Alps Motif

[Olivie Inoue]

Thecurving of a phospholipid bilayer, for example into a liposome, causes disturbances to the packing of the lipids on the side of the bilayer that has the larger surface area (the outside of a liposome for example). The less "ordered" or "looser" packing of the lipids is recognized by ALPS motifs.

ALPS motifs are 20 to 40 amino acid long portions of proteins that have important collections of types of amino acid residues. Bulky hydrophobic amino acid residues, such as Phenylalanine, Leucine, and Tryptophan are present every 3 or 4 positions, with many polar but uncharged amino acid residues such as Glycine, Serine and Threonine between. The ALPS is unstructured in solution but folds as an alpha helix when associated with the membrane bilayer, such that the hydrophobic residues insert between loosely packed lipids and the polar residues point toward the aqueous cytoplasm.

Heterogeneities (e.g., membrane proteins and lipid domains) and deformations (e.g., highly curved membrane regions) in biological lipid membranes cause lipid packing defects that may trigger functional sorting of lipids and membrane-associated proteins. To study these phenomena in a controlled and efficient way within molecular simulations, we developed an external field protocol that artificially enhances packing defects in lipid membranes by enforcing local thinning of a flat membrane region. For varying lipid compositions, we observed strong thinning-induced depletion or enrichment, depending on the lipid's intrinsic shape and its effect on a membrane's elastic modulus. In particular, polyunsaturated and lysolipids are strongly attracted to regions high in packing defects, whereas phosphatidylethanolamine (PE) lipids and cholesterol are strongly repelled from it. Our results indicate that externally imposed changes in membrane thickness, area, and curvature are underpinned by shared membrane elastic principles. The observed sorting toward the thinner membrane region is in line with the sorting expected for a positively curved membrane region. Furthermore, we have demonstrated that the amphipathic lipid packing sensor (ALPS) protein motif, a known curvature and packing defect sensor, is effectively attracted to thinner membrane regions. By extracting the force that drives amphipathic molecules toward the thinner region, our thinning protocol can directly quantify and score the lipid packing sensing of different amphipathic molecules. In this way, our protocol paves the way toward high-throughput exploration of potential defect- and curvature-sensing motifs, making it a valuable addition to the molecular simulation toolbox.

Figure 2. Membrane thinning induces lipid sorting. (A) Chemical structures and MARTINI's coarse-grained representations of POPC, POPE, CHOL, LysoPC, and PLiPC. (B) Histograms of the POPC, POPE, CHOL, LysoPC, and PLiPC content in 50 bins along the x-axis over 4 μs of simulation. Data are averages over five replica runs. As a reference, the black dashed line depicts the initial distribution of 30 mol%. Membrane snapshots show the final configuration, with 70 mol% POPC in transparent gray and 30 mol% of mixed-in lipid in orange. The full trajectories are provided as movies in the Supplementary Material. (C) Quantification of lipid mixing after 4 μs of simulation for every lipid composition in the thin zone (cyan) and the normal zone (black). Data is normalized to 30 mol%.

Copyright 2020 van Hilten, Stroh and Risselada. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Membrane curvature is involved in numerous biological pathways like vesicle trafficking, endocytosis or nuclear pore complex assembly. In addition to its topological role, membrane curvature is sensed by specific proteins, enabling the coordination of biological processes in space and time. Amongst membrane curvature sensors are the ALPS (Amphipathic Lipid Packing Sensors). ALPS motifs are short peptides with peculiar amphipathic properties. They are found in proteins targeted to distinct curved membranes, mostly in the early secretory pathway. For instance, the ALPS motif of the golgin GMAP210 binds trafficking vesicles, while the ALPS motif of Nup133 targets nuclear pores. It is not clear if, besides curvature sensitivity, ALPS motifs also provide target specificity, or if other domains in the surrounding protein backbone are involved. To elucidate this aspect, we studied the subcellular localization of ALPS motifs outside their natural protein context. The ALPS motifs of GMAP210 or Nup133 were grafted on artificial fluorescent probes. Importantly, ALPS motifs are held in different positions and these contrasting architectures were mimicked by the fluorescent probes. The resulting chimeras recapitulated the original proteins localization, indicating that ALPS motifs are sufficient to specifically localize proteins. Modulating the electrostatic or hydrophobic content of Nup133 ALPS motif modified its avidity for cellular membranes but did not change its organelle targeting properties. In contrast, the structure of the backbone surrounding the helix strongly influenced targeting. In particular, introducing an artificial coiled-coil between ALPS and the fluorescent protein increased membrane curvature sensitivity. This coiled-coil domain also provided membrane curvature sensitivity to the amphipathic helix of Sar1. The degree of curvature sensitivity within the coiled-coil context remains correlated to the natural curvature sensitivity of the helices. This suggests that the chemistry of ALPS motifs is a key parameter for membrane curvature sensitivity, which can be further modulated by the surrounding protein backbone.

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In addition to the lack of knowledge about Nup84, we have little structural information about yeast Nup133 aside from the C-terminal heel domain, which only has modest similarity to its human homolog23,24 (Fig. 1b). Also, completing the structure of the Y complex from a single species should help understand the differences in the NPC structure across species33,34,35. As a member of the cytoplasmic and nucleoplasmic rings in the NPC, the Y complex coats the peripheral inner and outer nuclear membrane (INM, ONM, respectively) bordering the circular openings in the NE36. It remains unclear how the yeast Y complex interacts with the INM and ONM and how it links to the inner NPC ring. The ArfGAP1 lipid packing sensor (ALPS) motif in the human homolog of Nup133 is thought to anchor the Y complex to the INM and ONM22,37,38. The hsNup133 ALPS motif is critical for interphase assembly in metazoa, a process which is thought to be similar to NPC assembly in organisms with closed mitosis like S. cerevisiae38,39. Whether or not the ALPS motif is conserved in S. cerevisiae has been debated in the literature, due to the lack of high-resolution structural or functional studies on yeast Nup13322,25,37.

Here we report the structures of the Nup84-Nup133 C-terminal α-helical domain and Nup133 N-terminal β-propeller from S. cerevisiae. The structures were obtained using nanobodies, single domain antibodies derived from alpacas40, as crystallization chaperones. This completes the entire structure of the Y complex from S. cerevisiae, allowing us to create a complete composite model of the Y complex assembly from a single species. Additionally, we show that Nup133 has a functional ALPS motif through liposome interaction studies. This model of the Y complex delineates key hinge points and possible motion ranges in the Y complex stalk and establishes the position of Nup133 with its ALPS motif placed adjacent to the membrane in the NPC assembly.

When superimposing each ACE1 structure, one can easily detect their structural similarity (Fig. 2). Most of the trunk helices run perpendicular to the long axis of the protein, while the crown helices tip upwards. The typically longer tail helices lean downwards, away from the trunk. Comparing the tail modules across Nups, they are visibly rotated to different degrees relative to each trunk, highlighting the flexibility at the trunk-tail interface. While Sec31 and Sec16 both do not have a tail, they follow the same helical topology in the trunk and crown as the Nups.

a Structures of scNup133NTD (purple) and hsNup133NTD (orange). The missing DA34 loop in the structures are indicated by a black dotted line. Helical wheel diagram for each loop is shown with polar residues (teal), nonpolar (orange), and glycine (gray) colored. Arrow indicates predicted hydrophobic face and length of arrow scales with strength of the hydrophobic moment. b Liposome floatation assay with Nup133NTD. Panels show Coomassie stained SDS-PAGE fractions isolated from the gradients. A cartoon of fractions is shown on the right. c Negative stain electron microscopy images of liposomes preincubated with Nup133NTD (upper) and Nup133NTD ΔALPS (lower). Scale bar at the bottom left of each panel. Panels are representative images taken from two independent experiments.

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