Brownian Dynamics Simulation of Nucleocytoplasmic Transport: A Coarse-Grained Model for the Functional State of the Nuclear Pore Complex

The nuclear pore complex (NPC) regulates molecular traffic across the nuclear envelope (NE). Selective transport happens on the order of milliseconds and the length scale of tens of nanometers; however, the transport mechanism remains elusive. Central to the transport process is the hydrophobic interactions between karyopherins (kaps) and Phe-Gly (FG) repeat domains. Taking into account the polymeric nature of FG-repeats grafted on the elastic structure of the NPC, and the kap-FG hydrophobic affinity, we have established a coarse-grained model of the NPC structure that mimics nucleocytoplasmic transport. To establish a foundation for future works, the methodology and biophysical rationale behind the model is explained in details. The model predicts that the first-passage time of a 15 nm cargo-complex is about 2.6±0.13 ms with an inverse Gaussian distribution for statistically adequate number of independent Brownian dynamics simulations. Moreover, the cargo-complex is primarily attached to the channel wall where it interacts with the FG-layer as it passes through the central channel. The kap-FG hydrophobic interaction is highly dynamic and fast, which ensures an efficient translocation through the NPC. Further, almost all eight hydrophobic binding spots on kap-β are occupied simultaneously during transport. Finally, as opposed to intact NPCs, cytoplasmic filaments-deficient NPCs show a high degree of permeability to inert cargos, implying the defining role of cytoplasmic filaments in the selectivity barrier

Computational Biophysical Model of the Nuclear Pore Complex: Insights from Statistical and Big-Data Analysis

The nuclear pore complexes (NPCs) are the sole channels known on the nuclear envelope in eukaryotes through which nucleocytoplasmic traffic is selectively and efficiently conducted. While the NPC has been the subject of extensive research for the past six decades and many of its structural, biochemical, and biophysical details are revealed, mechanistics of selective transport yet to be known. The main game-player in conducting the selective transport is a class of the NPC proteins rich in Phe-Gly (FG)-repeat domains that are localized mainly to the channel interior. FG-repeat domains are natively unfolded and thus belong to the family of intrinsically disordered proteins (IDPs). The conformational behavior of FG-repeats remains unknown because of lack of detailed information about their microdynamics, leaving plenty of room for speculation on how the selectivity barrier forms inside the NPC. To tackle the microdynamics of FG-repeats, here I used polymer physics’ principles to develop a coarse-grained computational biophysical model, incorporating the full sequence of all amino-acids. The simulations were run under Brownian dynamics approach to generate long time-evolution of FG-repeat domains. Under known physiological conditions and geometrical constraints, FG-repeats form a spatially nonuniform cohesive meshwork, percolating in radial and axial directions, with a dense hydrophobic zone in the middle and a low-density zone near the wall. The FG-meshwork is extremely dynamic, resembling a jerking plug with a fluctuating concentration in radial direction. Being porous with the dominant pore sizes of 4 and 6 nm, this dynamic meshwork is permeable to the active cargos in a hydrophobic, and to a lesser extent, charge, stimuli-responsive manner, but strongly impermeable to inert cargos having the same size. An active cargo creates a big deformation inside the FG-meshwork, but because of rapid Brownian motions of the FG-repeats, it reconstructs itself in in a cargo size and shape dependent manner, suggesting the individual FG-repeats undergo reversible collapse. Significantly, the reconstruction process follows a saturating exponential pattern with rapid and slow phases. The characteristic time of reconstruction is a function of cargo size and shape, and is generally smaller for the elongated cargos compared to globular cargos having the same surface chemistry. Next, I used computational microrheology via many-particle tracking without external probe to investigate the full mechanical spectrum of FG-repeats under different physical and geometrical conditions, including FG-repeats’ composition, FG-repeats’ length, geometrical confinement, shuttling cargo, and end-tethering. The results reveal that FG-repeats show a non-Newtonian behavior as manifested in their shear-thinning viscosity. The viscoelastic response of FG-repeats is strongly frequency-dependent, and is consistent with the function of the permeability barrier at different frequencies, or equivalently, at different timescales. At low frequencies, equivalent to timescale of nucleocytoplasmic transport, FG-repeats form a pseudo solid-like meshwork. At high frequencies, equivalent to the timescale of thermal diffusion of small molecules, FG-repeats behave like a predominantly viscous liquid. The end-tethering is determined to be the most influential factor in shaping the mechanical spectrum of the FG-repeats. When the end-tethering is lifted and FG-repeats get free in the space, they invariably behave as a non-Newtonian viscous liquid over all frequencies. The other factors investigated are geometrical confinement, FG-repeats composition, i.e. hydrophobicity and charge content, length of FG-repeats, and shuttling cargo. Although these factors might shift the value of viscoelastic response in the frequency domain, they do not change the physics of the response, as end-tethering does. Comparing the viscoelastic response of FG-repeats with a general polymer melt show a remarkable consistency between FG-repeats and polymer melt over the range of frequencies accessible to the current model. Ultimately, the answer to the question of “do FG-repeats form gel?” is discussed in detail

Computational Biophysical Model of the Nuclear Pore Complex: Insights from Statistical and Big-Data Analysis

The nuclear pore complexes (NPCs) are the sole channels known on the nuclear envelope in eukaryotes through which nucleocytoplasmic traffic is selectively and efficiently conducted. While the NPC has been the subject of extensive research for the past six decades and many of its structural, biochemical, and biophysical details are revealed, mechanistics of selective transport yet to be known. The main game-player in conducting the selective transport is a class of the NPC proteins rich in Phe-Gly (FG)-repeat domains that are localized mainly to the channel interior. FG-repeat domains are natively unfolded and thus belong to the family of intrinsically disordered proteins (IDPs). The conformational behavior of FG-repeats remains unknown because of lack of detailed information about their microdynamics, leaving plenty of room for speculation on how the selectivity barrier forms inside the NPC. To tackle the microdynamics of FG-repeats, here I used polymer physics’ principles to develop a coarse-grained computational biophysical model, incorporating the full sequence of all amino-acids. The simulations were run under Brownian dynamics approach to generate long time-evolution of FG-repeat domains. Under known physiological conditions and geometrical constraints, FG-repeats form a spatially nonuniform cohesive meshwork, percolating in radial and axial directions, with a dense hydrophobic zone in the middle and a low-density zone near the wall. The FG-meshwork is extremely dynamic, resembling a jerking plug with a fluctuating concentration in radial direction. Being porous with the dominant pore sizes of 4 and 6 nm, this dynamic meshwork is permeable to the active cargos in a hydrophobic, and to a lesser extent, charge, stimuli-responsive manner, but strongly impermeable to inert cargos having the same size. An active cargo creates a big deformation inside the FG-meshwork, but because of rapid Brownian motions of the FG-repeats, it reconstructs itself in in a cargo size and shape dependent manner, suggesting the individual FG-repeats undergo reversible collapse. Significantly, the reconstruction process follows a saturating exponential pattern with rapid and slow phases. The characteristic time of reconstruction is a function of cargo size and shape, and is generally smaller for the elongated cargos compared to globular cargos having the same surface chemistry. Next, I used computational microrheology via many-particle tracking without external probe to investigate the full mechanical spectrum of FG-repeats under different physical and geometrical conditions, including FG-repeats’ composition, FG-repeats’ length, geometrical confinement, shuttling cargo, and end-tethering. The results reveal that FG-repeats show a non-Newtonian behavior as manifested in their shear-thinning viscosity. The viscoelastic response of FG-repeats is strongly frequency-dependent, and is consistent with the function of the permeability barrier at different frequencies, or equivalently, at different timescales. At low frequencies, equivalent to timescale of nucleocytoplasmic transport, FG-repeats form a pseudo solid-like meshwork. At high frequencies, equivalent to the timescale of thermal diffusion of small molecules, FG-repeats behave like a predominantly viscous liquid. The end-tethering is determined to be the most influential factor in shaping the mechanical spectrum of the FG-repeats. When the end-tethering is lifted and FG-repeats get free in the space, they invariably behave as a non-Newtonian viscous liquid over all frequencies. The other factors investigated are geometrical confinement, FG-repeats composition, i.e. hydrophobicity and charge content, length of FG-repeats, and shuttling cargo. Although these factors might shift the value of viscoelastic response in the frequency domain, they do not change the physics of the response, as end-tethering does. Comparing the viscoelastic response of FG-repeats with a general polymer melt show a remarkable consistency between FG-repeats and polymer melt over the range of frequencies accessible to the current model. Ultimately, the answer to the question of “do FG-repeats form gel?” is discussed in detail

The interaction of CRM1 and the nuclear pore protein Tpr.

While much has been devoted to the study of transport mechanisms through the nuclear pore complex (NPC), the specifics of interactions and binding between export transport receptors and the NPC periphery have remained elusive. Recent work has demonstrated a binding interaction between the exportin CRM1 and the unstructured carboxylic tail of Tpr, on the nuclear basket. Strong evidence suggests that this interaction is vital to the functions of CRM1. Using molecular dynamics simulations and a newly refined method for determining binding regions, we have identified nine candidate binding sites on CRM1 for C-Tpr. These include two adjacent to RanGTP--from which one is blocked in the absence of RanGTP--and three next to the binding region of the cargo Snurportin. We report two additional interaction sites between C-Tpr and Snurportin, suggesting a possible role for Tpr import into the nucleus. Using bioinformatics tools we have conducted conservation analysis and functional residue prediction investigations to identify which parts of the obtained binding sites are inherently more important and should be highlighted. Also, a novel measure based on the ratio of available solvent accessible surface (RASAS) is proposed for monitoring the ligand/receptor binding process

Looking "Under the Hood" of Cellular Mechanotransduction with Computational Tools: A Systems Biomechanics Approach across Multiple Scales.

Signal modulation has been developed in living cells throughout evolution to promote utilizing the same machinery for multiple cellular functions. Chemical and mechanical modules of signal transmission and transduction are interconnected and necessary for organ development and growth. However, due to the high complexity of the intercommunication of physical intracellular connections with biochemical pathways, there are many missing details in our overall understanding of mechanotransduction processes, i.e., the process by which mechanical signals are converted to biochemical cascades. Cell-matrix adhesions are mechanically coupled to the nucleus through the cytoskeleton. This modulated and tightly integrated network mediates the transmission of mechanochemical signals from the extracellular matrix to the nucleus. Various experimental and computational techniques have been utilized to understand the basic mechanisms of mechanotransduction, yet many aspects have remained elusive. Recently, in silico experiments have made important contributions to the field of mechanobiology. Herein, computational modeling efforts devoted to understanding integrin-mediated mechanotransduction pathways are reviewed, and an outlook is presented for future directions toward using suitable computational approaches and developing novel techniques for addressing important questions in the field of mechanotransduction

The Interaction of CRM1 and the Nuclear Pore Protein Tpr

<div><p>While much has been devoted to the study of transport mechanisms through the nuclear pore complex (NPC), the specifics of interactions and binding between export transport receptors and the NPC periphery have remained elusive. Recent work has demonstrated a binding interaction between the exportin CRM1 and the unstructured carboxylic tail of Tpr, on the nuclear basket. Strong evidence suggests that this interaction is vital to the functions of CRM1. Using molecular dynamics simulations and a newly refined method for determining binding regions, we have identified nine candidate binding sites on CRM1 for C-Tpr. These include two adjacent to RanGTP – from which one is blocked in the absence of RanGTP – and three next to the binding region of the cargo Snurportin. We report two additional interaction sites between C-Tpr and Snurportin, suggesting a possible role for Tpr import into the nucleus. Using bioinformatics tools we have conducted conservation analysis and functional residue prediction investigations to identify which parts of the obtained binding sites are inherently more important and should be highlighted. Also, a novel measure based on the ratio of available solvent accessible surface (RASAS) is proposed for monitoring the ligand/receptor binding process.</p></div