Biology subjects

Mugnai, M. L.

Publications and source records attributed to Mugnai, M. L..

12 recordsLinked to original sources

bioRxiv2025

Lever arm Flexibility Controls the extent of (Un)coupling to the Motor Domain in Myosin Motors

Dimeric myosin motors, with both heads simultaneously bound to filamentous actin, are in a frustrated conformation. The lever arm of each head would prefer to orient forward, but the inter-head tension hinders the relaxation to the state favored by a myosin monomer. Here, we investigate theoretically the impact of lever arm stiffness and coupling to the head domain using a polymer model. The theory for MV and MVI qualitatively reproduce the salient experimental observations. Furthermore, we construct chimeras in which the lever arm and head domains are swapped, and predict that the fluctuations of the LH lever arm of MVI are strongly impacted by the stiffness of the lever arm. Finally, by continuously and independently varying the lever arm persistence length and the strength of its coupling to the head domain we predict their roles in altering the average geometry and conformational flexibility of the dimer. We explore conditions under which LH lever arm flexibility and angle with respect to F-actin lead to coupling to the motor domain. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/669198v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1c27772org.highwire.dtl.DTLVardef@3015a9org.highwire.dtl.DTLVardef@de48eaorg.highwire.dtl.DTLVardef@1853176_HPS_FORMAT_FIGEXP M_FIG For Table of Contents Use Only C_FIG

biophysics↗

bioRxiv2025

Water-mediated interactions between glycans are weakly repulsive and unexpectedly long-ranged

Glycans on the cell surface play an essential role in mediating cell-cell interactions as well as immune response. Despite their importance, interactions between them have not been fully characterized. Here, we reveal, using all-atom molecular dynamics simulations and free energy calculations, that water-mediated interactions between a pair of N-glycans without net charge are weakly repulsive with a range that exceeds their sizes. Unexpectedly, the effective glycan-glycan interactions decay logarithmically as the separation between them increases. Strikingly, this finding coincides exactly with the predicted interaction, which is entropic in origin, between two star polymers, consisting of long flexible polymers grafted onto colloidal particles. The weak repulsive interaction, which extends beyond the size of a glycan, is sensitive to the relative orientation of the glycans. The effective long-range repulsive interaction vanishes if the charges on water are turned off, thus establishing that electrostatic interactions, arising in part due to the persistent hydrogen bonds between water and the glycans, are responsible for the inter-glycan repulsion.

biophysics↗

bioRxiv2023

Sizes, Conformational fluctuations, and SAXS profiles for Intrinsically Disordered Proteins

The preponderance of Intrinsically Disordered Proteins (IDPs) in the eukaryotic proteome, and their ability to interact with each other, folded proteins, RNA, and DNA for functional purposes, have made it important to quantitatively characterize their biophysical properties. Toward this end, we developed the transferable Self-Organized Polymer (SOP-IDP) model to calculate the properties of several IDPs. The values of the radius of gyration (Rg) obtained from SOP-IDP simulations are in excellent agreement (correlation coefficient of 0.96) with those estimated from SAXS experiments. For AP180 and Epsin, the predicted values of the hydrodynamic radii (Rhs) are in quantitative agreement with those from Fluorescence Correlation Spectroscopy (FCS) experiments. Strikingly, the calculated SAXS spectra for thirty-six IDPs are also nearly superimposable on the experimental profiles. The dependence of Rg and the mean end-to-end distance (Ree) on chain length, N, follows Florys scaling law, R {approx} aN 0.588 ( = g, and e), suggesting that globally IDPs behave as synthetic polymers in a good solvent. The values of ag, and ae are 0.20 nm and 0.48 nm respectively. Surprisingly, finite size corrections to scaling, expected on theoretical grounds, are negligible for Rg and Ree. In contrast, only by accounting for the finite sizes of the IDPs, the dependence of experimentally measurable Rh on N can be quantitatively explained using{nu} = 0.588. Although Flory scaling law captures the estimates for Rg, Ree, and Rh accurately, the spread of the simulated data around the theoretical curve is suggestive of of sequence-specific features that emerge through a fine-grained analysis of the conformational ensembles using hierarchical clustering. Typically, the ensemble of conformations partitiones into three distinct clusters, having different equilibrium populations and structural properties. Without any further readjustments to the parameters of the SOP-IDP model, we also obtained excellent agreement with paramagnetic relaxation enhancement (PRE) measurements for -synuclein. The transferable SOP-IDP model sets the stage for several applications, including the study of phase separation in IDPs and interactions with nucleic acids.

biophysics↗

bioRxiv2021

Sequence determines the switch in the fibril forming regions in the low complexity FUS protein and its variants

Residues spanning distinct regions of the low-complexity domain of the RNA-binding protein, Fused in Sarcoma (FUS-LC), form fibril structures with different core morphologies. NMR experiments show that the 214 residue FUS-LC forms a fibril with an S-bend (core-1, residues 39-95), while the rest of the protein is disordered. In contrast, the fibrils of the C-terminal variant (FUS-LC-C; residues 111-214) has a U-bend topology (core-2, residues 112-150). Absence of the U-bend in FUS-LC implies that the two fibril cores do not coexist. Computer simulations show that these perplexing findings could be understood in terms of the population of sparsely-populated fibril-like excited states in the monomer. The propensity to form core-1 is higher compared to core-2. We predict that core-2 forms only in truncated variants that do not contain the core-1 sequence. At the monomer level, sequence-dependent enthalpic effects determine the relative stabilities of the core-1 and core-2 topologies. TOC graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=164 SRC="FIGDIR/small/451535v2_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@f81959org.highwire.dtl.DTLVardef@4859f3org.highwire.dtl.DTLVardef@96aecorg.highwire.dtl.DTLVardef@4ab94e_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

On the emergence of orientational order in folded proteins with implications for allostery

The beautiful structures of single and multi-domain proteins are clearly ordered in some fashion but cannot be readily classified using group theory methods that are successfully used to describe periodic crystals. For this reason, protein structures are considered to be aperiodic, and may have evolved this way for functional purposes, especially in instances that require a combination of softness and rigidity within the same molecule. By analyzing the solved protein structures, we show that orientational symmetry is broken in the aperiodic arrangement of the secondary structural elements (SSEs), which we deduce by calculating the nematic order parameter, P2. We find that the folded structures are nematic droplets with a broad distribution of P2. We argue that non-zero values of P2, leads to an arrangement of the SSEs that can resist external stresses forces, which is a requirement for allosteric proteins. Such proteins, which resist mechanical forces in some regions while being flexible in others, transmit signals from one region of the protein to another (action at a distance) in response to binding of ligands (oxygen, ATP or other small molecules).

biophysics↗

bioRxiv2020

Molecular Transfer Model for pH effects on Intrinsically Disordered Proteins: Theory and Applications

We present a theoretical method to study how changes in pH shape the heterogeneous conformational ensemble explored by intrinsically disordered proteins (IDPs). The theory is developed in the context of coarse-grained models, which enable a fast, accurate, and extensive exploration of conformational space at a given protonation state. In order to account for pH effects, we generalize the Molecular Transfer Model (MTM), in which conformations are re-weighted using the transfer free energy, which is the free energy necessary for bringing to equilibrium in a new environment a "frozen" conformation of the system. Using the semi-grand ensemble, we derive an exact expression of the transfer free energy, which amounts to the appropriate summation over all the protonation states. Because the exact result is computationally too demanding to be useful for large polyelectrolytes or IDPs, we introduce a mean-field (MF) approximation of the transfer free energy. Using a lattice model, we compare the exact and MF results for the transfer free energy and a variety of observables associated with the model IDP. We find that the precise location of the charged groups (the sequence), and not merely the net charge, determines the structural properties. We demonstrate that some of the limitations previously noted for MF theory in the context of globular proteins are mitigated when disordered polymers are studied. The excellent agreement between the exact and MF results poises us to use the method presented here as a computational tool to study the properties of IDPs and other biological systems as a function of pH.

biophysics↗

bioRxiv2020

Role of Long-range Allosteric Communication in Determining the Stability and Disassembly of SARS-COV-2 in Complex with ACE2

Severe acute respiratory syndrome (SARS) and novel coronavirus disease (COVID-19) are caused by two closely related beta-coronaviruses, SARS-CoV and SARS-CoV-2, respectively. The envelopes surrounding these viruses are decorated with spike proteins, whose receptor binding domains (RBDs) initiate invasion by binding to the human angiotensin-converting enzyme 2 (ACE2). Subtle changes at the interface with ACE2 seem to be responsible for the enhanced affinity for the receptor of the SARS-CoV-2 RBD compared to SARS-CoV RBD. Here, we use Elastic Network Models (ENMs) to study the response of the viral RBDs and ACE2 upon dissassembly of the complexes. We identify a dominant detachment mode, in which the RBD rotates away from the surface of ACE2, while the receptor undergoes a conformational transition which stretches the active-site cleft. Using the Structural Perturbation Method, we determine the network of residues, referred to as the Allostery Wiring Diagram (AWD), which drives the large-scale motion activated by the detachment of the complex. The AWD for SARS-CoV and SARS-CoV-2 are remarkably similar, showing a network that spans the interface of the complex and reaches the active site of ACE2, thus establishing an allosteric connection between RBD binding and receptor catalytic function. Informed in part by the AWD, we used Molecular Dynamics simulations to probe the effect of interfacial mutations in which SARS-CoV-2 residues are replaced by their SARS-CoV counterparts. We focused on a conserved glycine (G502 in SARS-CoV-2, G488 in SARS-CoV) because it belongs to a region that initiates the dissociation of the complex along the dominant detachment mode, and is prominent in the AWD. Molecular Dynamics simulations of SARS-CoV-2 wild-type and G502P mutant show that the affinity for the human receptor of the mutant is drastically diminished. Our results suggest that in addition to residues that are in direct contact with the interface those involved in long range allosteric communication are also a determinant of the stability of the RBD-ACE2 complex.

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bioRxiv2020

Effects of Gold Nanoparticles on the Stepping Trajectories of Kinesin

Substantial increase in the temporal resolution of the stepping of dimeric molec- ular motors is possible by tracking the position of a large gold nanoparticle (GNP) attached to a labeled site on one of the heads. This technique was used to measure the stepping trajectories of conventional kinesin (Kin1) using the time dependent position of the GNP as a proxy. The trajectories revealed that the detached head always passes to the right of the head that is tightly bound to the microtubule (MT) during a step. In interpreting the results of such experiments, it is implicitly assumed that the GNP does not significantly alter the diffusive motion of the detached head. We used coarse-grained simulations of a system consisting of the MT-Kin1 complex with and without attached GNP to investigate how the stepping trajectories are affected. The two significant findings are: (1) The GNP does not faithfully track the position of the stepping head. (2) The rightward bias is typically exaggerated by the GNP. Both these findings depend on the precise residue position to which the GNP is attached. Surprisingly, we predict that the stepping trajectories of kinesin are not significantly affected if, in addition to the GNP, a 1 m diameter cargo is attached to the coiled coil. Our simulations suggest the effects of the large probe have to be considered when inferring the stepping mechanisms using GNP tracking experiments.

biophysics↗

bioRxiv2019

Step-wise Hydration of Magnesium by Four Water Molecules Precedes Phosphate Release in a Myosin Motor

Molecular motors, such as myosin, kinesin, and dynein, convert the energy released by the hydrolysis of ATP into mechanical work, which allows them to undergo directional motion on cytoskeletal tracks. This process is achieved through synchronization between the catalytic activity of the motor and the associated changes in its conformation. A pivotal step in the chemomechanical transduction in myosin motors occurs after they bind to the actin filament, which triggers the release of phosphate (Pi, product of ATP hydrolysis) and the rotation of the lever arm. Here, we investigate the mechanism of phosphate release in myosin VI, which has been debated for over two decades, using extensive molecular dynamics simulations involving multiple trajectories each several s long. Because the escape of phosphate is expected to occur on time-scales on the order of milliseconds in myosin VI, we observed Pi release only if the trajectories were initiated with a rotated phosphate inside the nucleotide binding pocket. The rotation provided the needed perturbation that enabled successful expulsions of Pi in several trajectories. Analyses of these trajectories lead to a robust mechanism of Pi release in the class of motors belonging to the myosin super family. We discovered that although Pi populates the traditional \"back door\" route, phosphate exits through various other gateways, thus establishing the heterogeneity in the escape routes. Remarkably, we observe that the release of phosphate is preceded by a step-wise hydration of the ADP-bound magnesium ion. In particular, the release of the anion occurred only after four water molecules hydrate the cation (Mg2+). By performing comparative structural analyses, we suggest that the hydration of magnesium is the key step in the phosphate release in a number of ATPases and GTPases that share a similar structure in the nucleotide binding pocket. Thus, nature may have evolved hydration of Mg2+ by discrete water molecules as a general molecular switch for Pi release, which is a universal step in the catalytic cycle of many machines which share little sequence or structural similarity.

biophysics↗

bioRxiv2019

Processivity and Velocity for Motors Stepping on Periodic Tracks

Processive molecular motors enable cargo transportation by assembling into dimers capable of taking several consecutive steps along a cytoskeletal filament. In the well-accepted hand-over-hand stepping mechanism the trailing motor detaches from the track and binds the filament again in leading position. This requires fuel consumption in the form of ATP hydrolysis, and coordination of the catalytic cycles between the leading and the trailing heads. However, alternative stepping mechanisms exist, including inchworm-like movements, backward steps, and foot stomps. Whether all of these pathways are coupled to ATP hydrolysis remains to be determined. Here, in order to establish the principles governing the dynamics of processive movement, we present a theoretical framework which includes all of the alternative stepping mechanisms. Our theory bridges the gap between the elemental rates describing the biochemical and structural transitions in each head, and the experimentally measurable quantities, such as velocity, processivity, and probability of backward stepping. Our results, obtained under the assumption that the track is periodic and infinite, provide expressions which hold regardless of the topology of the network connecting the intermediate states, and are therefore capable of describing the function of any molecular motor. We apply the theory to myosin VI, a motor that takes frequent backward steps, and moves forward with a combination of hand-over-hand and inchworm-like steps. Our model reproduces quantitatively various observables of myosin VI motility measured experimentally from two groups. The theory is used to predict the gating mechanism, the pathway for backward stepping, and the energy consumption as a function of ATP concentration.\n\nSignificance StatementMolecular motors harness the energy released by ATP hydrolysis to transport cargo along cytoskeletal filaments. The two identical heads in the motor step alternatively on the polar track by communicating with each other. Our goal is to elucidate how the coordination between the two heads emerges from the catalytic cycles. To do so, we created a theoretical framework that allows us to relate the measurable features of motility, such as motor velocity, with the biochemical rates in the leading and trailing heads, thereby connecting biochemical activity and motility. We illustrate the efficacy of the theory by analyzing experimental data for myosin VI, which takes frequent backward steps, and moves forward by a hand-over-hand and inchworm-like steps.

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bioRxiv2018

Sequence effects on size, shape, and structural heterogeneity in Intrinsically Disordered Proteins

Intrinsically disordered proteins (IDPs) lack well-defined three-dimensional structures, thus challenging the archetypal notion of structure-function relationships. Determining the ensemble of conformations that IDPs explore under physiological conditions is the first step towards understanding their diverse cellular functions. Here, we quantitatively characterize the structural features of IDPs as a function of sequence and length using coarse-grained simulations. For diverse IDP sequences, with the number of residues (NT) ranging from 24 to 441, our simulations not only reproduce the radii of gyration (Rg) obtained from experiments, but also predict the full scattering intensity profiles in very good agreement with Small Angle X-ray Scattering experiments. The Rg values are well-described by the standard Flory scaling law, [Formula], with v {approx} 0.588, making it tempting to assert that IDPs behave as polymers in a good solvent. However, clustering analysis reveals that the menagerie of structures explored by IDPs is diverse, with the extent of heterogeneity being highly sequence-dependent, even though ensemble-averaged properties, such as the dependence of Rg on chain length, may suggest synthetic polymer-like behavior in a good solvent. For example, we show that for the highly charged Prothymosin-, a substantial fraction of conformations is highly compact. Even if the sequence compositions are similar, as is the case for -Synuclein and a truncated construct from the Tau protein, there are substantial differences in the conformational heterogeneity. Taken together, these observations imply that metrics based on net charge or related quantities alone, cannot be used to anticipate the phases of IDPs, either in isolation or in complex with partner IDPs or RNA. Our work sets the stage for probing the interactions of IDPs with each other, with folded protein domains, or with partner RNAs, which are critical for describing the structures of stress granules and biomolecular condensates with important cellular functions. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/427476v2_ufig1.gif" ALT="Figure 1"> View larger version (12K): org.highwire.dtl.DTLVardef@1b50bd9org.highwire.dtl.DTLVardef@11cf542org.highwire.dtl.DTLVardef@29ebb5org.highwire.dtl.DTLVardef@31e965_HPS_FORMAT_FIGEXP M_FIG Graphical TOC Entry C_FIG

biophysics↗

bioRxiv2018

Dynamics of Allosteric Transitions in Dynein

1Cytoplasmic Dynein, a motor with an unusual architecture made up of a motor domain belonging to the AAA+ family, walks on microtubule towards the minus end. Prompted by the availability of structures in different nucleotide states, we performed simulations based on a new coarse-grained model to illustrate the molecular details of the dynamics of allosteric transitions in the motor. The simulations show that binding of ATP results in the closure of the cleft between the AAA1 and AAA2, which in turn triggers conformational changes in the rest of the motor domain, thus poising dynein in the pre-power stroke state. Interactions with the microtubule, which are modeled implicitly, substantially enhances the rate of ADP release, and formation of the post-power stroke state. The dynamics associated with the key mechanical element, the linker (LN) domain, which changes from a straight to a bent state and vice versa, are highly heterogeneous suggestive of multiple routes in the pre power stroke to post power stroke transition. We show that persistent interactions between the LN and the insert loops in the AAA2 domain prevent the formation of pre-power stroke state when ATP is bound to AAA3, thus locking dynein in a non-functional repressed state. Motility in such a state may be rescued by applying mechanical force to the LN domain. Taken together, these results show how the intricate signaling dynamics within the motor domain facilitate the stepping of dynein.

biophysics↗