Search bioRxiv⌕ Search

Biology subjects

Timme, M.

Publications and source records attributed to Timme, M..

4 recordsLinked to original sources

Ceramides disrupt hexokinase HKI-VDAC complex assembly and modulate VDAC tilting

Ceramides bind voltage-dependent anion channels (VDACs) to promote mitochondrial apoptosis. However, the underlying mechanism is unclear. Ceramide binding relies on a charged, bilayer-facing glutamate that also controls complex formation between VDACs and pro-survival hexokinase HKI, suggesting that ceramides may exert their pro-apoptotic activity by displacing HKI from VDACs. Here, we challenged this model using coarse-grain molecular dynamics simulations. We show that ceramides impair VDAC-HKI complex assembly, but not via direct competition with HKI for binding to the crucial glutamate. Instead, we find that ceramides disrupt the membrane thinning capacity of polar channel residues adjacent to the glutamate, thereby congesting a low-energy passageway used by HKI for efficient complex formation. We also demonstrate that VDACs are tilted in the membrane and that ceramides act as modulators of channel tilt by neutralizing the hydrophobic mismatch imposed by the polar channel residues. We postulate that this ceramide action further modulates the oligomerization propensity of VDACs. Altogether, our data reveal a mechanistic basis for how ceramides may trigger apoptotic cell death.

biophysics↗

Key determinants of VDAC-hexokinase I complex assembly revealed by a minimal vesicle-based interaction assay

Binding of hexokinase HKI to mitochondrial voltage-dependent anion channels (VDACs) regulates the metabolic fate of glucose and promotes cell survival in hyperglycolytic tumors. Computer simulations indicated that complex assembly relies on intimate contacts between the N-terminal -helix of HKI and a charged bilayer-facing glutamate on the outer wall of VDACs. Protonation of this residue blocks complex formation in silico, explaining the release of HKI from mitochondria observed upon cytosolic acidification. To validate these findings, we here developed an in vitro assay for interrogating HKI binding to VDAC1 reconstituted in vesicles captured on a functionalized surface. TIRF-based quantitative interaction studies with a fluorescent peptide comprising the N-terminal -helix of HKI revealed a crucial role of the bilayer-facing glutamate in complex assembly and recapitulated an exquisite sensitivity of HKI-VDAC binding to fluctuations in pH. Our assay opens up important opportunities to investigate the impact of membrane environment on HKI-VDAC complex assembly and may benefit the development of therapeutics that target pathogenic imbalances in this process.

biophysics↗

Hexokinase-I directly binds to a charged membrane-buried glutamate of mitochondrial VDAC1 and VDAC2

Binding of hexokinase HKI to mitochondrial voltage-dependent anion channels (VDACs) has far-reaching physiological implications. However, the structural basis of this interaction is unclear. Combining computer simulations with experiments in cells, we here show that complex assembly relies on intimate contacts between the N-terminal -helix of HKI and a charged membrane-buried glutamate on the outer wall of VDAC1 and VDAC2. Protonation of this residue blocks complex formation in silico while acidification of the cytosol causes a reversable release of HKI from mitochondria. Membrane insertion of HKI occurs adjacent to the bilayer-facing glutamate where a pair of polar channel residues mediates a marked thinning of the cytoplasmic leaflet. Disrupting the membrane thinning capacity of VDAC1 dramatically impairs its ability to bind HKI in silico and in cells. Our data reveal key topological and mechanistic insights into HKI-VDAC complex assembly that may benefit the development of therapeutics to counter pathogenic imbalances in this process.

biochemistry↗

Geometric constraints in protein folding

The intricate three-dimensional geometries of protein tertiary structures underlie protein function and emerge through a folding process from one-dimensional chains of amino acids. The exact spatial sequence and con1guration of amino acids, the biochemical environment and the temporal sequence of distinct interactions yield a complex folding process that cannot yet be easily tracked for all proteins. To gain qualitative insights into the fundamental mechanisms behind the folding dynamics and generic features of the folded structure, we propose a simple model of structure formation that takes into account only fundamental geometric constraints and otherwise assumes randomly paired connections. We find that despite its simplicity, the model results in a network ensemble consistent with key overall features of the ensemble of Protein Residue Networks we obtained from more than 1000 biological protein geometries as available through the Protein Data Base. Speci1cally, the distribution of the number of interaction neighbors a unit (amino acid) has, the scaling of the structures spatial extent with chain length, the eigenvalue spectrum and the scaling of the smallest relaxation time with chain length are all consistent between model and real proteins. These results indicate that geometric constraints alone may already account for a number of generic features of protein tertiary structures.\n\nAuthor summaryHow proteins fold constitutes one of the most persistent, broad, and exciting open research questions at the intersection of biology, chemistry, and physics. Which mechanisms induce a one-dimensional sequence of amino acids to form into a complex three-dimensional (3D) structure? Proteins in their active 3D structure impact most of the basic processes inside cells, including gene regulation, cell metabolism, and the creation of protein structures themselves. Yet, a general rule about which conditions lead to which speci1c 3D protein structures remains unknown to date.\n\nHere, we demonstrate how a simple model that takes only fundamental geometric constraints into account and otherwise assumes randomly paired connections, naturally generates an ensemble of folded structures that exhibits many of its coarse scale features consistent with those of protein residue networks resulting from tertiary structures of biological proteins. Speci1cally, we tested a set of more than 1000 biological proteins and model structures and extracted a range of ensemble properties, including the spatial extension with chain size, the distribution of the number of interacting neighbors in the folded structure, the spectrum of Laplacian eigenvalues, and the distribution of the dominant non-trivial eigenvalue. We found that all of those properties are consistent between the ensemble of biological protein residue networks and the networks emerging in a self-organized way from the simple model.\n\nThese results indicate that coarse ensemble properties of 3D protein structures are already induced by geometric constraints alone such that only finer scales of the folded structures of individual proteins are speci1cally controlled by the details of their amino acid sequences. Such simple models provide a new angle of analyzing protein structures at the coarse scale of ensembles and may help understand core mechanisms underlying the complex folding process.

biophysics↗