Biochemical Characterization of a Human Septin Octamer

Fischer, Martin; Frank, Dominik; Rösler, Reinhild; Johnsson, Nils; Gronemeyer, Thomas

doi:10.3389/fcell.2022.771388

Cited by 10 publications

(7 citation statements)

References 42 publications

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“…We used The role of nucleotide hydrolysis in septins is still poorly understood and subject of debate (Grupp & Gronemeyer, 2022). GTP hydrolysis was shown by biochemical assays for members of the Momany 1a and 2b groups, but hydrolysis products are not released to the surrounding medium by native hexameric or octameric complexes (Fischer, Frank, Rösler, Johnsson, & Gronemeyer, 2022). It is meanwhile commonly accepted that septins evolved from a common ancestor (Pan, Malmberg, & Momany, 2007) and during evolution some septins seem to have lost their ability to hydrolyze GTP.…”

Section: Resultsmentioning

confidence: 99%

An in silico approach to determine inter‐subunit affinities in human septin complexes

2023

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The septins are a conserved family of filament‐forming guanine nucleotide binding proteins, often named the fourth component of the cytoskeleton. Correctly assembled septin structures are required for essential intracellular processes such as cytokinesis, vesicular transport, polarity establishment, and cellular adhesion. Structurally, septins belong to the P‐Loop NTPases but they do not mediate signals to effectors through GTP binding and hydrolysis. GTP binding and hydrolysis are believed to contribute to septin complex integrity, but biochemical approaches addressing this topic are hampered by the stability of septin complexes after recombinant expression and the lack of nucleotide‐depleted complexes. To overcome this limitation, we used a molecular dynamics‐based approach to determine inter‐subunit binding free energies in available human septin dimer structures and in their apo forms, which we generated in silico. The nucleotide in the GTPase active subunits SEPT2 and SEPT7, but not in SEPT6, was identified as a stabilizing element in the G interface. Removal of GDP from SEPT2 and SEPT7 results in flipping of a conserved Arg residue and disruption of an extensive hydrogen bond network in the septin unique element, concomitant with a decreased inter‐subunit affinity. Based on these findings we propose a singular “lock‐hydrolysis” mechanism stabilizing human septin filaments.

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Section: Resultsmentioning

confidence: 99%

An in silico approach to determine inter‐subunit affinities in human septin complexes

2023

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“…Septins are GTP‐binding proteins of the cytoskeleton. They assemble into heteromeric hexamers or octamers, and those oligomers polymerize into non‐polar filaments (Cavini et al, 2021; Fischer, Frank, Rösler, Johnsson, & Gronemeyer, 2022; Spiliotis & McMurray, 2020; Spiliotis & Nakos, 2021; Woods & Gladfelter, 2021). Biochemical experiments with purified components show that septin filaments bind directly to lipid membranes with positive curvature (Beber et al, 2019; Bridges, Jentzsch, Oakes, Occhipinti, & Gladfelter, 2016; Cannon, Woods, Crutchley, & Gladfelter, 2019).…”

Section: Septins and Cell–cell Junctions Of Ecsmentioning

confidence: 99%

Septin and actin contributions to endothelial cell–cell junctions and monolayer integrity

et al. 2022

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Septins in endothelial cells (ECs) have important roles supporting the integrity of the endothelial monolayer. Cell–cell junctions in EC monolayers are highly dynamic, with continuous retractions and protrusions. Depletion of septins in ECs leads to disruption of cell–cell junctions, which are composed of VE‐cadherin and other junctional proteins. In EC monolayers, septins are concentrated at the plasma membrane at sites of cell–cell contact, in curved‐ and scallop‐shaped patterns. These membrane‐associated septin accumulations are located in regions of positive membrane curvature, and those regions are often associated with and immediately adjacent to actin‐rich protrusions with negative membrane curvature. EC septins associate directly with plasma membrane lipids, based on findings with site‐specific mutations of septins in ECs, which is consistent with biochemical and cell biological studies in other systems. Loss of septins leads to disruption of the EC monolayer, and gaps form between cells. The number and breadth of cell–cell contacts and junctions decreases, and the number and frequency of retractions, ruffles, and protrusions at cell edges also decreases. In addition, loss of septins leads to decreased amounts of F‐actin at the cortical membrane, along with increased amounts of F‐actin in stress fibers of the cytoplasm. Endothelial monolayer disruption from loss of septins is also associated with decreased transendothelial electric resistance (TEER) and increased levels of transendothelial migration (TEM) by immune and cancer cells, owing to the gaps in the monolayer. A current working model is that assembly of septin filaments at regions of positive membrane curvature contributes to a mechanical footing or base for actin‐based protrusive forces generated at adjoining regions of the membrane. Specific molecular interactions between the septin and actin components of the cytoskeleton may also be important contributors. Regulators of actin assembly may promote and support the assembly of septin filaments at the membrane, as part of a molecular feedback loop between the assembly of septin and actin filaments.

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“…The role of nucleotide hydrolysis in septins is still poorly understood and subject of debate (Grupp and Gronemeyer 2022). GTP hydrolysis was shown by biochemical assays for members of the Momany 1a and 2b groups, but hydrolysis products are not released to the surrounding medium by native hexameric or octameric complexes (Fischer et al 2022). It is meanwhile commonly accepted that septins evolved from a common ancestor (Pan, Malmberg, and Momany 2007) and during evolution some septins seem to have lost their ability to hydrolyze GTP.…”

Section: For All Rmsd Values and Rmsd Plots)mentioning

confidence: 99%

Anin silicoapproach to determine inter-subunit affinities in human septin complexes

Grupp

Lemkul

Gronemeyer

2022

Preprint

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The septins are a conserved family of filament-forming guanine nucleotide binding proteins, often named the fourth component of the cytoskeleton. Correctly assembled septin structures are required for essential intracellular processes such as cytokinesis, vesicular transport, polarity establishment, and cellular adhesion. Structurally, septins belong to the P-Loop NTPases but they do not mediate signals to effectors through GTP binding and hydrolysis. GTP binding and hydrolysis are believed to contribute to septin complex integrity, but biochemical approaches addressing this topic are hampered by the stability of septin complexes after recombinant expression and the lack of nucleotide-depleted complexes. To overcome this limitation, we used a molecular dynamics-based approach to determine inter-subunit binding free energies in available human septin dimer structures and in their apo forms, which we generated in silico. The nucleotide in the GTPase active subunits SEPT2 and SEPT7, but not in SEPT6, was identified as a stabilizing element in the G interface as it is coordinated at its ribose ring to conserved amino acids. Removal of GDP from SEPT2 and SEPT7 results in flipping of a conserved Arg residue and disruption of an extensive hydrogen bond network in the septin unique element, concomitant with a decreased inter-subunit affinity.

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Biochemical Characterization of a Human Septin Octamer

Cited by 10 publications

References 42 publications

An in silico approach to determine inter‐subunit affinities in human septin complexes

An in silico approach to determine inter‐subunit affinities in human septin complexes

Septin and actin contributions to endothelial cell–cell junctions and monolayer integrity

Anin silicoapproach to determine inter-subunit affinities in human septin complexes

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