How Anionic Lipids Affect Spatiotemporal Properties of KRAS4B on Model Membranes

Ngo, Van; Sarkar, Sumantra; Neale, Chris; Garcı́a, Angel E.

doi:10.1021/acs.jpcb.0c02642

Cited by 18 publications

(26 citation statements)

References 105 publications

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“…Per-residue contact data were plotted using Grace (http://plasma-gate.weiz-mann.ac.il/Grace/). Lipid binding preference was calculated from whole-protein contact data using the following equation, based on that previously described by Ngo et al (66):…”

Section: Analysis Of Protein-lipid Contacts and Lipid Binding Preferencementioning

confidence: 99%

Modeling of full-length Piezo1 suggests importance of the proximal N-terminus for dome structure

Chong

Vecchis

Hyman

et al. 2021

Biophysical Journal

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Piezo1 forms a mechanically activated calcium-permeable nonselective cation channel that is functionally important in many cell types. Structural data exist for C-terminal regions, but we lack information about N-terminal regions and how the entire channel interacts with the lipid bilayer. Here, we use computational approaches to predict the three-dimensional structure of the full-length Piezo1 and simulate it in an asymmetric membrane. A number of novel insights are suggested by the model: 1) Piezo1 creates a trilobed dome in the membrane that extends beyond the radius of the protein, 2) Piezo1 changes the lipid environment in its vicinity via preferential interactions with cholesterol and phosphatidylinositol 4,5-bisphosphate (PIP 2 ) molecules, and 3) cholesterol changes the depth of the dome and PIP 2 binding preference. In vitro alteration of cholesterol concentration inhibits Piezo1 activity in a manner complementing some of our computational findings. The data suggest the importance of N-terminal regions of Piezo1 for dome structure and membrane cholesterol and PIP 2 interactions.

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Section: Analysis Of Protein-lipid Contacts and Lipid Binding Preferencementioning

confidence: 99%

Modeling of full-length Piezo1 suggests importance of the proximal N-terminus for dome structure

Chong

Vecchis

Hyman

et al. 2021

Biophysical Journal

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“…These results suggest that the two (Asn-Lys) or six (Asn-Lys-Ile-Glu-Ser-Gly) N-terminal residues in the Rab4a HVR linker domain contribute to limiting the intrinsic tethering potency of the G-domain through their interactions with the polar lipid headgroups. Considering that recent computational studies by molecular dynamics simulations of membrane-bound K-Ras, belonging to the Ras superfamily, reported the multiple orientation states of the K-Ras G-domain on the membrane surface that are affected by the C-terminal HVR linker and anionic membrane lipids ( Prakash et al, 2016 ; Prakash and Gorfe, 2019 ; Neale and García, 2020 ; Ngo et al, 2020 ), it is conceivable that the hydrophilic interactions between the N-terminal residues of the Rab4a HVR linker and anionic lipids may guide the G-domain into the specific “tethering-incompetent” membrane orientation.…”

Section: Resultsmentioning

confidence: 99%

Membrane Tethering Potency of Rab-Family Small GTPases Is Defined by the C-Terminal Hypervariable Regions

Ueda

Tamura

Mima

2020

Front. Cell Dev. Biol.

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Membrane tethering is a crucial step to determine the spatiotemporal specificity of secretory and endocytic trafficking pathways in all eukaryotic endomembrane systems. Recent biochemical studies by a chemically-defined reconstitution approach reveal that, in addition to the structurally-diverse classic tethering factors such as coiledcoil tethering proteins and multisubunit tethering complexes, Rab-family small GTPases also retain the inherent membrane tethering functions to directly and physically bridge two distinct lipid bilayers by themselves. Although Rab-mediated membrane tethering reactions are fairly efficient and specific in the physiological context, its mechanistic basis is yet to be understood. Here, to explore whether and how the intrinsic tethering potency of Rab GTPases is controlled by their C-terminal hypervariable region (HVR) domains that link the conserved small GTPase domains (G-domains) to membrane anchors at the C-terminus, we quantitatively compared tethering activities of two representative Rab isoforms in humans (Rab5a, Rab4a) and their HVR-deleted mutant forms. Strikingly, deletion of the HVR linker domains enabled both Rab5a and Rab4a isoforms to enhance their intrinsic tethering potency, exhibiting 5-to 50-fold higher initial velocities of tethering for the HVR-deleted mutants than those for the full-length, wild-type Rabs. Furthermore, we revealed that the tethering activity of full-length Rab5a was significantly reduced by the omission of anionic lipids and cholesterol from membrane lipids and, however, membrane tethering driven by HVR-deleted Rab5a mutant was completely insensitive to the headgroup composition of lipids. Reconstituted membrane tethering assays with the C-terminally-truncated mutants of Rab4a further uncovered that the N-terminal residues in the HVR linker, located adjacent to the G-domain, are critical for regulating the intrinsic tethering activity. In conclusion, our current findings establish that the nonconserved, flexible C-terminal HVR linker domains define membrane tethering potency of Rab-family small GTPases through controlling the close attachment of the globular G-domains to membrane surfaces, which confers the active tethering-competent state of the G-domains on lipid bilayers.

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“…These results suggest that the two (Asn-Lys) or six (Asn-Lys-Ile-Glu-Ser-Gly) N-terminal residues in the Rab4a HRV linker domain contribute to limiting the intrinsic tethering potency of the G-domain through their interactions with the polar lipid headgroups. Considering that recent computational studies by molecular dynamics simulations of membrane-bound K-Ras, belonging to the Ras superfamily, reported the multiple orientation states of the K-Ras G-domain on the membrane surface that are affected by the C-terminal HVR linker and anionic membrane lipids (Prakash et al, 2016;Prakash and Gorfe, 2019;Neale and Garcia, 2020;Ngo et al, 2020), it is conceivable that the hydrophilic interactions between the N-terminal residues of the Rab4a HVR linker and anionic lipids may guide the G-domain into the specific "tetheringincompetent" membrane orientation.…”

Section: Critical Residues In the Hvr Linker Domain Of Rab4a For Regumentioning

confidence: 99%

Membrane tethering potency of Rab-family small GTPases is defined by the C-terminal hypervariable regions

Ueda

Tamura

Mima

2020

Preprint

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AbstractMembrane tethering is a crucial step to determine the spatiotemporal specificity of secretory and endocytic trafficking pathways in all eukaryotic endomembrane systems. Recent biochemical studies by a chemically-defined reconstitution approach reveal that, in addition to the structurally-diverse classic tethering factors such as coiled-coil tethering proteins and multisubunit tethering complexes, Rab-family small GTPases also retain the inherent membrane tethering functions to directly and physically bridge two distinct lipid bilayers by themselves. Although Rab-mediated membrane tethering reactions are fairly efficient and specific in the physiological context, its mechanistic basis is yet to be understood. Here, to explore whether and how the intrinsic tethering potency of Rab GTPases is controlled by their C-terminal hypervariable region (HVR) domains that link the conserved small GTPase domains (G-domains) to membrane anchors at the C-terminus, we quantitatively compared tethering activities of two representative Rab isoforms in humans (Rab5a, Rab4a) and their HVR-deleted mutant forms. Strikingly, deletion of the HVR linker domains enabled both Rab5a and Rab4a isoforms to drastically enhance their intrinsic tethering potency, exhibiting 5- to 50-fold higher initial velocities of tethering for the HVR-deleted mutants than those for the full-length, wild-type Rabs. Furthermore, we revealed that the tethering activity of full-length Rab5a was significantly reduced by the omission of anionic lipids and cholesterol from membrane lipids and, however, membrane tethering driven by HVR-deleted Rab5a mutant was completely insensitive to the headgroup composition of lipids. In conclusion, our current findings establish that the non-conserved, flexible C-terminal HVR linker domains define membrane tethering potency of Rab-family small GTPases through controlling the close attachment of the globular G-domains to membrane surfaces, which confers the active tethering-competent state of the G-domains on lipid bilayers.

show abstract

How Anionic Lipids Affect Spatiotemporal Properties of KRAS4B on Model Membranes

Cited by 18 publications

References 105 publications

Modeling of full-length Piezo1 suggests importance of the proximal N-terminus for dome structure

Modeling of full-length Piezo1 suggests importance of the proximal N-terminus for dome structure

Membrane Tethering Potency of Rab-Family Small GTPases Is Defined by the C-Terminal Hypervariable Regions

Membrane tethering potency of Rab-family small GTPases is defined by the C-terminal hypervariable regions

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