Seipin transmembrane segments critically function in triglyceride nucleation and lipid droplet budding from the membrane

ABSTRACTLipid droplets (LDs) are organelles formed in the endoplasmic reticulum (ER) to store triacylglycerol (TG) and sterol esters. The ER protein seipin is key for LD biogenesis. Seipin forms a cage-like structure, with each seipin monomer containing a conserved hydrophobic helix (HH) and two transmembrane (TM) segments. How the different parts of seipin function in TG nucleation and LD budding is poorly understood. Here, we utilized molecular dynamics simulations of human seipin, along with cell-based experiments, to study seipin’s functions in protein-lipid interactions, lipid diffusion, and LD maturation. All-atom (AA) simulations indicate that most seipin TM segment residues located in the phospholipid (PL) tail region of the bilayer attract TG. We also find seipin TM segments control lipid diffusion and permeation into the protein complex. Simulating larger, growing LDs with coarse-grained (CG) models, we find that the seipin TM segments form a constricted neck structure to facilitate conversion of a flat oil lens into a budding LD. Using cell experiments and simulations, we also show that conserved, positively charged residues at the end of seipin’s TM segments affect LD maturation. We propose a model in which seipin TM segments critically function in TG nucleation and LD growth.

Tubular Assembly Formation Induced by Leucine Alignment along the Hydrophobic Helix of Amphiphilic Polypeptides

The introduction of α-helical structure with a specific helix–helix interaction into an amphipathic molecule enables the determination of the molecular packing in the assembly and the morphological control of peptide assemblies. We previously reported that the amphiphilic polypeptide SL12 with a polysarcosine (PSar) hydrophilic chain and hydrophobic α-helix (l-Leu-Aib)6 involving the LxxxLxxxL sequence, which induces homo-dimerization due to the concave–convex interaction, formed a nanotube with a uniform 80 nm diameter. In this study, we investigated the importance of the LxxxLxxxL sequence for tube formation by comparing amphiphilic polypeptide SL4A4L4 with hydrophobic α-helix (l-Leu-Aib)2-(l-Ala-Aib)2-(l-Leu-Aib)2 and SL12. SL4A4L4 formed spherical vesicles and micelles. The effect of the LxxxLxxxL sequence elongation on tube formation was demonstrated by studying assemblies of PSar-b-(l-Ala-Aib)-(l-Leu-Aib)6-(l-Ala-Aib) (SA2L12A2) and PSar-b-(l-Leu-Aib)8 (SL16). SA2L12A2 formed nanotubes with a uniform 123 nm diameter, while SL16 assembled into vesicles. These results showed that LxxxLxxxL is a necessary and sufficient sequence for the self-assembly of nanotubes. Furthermore, we fabricated a double-layer nanotube by combining two kinds of nanotubes with 80 and 120 nm diameters—SL12 and SA2L12A2. When SA2L12A2 self-assembled in SL12 nanotube dispersion, SA2L12A2 initially formed a rolled sheet, the sheet then wrapped the SL12 nanotube, and a double-layer nanotube was obtained.

The client-binding domain of the cochaperone Sgt2 has a helical-hand structure that binds a short hydrophobic helix

The targeting and insertion of tail-anchored (TA) integral membrane proteins (IMP) into the correct membrane is critical for cellular homeostasis. The fungal protein Sgt2, and its human homolog SGTA, binds hydrophobic clients and is the entry point for targeting of ER-bound TA IMPs. Here we reveal molecular details that underlie the mechanism of Sgt2 binding to TA IMP clients. We establish that the Sgt2 C-terminal region is flexible but conserved and sufficient for client binding. A molecular model for this domain reveals a helical hand forming a hydrophobic groove, consistent with a higher affinity for TA IMP clients with hydrophobic faces and a minimal length of 11 residues. This work places Sgt2 into a broader family of TPR-containing co-chaperone proteins.

Structural basis of recognition and destabilization of histone H2B ubiquitinated nucleosome by DOT1L histone H3 Lys79 methyltransferase

DOT1L is a histone H3 Lys79 methyltransferase whose activity is stimulated by histone H2B Lys120 ubiquitination, suggesting cross-talk between histone H3 methylation and H2B-ubiquitination. Here, we present cryo-EM structures of DOT1L complex with unmodified and H2B-ubiquitinated nucleosomes, showing that DOT1L recognizes H2B-ubiquitin and the H2A/H2B acidic patch through a C-terminal hydrophobic helix and an arginine anchor in DOT1L respectively. Furthermore, the structures combined with single-molecule FRET experiment show that H2B-ubiquitination enhances a non-catalytic function of DOT1L destabilizing nucleosome. These results establish the molecular basis of the cross-talk between H2B ubiquitination and H3 Lys79 methylation as well as nucleosome destabilization by DOT1L.

Triterpenoids manipulate a broad range of virus-host fusion via wrapping the HR2 domain prevalent in viral envelopes

A trimer-of-hairpins motif has been identified in triggering virus-cell fusion within a variety of viral envelopes. Chemically manipulating such a motif represents current repertoire of viral fusion inhibitors. Here, we report that triterpenoids, a class of natural products, antagonize this trimer-of-hairpins via its constitutive heptad repeat-2 (HR2), a prevalent α-helical coil in class I viral fusion proteins. Triterpenoids inhibit the entry of Ebola, Marburg, HIV, and influenza A viruses with distinct structure-activity relationships. Specifically, triterpenoid probes capture the viral envelope via photocrosslinking HR2. Profiling the Ebola HR2-triterpenoid interactions using amino acid substitution, surface plasmon resonance, and nuclear magnetic resonance revealed six residues accessible to triterpenoids, leading to wrapping of the hydrophobic helix and blocking of the HR1-HR2 interaction critical in the trimer-of-hairpins formation. This finding was also observed in the envelopes of HIV and influenza A viruses and might potentially extend to a broader variety of viruses, providing a mechanistic insight into triterpenoid-mediated modulation of viral fusion.

Triterpenoids Manipulate a Broad Range of Virus-Host Fusion via Wrapping the HR2 Domain Prevalent in Viral Envelopes

Recent years have witnessed a breakthrough in identification of a trimer-of-hairpins motif within viral envelopes that triggers a broad range of virus-host fusion. Identifying a domain capable of controlling virus-host fusion remains a challenge due to sequence diversity, heavy glycan shielding and multiple conformations. Here, we report that HR2, a prevalent heptad repeat sequence comprising an alpha-helical coil anchored in viral membranes, is an accessible site to triterpenes, a class of widely distributed natural products. Triterpenes and their derivatives inhibit the entry of Ebola, HIV, and influenza A viruses with distinct structure-activity relationships. Specifically, triterpenoid probes, upon activation by ultraviolet light, capture the viral envelope via crosslinking the HR2 coil. Profiling the Ebola HR2 sequence using amino acid substitution, surface plasmon resonance (SPR) and nuclear magnetic resonance (NMR) spectroscopy disclosed six constitutive residues that are accessible to triterpenoids, leading to wrapping of the hydrophobic helix by triterpenoids and blocking of the HR1-HR2 interaction, which is critical in the trimer-of-hairpins formation. This finding was also observed in the envelopes of HIV and influenza A viruses and might potentially extend to a broader variety of viruses. Our findings might translate into a shared mechanism that host utilize natural product triterpenoids to antagonize membrane fusion of respective viruses, complementing the current repertoire of antiviral agents.

ROSET Model of TonB Action in Gram-Negative Bacterial Iron Acquisition

Therotationalsurveillance andenergytransfer (ROSET) model of TonB action suggests a mechanism by which the electrochemical proton gradient across the Gram-negative bacterial inner membrane (IM) promotes the transport of iron through ligand-gated porins (LGP) in the outer membrane (OM). TonB associates with the IM by an N-terminal hydrophobic helix that forms a complex with ExbBD. It also contains a central extended length of rigid polypeptide that spans the periplasm and a dimericC-terminal-ββαβ-domain (CTD) with LysM motifs that binds the peptidoglycan (PG) layer beneath the OM bilayer. The TonB CTD forms a dimer with affinity for both PG- and TonB-independent OM proteins (e.g., OmpA), localizing it near the periplasmic interface of the OM bilayer. Porins and other OM proteins associate with PG, and this general affinity allows the TonB CTD dimer to survey the periplasmic surface of the OM bilayer. Energized rotational motion of the TonB N terminus in the fluid IM bilayer promotes the lateral movement of the TonB-ExbBD complex in the IM and of the TonB CTD dimer across the inner surface of the OM. When it encounters an accessible TonB box of a (ligand-bound) LGP, the monomeric form of the CTD binds and recruits it into a 4-stranded β-sheet. Because the CTD is rotating, this binding reaction transfers kinetic energy, created by the electrochemical proton gradient across the IM, through the periplasm to the OM protein. The equilibration of the TonB C terminus between the dimeric and monomeric forms that engage in different binding reactions allows the identification of iron-loaded LGP and then the internalization of iron through their trans-outer membrane β-barrels. Hence, the ROSET model postulates a mechanism for the transfer of energy from the IM to the OM, triggering iron uptake.

The positive inside rule is stronger when followed by a transmembrane helix.

The translocon recognizes transmembrane helices with sufficient level of hydrophobicity and inserts them into the membrane. However, sometimes less hydrophobic helices are also recognized. Positive inside rule, orientational preferences of and specific interactions with neighboring helices have been shown to aid in the recognition of these helices, at least in artificial systems. To better understand how the translocon inserts marginally hydrophobic helices, we studied three \red{naturally occurring marginally hydrophobic} helices, which were previously shown to require the subsequent helix for efficient translocon recognition. We find no evidence for specific interactions when we scan all residues in the subsequent helices. Instead, we identify arginines located at the N-terminal part of the subsequent helices that are crucial for the recognition of the marginally hydrophobic transmembrane helices, indicating that the positive inside rule is important. However, in two of the constructs these arginines do not aid in the recognition without the rest of the subsequent helix, i.e. the positive inside rule alone is not sufficient. Instead, the improved recognition of marginally hydrophobic helices can here be explained as follows; the positive inside rule provides an orientational preference of the subsequent helix, which in turn allows the marginally hydrophobic helix to be inserted, i.e. the effect of the positive inside rule is stronger if positively charged residues are followed by a transmembrane helix. Such a mechanism can obviously not aid C-terminal helices and consequently we find that the terminal helices in multi-spanning membrane proteins are more hydrophobic than internal helices.