The neuronal dynamin1 functions in the release of synaptic vesicles by orchestrating the process of GTPase dependent membrane fission. Dynamin1 associates with the plasma membrane-localized phosphatidylinositol-4,5-bisphosphate (PIP2) through the centrally-located pleckstrin homology domain (PHD). The PHD is dispensable as fission (in model membranes) can be managed, even when the PHD-PIP2 interaction is replaced by a generic polyhistidine- or polylysine-lipid interaction. However, the absence of the PHD renders a dramatic dampening of the rate of fission. These observations suggest that the PHD-PIP2 containing membrane interaction could have evolved to expedite fission to fulfill the requirement of rapid kinetics of synaptic vesicle recycling. Here, we use a suite of multiscale modeling approaches to explore PHD-membrane interactions. Our results reveal that (a) the binding of PHD to PIP2-containing membranes modulates the lipids towards fission-favoring conformations and softens the membrane, and (b) PHD associates with membrane in multiple orientations using variable loops as pivots. We identify a new loop (VL4), which acts as an auxiliary pivot and modulates the orientation flexibility of PHD on the membrane — a mechanism we believe may be important for high fidelity dynamin collar assembly. Together, these insights provide a molecular-level understanding of the catalytic role of PHD in dynamin-mediated membrane fission. [Media: see text] [Media: see text] [Media: see text] [Media: see text]

Classical dynamins are best understood for their ability to generate vesicles by membrane fission. During clathrin-mediated endocytosis (CME), dynamin is recruited to the membrane through multivalent protein and lipid interactions between its proline-rich domain (PRD) with SRC Homology 3 (SH3) domains in endocytic proteins and its pleckstrin-homology domain (PHD) with membrane lipids. Variable loops (VL) in the PHD bind lipids and partially insert into the membrane thereby anchoring the PHD to the membrane. Recent molecular dynamics (MD) simulations reveal a novel VL4 that interacts with the membrane. Importantly, a missense mutation that reduces VL4 hydrophobicity is linked to an autosomal dominant form of Charcot-Marie-Tooth (CMT) neuropathy. We analyzed the orientation and function of the VL4 to mechanistically link data from simulations with the CMT neuropathy. Structural modeling of PHDs in the cryo-electron microscopy (cryo-EM) cryoEM map of the membrane-bound dynamin polymer confirms VL4 as a membrane-interacting loop. In assays that rely solely on lipid-based membrane recruitment, VL4 mutants with reduced hydrophobicity showed an acute membrane curvature-dependent binding and a catalytic defect in fission. Remarkably, in assays that mimic a physiological multivalent lipid- and protein-based recruitment, VL4 mutants were completely defective in fission across a range of membrane curvatures. Importantly, expression of these mutants in cells inhibited CME, consistent with the autosomal dominant phenotype associated with the CMT neuropathy. Together, our results emphasize the significance of finely tuned lipid and protein interactions for efficient dynamin function.

Characterization of different conformational states of proteins is essential to understand their stability and activity. Biophysical techniques aid in analysing these conformational states and molecular fluorescence is one of the most reliable and quickly accessible methods. Apart from the intrinsic fluorescence of proteins, external fluorescence dyes such as TNS, ANS, nile red and thioflavin are also used to characterize partially unfolded, aggregated and fibrillar states of proteins, though their exact molecular-level interactions with proteins are yet to be completely unravelled. The present study attempts to investigate the binding of TNS molecules on different conformational states of proteins. Unconstrained molecular dynamics simulation of 50 molecules of TNS with the native state of BSA, native and two partially unfolded states of RNase A and α-lactalbumin in water was carried out. Dynamics simulation of TNS alone in different solvents such as water, ethanol, DMF and DMSO was also performed. Binding environments in all the proteins and the solvents were analysed in terms of H-bonding interactions, order of contacts, amino acid specificity and conformational changes of TNS, and correlated with experimentally observed fluorescence changes of the dye. The results suggest that TNS forms aggregates in water whereas in non-aqueous solvents the order of aggregates is lower which might result in an enhancement of its fluorescence intensity. Further, TNS preferably interacts with basic and aromatic amino acid residues of the proteins. In RNase A and α-lactalbumin, most of the TNS molecules tend to form aggregates even with the unfolded conformations of the proteins. However in BSA, the number of aggregated TNS molecules is less and TNS molecules in monomeric form are found in the hydrophobic crevices of the protein. This might result in an enhancement of the fluorescence in BSA compared to the other proteins. The distributions of angles and dihedrals of TNS in different environments suggest that the bending movement between the naphthyl and tolyl rings is constrained whereas significant planar rotations could be observed both in solvents and in protein-bound states.

The neuronal dynamin1 functions in the release of synaptic vesicles by orchestrating a process of GTPase-dependent membrane fission. Dynamin1 associates with the plasma membrane-localized phosphatidylinositol-4,5bisphosphate (PIP2) through the centrallylocated pleckstrin homology domain (PHD). The PHD is dispensable as fission can be managed even when the PHD-PIP2 interaction is replaced by a generic polyhistidine-or polylysine-lipid interaction. Remarkably however, the absence of the PHD renders a dramatic dampening of the rate of fission. These observations suggest that the PHD-PIP2 interaction could have evolved to expedite fission to fulfill the requirement of rapid kinetics of synaptic vesicle recycling. Here, we use a suite of multiscale modeling approaches that combine atomistic molecular dynamics simulations, mixed-resolution membrane mimetic models, coarse-grained molecular simulations and advanced free-energy sampling (metadynamics) methods to explore PHDmembrane interactions. Our results reveal that; (a) the binding of PHD to PIP2-containing membranes modulates the lipids towards fission-favoring conformations and overall softens the membrane thus rendering it pliable

One of the mysteries in studying the molecular “Origin of Life” is the emergence of RNA and RNA-based life forms, where non-enzymatic polymerization of nucleotides is a crucial hypothesis in formation of large RNA chains. The non-enzymatic polymerization can be mediated by various environmental settings such as cycles of hydration and dehydration, temperature variations and proximity to a variety of organizing matrices such as clay, salt, fatty acids, lipid membrane and mineral surface. In this work, we explore the influence of different phases of the lipid membrane towards nucleotide organization and polymerization in a simulated prebiotic setting. We calculate the free energy cost of localizing a mononucleotide, Uridine monophosphate (UMP), in distinct membrane settings and we perform all-atom molecular dynamics (MD) simulations to estimate the role of the monophasic and biphasic membrane in modifying the behavior of UMPs localization and their clustering mechanism. Based on the free-energy and diffusion data from our MD calculations, we develop a lattice based model to explore the thermodynamic limits of the observations made from the MD simulations. The mathematical model substantiates our hypothesis that the lipid layers can act as unique substrates for ‘catalyzing’ polymerization of mononucleotides due to the inherent spatiotemporal heterogeneity and phase change behavior.