute phase sublayer is higher than that of the dilute and semi-dilute phases on the central streamline and in the viscous bottom layer, showing weak drag reduction while the situation is opposite on the logarithmic layer of the boundary layer. The relevant research can provide a process strategy for fiber orientation optimization and rheological control in the industrial applications of suspension.

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