ICTS

1. Department of Biological Sciences, Columbia University, NY, USA
2. Department of Microbiology and Cell Biology Indian Institute of Science, Bangalore
    

Biochemical and structural studies of MutT1, a novel enzyme in Mycobacterium smegmatis: hydrolysis of diadenosine polyphosphates

Ap4A is the first one to be discovered among the members of diadenosine polyphosphates, a class of compounds denoted as Ap n A, where n=2-6. The intracellular Ap4A is an inevitable byproduct of cellular metabolism. It is synthesised in vitro (and presumably in vivo) predominantly by a side reaction of an aminoacyladenylate with an acceptor nucleotide, catalysed by various aminoacyl-tRNA synthetases. Increase in the intracellular level of Ap4A, due to the lack of the hydrolase responsible for its degradation, is believed to affect the mobility, cell metabolism and intracellular invasion capacity of many bacteria. With regard to mycobacteria, involvement of Ap4A in its survival and virulence inside its host is conceivable. Moreover, the presence of genes and their products involved in degradation of Ap4A in mycobaterial sp. suggest that the level of Ap4A in the cell should be regulated to avoid its possible detrimental consequences. Assuming a functional analogy of Ap4A in mycobacterial pathogenesis, the enzymes involved in the regulation of intracellular level of Ap4A can be targeted for the development of inhibitors.

Mycobacterium smegmatis MutT1 (MsMutT1) belongs to Nudix hydrolase superfamily of enzymes, a family which encompasses members with specificity towards various substrates. Recently, we have detected a novel Ap4A hydrolase like activity (catalysing the hydrolysis of Ap4A, Ap5A and Ap6A) in MsMutT1, which, until now, was known to hydrolyse 8-oxo- GTP and 8-oxo- dGTP and was, thus, thought to be involved in the sanitization of nucleotide pool. A detailed analysis of the structures of MsMutT1 in complex with a substrate of Ap4A hydrolases namely, Ap4A, the product, ATP and an inhibitory complex involving fluoride and magnesium ions along with the biochemical activity of this enzyme on various diadenosine polyphosphates (Ap4A, Ap5A and Ap6A), is presented here. 
 

Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai, India

Understanding the Structural role of β-tubulin mutations R306-C/H in the development of drug resistance to a ‘non-taxoid’ site drug, Laulimalide using molecular modelling

Laulimalide is 'non-taxoid site' microtubule stabilizing drug. It has promising anticancer activity against taxol resistant cancer cells because of high solublilty and less susceptibilty towards the P-gp pump. Laulimalide prefers the outer surface of β-tubulin and promote microtubule stabilization by linking two adjacent αβ-Tubulin heterodimers from parallel protofilament. It promotes tubulin polymerization in MDR cell line. However, recent study shows the resistance against laulimalide in βI-tubulin isotype due to single point mutation of R306C/H position in human ovarian cancer cells. However, the binding mode and mechanism of laulimalide resistance due to mutation of R306C/H in βI-tubulin isotype is not well understood due to lack of structural information. Therefore, we employed homology modelling, molecular docking and molecular dynamics simulations to understand the binding mode and interaction of laulimalide with wild type and mutants (R306C) human αβI-tubulin isotype heterodimer. Molecular docking study shows that, mutations affect the binding of laulimalide at the H9-H10 binding pocket compare to wild type αβI-tubulin. Further, analysis of molecular dynamics simulations results using root mean square deviation (RMSD), root mean square fluctations (RMSF) and radius of gyration (Rg) show that, mutations (R306C/H) affect the binding of laulimalide due to conformational changes in the H9-B8 loop of βI-tubulin protein. Such conformational changes in the binding pocket leads to decrease in the compactness of the mutant as compare to wild type βI-tubulin protein. Hence, our computational study shows that R306C/H mutations affects the laulimalide and βI-tubulin interaction which is responsible for the laulimalide-βI-tubulin association. This results will be helpful to understand the mechanism and role of R306C/H mutation in the development of laulimalide resistance as well as help to design potent analogues in future aganist drug resistant cancer.

Keywords: Laulimalide, multidrug resistance (MDR), molecular docking, molecular dynamics simulations.

Indian Institute of Science, Bangalore

Insights into the energetics of start codon selection during eukaryotic translation initiation by Molecular Dynamics Simulations

In order to start the initiation of translation in eukaryotic protein synthesis which is quite a complex process, the correct codon for initiation which is referred as start codon has to be recognized. Almost 12 proteins, known as the eukaryotic initiation factors (eIF) participate to arrange the translation process. Among them eIF1, eIF1A and eIF3, in complex with the small 40S ribosomal subunit, recruits the initiator tRNA (tRNAiMet ) with the GTP-bound factor eIF2 in a ternary complex (TC) with anticodon CAU to the peptidyl-site (P-site), resulting in the 43S pre-initiation complex (PIC). Then, the mRNA attaches to the 43S PIC together with eIF4 producing the 48S initiation complex (IC). The tRNA binds to the mRNA and codon scanning is started until the correct start codon, AUG, has been found. This selection of the correct start codon is the most crucial step in protein synthesis. When the initiator has to choose the right one among a set of alternatives, it might stop and consider the best. Though the scanning process and the role of various initiation factors have been studied well, still there remains a controversy. 48S IC, exists in two conformations, P-OUT (open state), where tRNA is not fully occupied in the P-site and P-IN (closed state) where tRNA is fully accommodated. The controversy is whether tRNA can scan the correct codon in P-OUT or every time it has to go to closed P-IN state for codon scanning. So we have calculated the free energy of binding of tRNA with the start codon and with different near cognate codons in P-IN state and will compare the same which has been done in P-OUT state. Thus from the energetic landscape of base-pairing of tRNA with different near cognate codons in both states, the scanning ability of tRNA in P-OUT state can be explored which will imply the occurrence of energetically expensive and time consuming transition between P-OUT and P-IN states.
 

Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai, India

Exploring the effects of microtubule & motor properties on microtubule bundle dynamics using stochastic simulations

Microtubule bundles are groups of microtubule filaments held together by crosslinkers and MAPs. A major function of these bundles is to provide structural support but they also contribute to force generation along with the actin network and this force is used to drive various cellular processes such as cell division, cell migration and neurite growth. Though microtubules and motors are being studied extensively in vitro and in vivo, studies on microtubule bundles are limited. This is due to the complex structure which is hard to construct in vitro and the lack of techniques to analyze the details of dynamics of the bundle & its components. We chose to use a computational approach to approximate the dynamics of the microtubule bundle and look at the effect of microtubule and motor properties. Recent models by Jakobs et al. 2015 and Zemel & Mogilner 2009 have looked at the effect of motors on microtubule dynamics but have not considered the various motor properties such as attachment-detachment kinetics, structural properties(eg. elasticity). We use the stochastic bungee spring model of motor proteins developed by Kunwar et al 2011 and apply it to the microtubule bundle. In our model, the bundle is crosslinked by motor proteins whose tails are permanently attached to a position on a microtubule while the head is movable and is bound to another microtubule. Preliminary results of the 2-dimensional bundle show qualitative conformance with the results from implicit simulation in literature. Currently, we are testing and running the 3- dimensional bundle simulation.
 

CSIR–Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad

Mechanistic insights into chiral proofreading during translation of the genetic code

Translational quality control involves the coordinated function of multiple cellular factors, including the ribosome. An important step during translation of the genetic code is the hydrolysis of a D-amino acid which has been erroneously esterified to a tRNA. This proofreading step is performed by a homodimeric enzyme named D-aminoacyl-tRNA deacylase (DTD). Ligand- bound crystal structures complemented with biophysical and biochemical analyses have revealed that an invariant, cross-subunit Gly-cisPro motif is responsible for DTD’s enantioselectivity. Furthermore, strict L-chiral rejection by the motif, rather than D-chiral selection, is the only design principle behind DTD’s chiral specificity. Consequently, DTD efficiently acts on Gly- tRNA(Gly), thereby causing the latter’s misediting. The “misediting paradox”, however, is resolved through protection of the cognate achiral substrate by the elongation factor, EF-Tu. Nevertheless, higher levels of DTD causes cellular toxicity due to depletion of Gly-tRNA(Gly). Our recent findings have revealed that DTD’s activity on glycine is advantageous because it is able to edit the non-cognate Gly-tRNA(Ala) species generated by alanyl-tRNA synthetase. Thus, DTD’s physiological role extends beyond “chiral proofreading”. We have also demonstrated that DTD is a primordial enzyme which functions at the RNA–protein interface and uses the 2′-OH of adenosine-76 of tRNA, instead of active site protein side chains, to perform catalysis.
 

Department of Physics, Indian Institute of Technology, Kanpur

Stochastic Thermodynamics of Ribosome: a graph theoretic perspective

Using graph theoretic approach, a stochastic mechano-chemical kinetic model for ribosome translation has been proposed. Towards understanding the system, we have computed many important thermodynamic quantities such as steady state probabilities, cycle fluxes, transition fluxes, one way cycle fluxes, cycle rate constants, cycle probabilities, mean time between coupled cycles, entropy production rate in terms of cycle rates, flux-force relation based on this framework. An advantage of graph theoretic framework may be highlighted by noting that cycle fluxes, cycle rate constants, cycle probabilities, mean time between coupled cycles, entropy production rate in terms of cycle rates, flux-force relation cannot be calculated using master equation approach. Also, it gives us a comprehensive overview of the general principles involved in free energy transduction. It may be asserted that free energy transduction is achieved by complete biochemical cycles and not by individual transitions. Therefore, it may be argued that this framework gives us a much superior and deeper understanding of our system. Given that each and every cellular process is governed by proteins which are synthesized by ribosome, the present study may give us interesting applications and impact our understanding of various cellular processes. As a part of the exercise, an attempt has been made to give a stronger uncertainty bound for cycles present in our network. Based on this bound, the dissipation in a particular cycle may be computed using corresponding uncertainty for the cycle which may be available from experiments.
 

1. IIT Kanpur, India
2. Sandia National Lab, Livermore, California, USA
    

Dynamics of a Microtubule tethered to a wall by molecular motors: Catch-Bond-like behavior

Microtubule (MT) is a polar nanotube formed by the polymerization of a dimer of two protein subunits, the alpha and beta tubulin. In a eukaryotic cell, MT is attached with the cortex of the cell by dynein motors where the heads of the dynein motors can bind with the MT while the tails of all the dyneins are anchored on the cell cortex. Thus dynein functions as an active linker between the MT and the cortex wall. Our stochastic kinetic model is motivated by this typical attachment between the MT and the cortex wall. We have studied the strength and stability of the attachment by applying an external tension on the attachment. The external tension can be applied by following two protocols, namely, force clamp and force ramp. During the force clamp protocol, a constant tension is applied to the bond and time taken by the system to get ruptured is defined as the lifetime of the attachment. We have computed the distribution of the lifetimes; nonmonotonic variation of the average lifetime, referred to as "catch-bond-like" behavior of the cortex-MT attachment, has been observed with the increase of the externally applied tension. In the case of force ramp protocol, the externally applied tension increases (linearly) with time until the attachment gets ruptured. We calculate the rupture force distribution which characterizes the strength of the bond.
 

1. Department of Physics, The LNM Institute of Information Technology, Sumel, Jaipur,  India
2. Centre for Condensed Matter Theory, Department of Physics, Indian Institute of Science, Bangalore
    

Overstretching of DNA: Emergence of new structural polymorphs and S-DNA

Single molecule force spectroscopy measurements [1-5] of DNA reveal remarkable changes in DNA structure in presence of external force and around 65-70 pN forces it exhibits a mechanical transition where its extension increases by 70%. Inspite of many experimental and theoretical studies over more than a decade, there remains a significant debate on the characteristic properties of the overstretched DNA [3-14]. Using extensive all atom molecular dynamics simulations we report that in the overstretched regime ds-B-DNA adopts a new elongated S-DNA [13-15] structure when it is stretched along the 3’ directions of the opposite strands whereas stretching along 5’ directions of the opposite strands leads to force induced melting form of the DNA. We next discuss the structural polymorphism of DNA exhibited through various conformations with a change in the known helical parameters. We further perform the structural characterization of the S-DNA by calculating various helical parameters. S-DNA is further characterized by changes in the number of H-bonds, entropy and free energy. We next find that the free energy barrier between the canonical and overstretched states of DNA is higher for the same termini pulling protocol in comparison to all other considered here. Our observations [15] not only reconcile with the available experimental findings qualitatively but also enhance the understanding of different overstretched DNA structures.
 

References:

1. S. B. Smith, L. Finzi, and C. Bustamante, Science 258, 1122 (1992).
2. S. B. Smith, Y. Cui, C. Bustamante, Science, 271, 795 (1996).
3. C. Bustamante, Z. Bryant, S. B. Smith, Nature 421, 423 (2003).
4. P. Cluzel, A. Lebrun, C. Heller, R. Lavery, J-L Viovy, D. Chatenay and F. Caron, Science 271, 792 (1996).
5. K. R. Chaurasiya, T. Paramanathan, M. J. McCauley and M. C. Williams, Physics of Life Reviews 7, 299 (2010).
6. M. C. Williams, I. Rouzina, V. A. Bloomfield, Acc. Chem. Res. 35, 159 (2002).
7. T. R. Strick, J. F. Allemand, D. Bensimon, V. Croquette, Biophys. J. 74, 2016 (1998).
8. J. Vlassakis, J. Williams, K. Hatch, C. Danilowicz, V. W. Coljee, M. Prentiss, J. Am. Chem. Soc. 130, 5004 (2008).
9. H. Clausen-Schaumann, M. Rief, C. Tolksdorf, H. E. Gaub, Biophys. J. 78, 1997 (2000).
10. N. Liu, T. Bu, Y. Song, W. Zhang, J. Li, J. Shen, H. Li, Langmuir 26, 9491 (2010).
11. I. Rouzina, V. A. Bloomfield, Biophys. J. 80, 882 (2001).
12. J. F. Leger, G. Romano, A. Sarkar, J. Robert, L. Bourdieu, D. Chatenay, J. F. Marko, Phys. Rev. Lett. 83, 1066 (1999).
13. A. Lebrun, and R. Lavery, Nucleic Acids Res. 24, 2260 (1996).
14. O. Krichevsky, Physics of Life Reviews 7, 350 (2010).
15. A. Garai, S. Mogurampelly, S. Bag, P. K. Maiti (Submitted for publication, 2017).
    
     

Dept. of Physics, IISc Bangalore

Insights into the fusion kinetics of human and HIV-1 multi-lipid asymmetric membrane models mediated by glycoprotein 41 (gp41) using coarse-grained molecular dynamics

The lethal Human Immunodeficiency Virus (HIV) exhibits cascade of complex structural transformations to induce fusion of target cell membrane and further replication within host cell which leads to Acquired Immuno-Deficiency Syndrome (AIDS). HIV attacks the T-cells of host via the spike like envelope proteins, a trimer of gp120 and gp41 subunits. HIV gp41 is a typical transmembrane (near C-terminal) protein with N-terminal having membrane fusogenic domain (fusion peptide). The fusion mechanism of gp41 was studied by different experimental methods. Despite those studies, exact molecular mechanisms of membrane fusion are still unknown with missing molecular details. In this context, coarse-grained molecular dynamics (MD) simulations was performed with MARTINI force field to study the kinetics of HIV gp41. Simulation of HIV gp41 properly placed between the equilibrated membranes of complex multimeric heterogeneous lipid bilayers mimicking human and HIV membranes for reasonable timescale definitely revealed the crucial residues (binding site) for activity of gp41. We have studied the kinetics of multi gp41 units (one to four) in the fusion mechanism of HIV vesicle and human membrane models. Interestingly, we observed gp41 works as an anchor between HIV and human membrane models. It induced higher curvature to the human bilayer and enhances stalk formation between the HIV and human membrane models. Thus, we strongly believe that the extensive systematic computational approach with gp41 and muti-lipid heterogenous membrane models in our study will certainly throw light on the mechanism of HIV and substantially support in designing potential inhibitor to eradicate AIDS.
 

Okinawa Institute of Science and Technology Graduate University (O.I.S.T.), Okinawa, Japan

Structural and functional studies of an essential histidine kinase from the Gram-positive pathogen S. aureus

Staphylococcus aureus causes a wide range of diseases and our interest is focused on the two-component system YycG-YycF that is essential for its growth. YycG/F are highly conserved in bacterial pathogens and are an attractive target for the development of novel antibiotics. We are conducting an integrated biochemical, biophysical and genetic approach to study the mechanism of YycG and F. In addition, we are screening a large natural compounds library using a luciferin-based assay to identify potential inhibitors or activators of YycG. Finally, we are attempting to solve the 3D structure of YycG and single domains by X-ray crystallography and NMR.
 

1. Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, 10032 USA
2. Department of Cell and Molecular Biology, Uppsala University, BMC, Box-596, Uppsala, Sweden
3. Integrated Program in Cellular, Molecular and Biomedical Studies, Columbia University, New York, 10032 USA
4. Department of Biological Sciences, Columbia University, NY 10027, USA
    

High-resolution termination complexes from Mycobacterium Smegmatis

Tuberculosis (TB) is an airborne disease and highly contagious. Although many drugs are available to treat TB, the fatality rate among TB patient is ~5-20% in different parts of the world. TB drugs employ diverse mechanisms to inhibit cell growth of Mycobacterium tuberculosis. Most of these drugs target the translation process. Mycobacterium smegmatis is a closely related non-pathogenic bacterium which is used as a safe model system in many laboratories to study pathogenesis and drug susceptibility of TB.

Here we discuss structures of the 70S ribosome in the translational termination phase from M. smegmatis obtained by cryo-electron microscopy (cryo-EM) combined with the single-particle reconstruction method. We have obtained near atomic-resolution cryo-EM maps of a termination complexes showing either release factor RF3 or both release factors RF2 and RF3 bound to the 70S ribosome. The cryo-EM maps have resolutions in the range of 3-3.5 Å and reveal many structural features distinct from other prokaryotic 70S ribosomes. These structural differences likely contribute toward the slow growth of Mycobacteria and their evolutionary ascendance. The structures obtained in this study provide a framework to design anti-TB drugs targeting the termination phase of translation.  
 

1. CSIR-Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad-007
2. CSIR-Institute of Genomics and Integrative Biology, New Delhi-007 
    

ECH & R-domains contribute to mycobacterial lipid diversity through unique variations

Polyketide synthases (PKS), non-ribosomal peptide synthetases (NRPS) along with fatty acid synthases (FAS) and the degradation machinery; FadAB along with enoyl-CoA hydratases/isomerases (ECH) help in maintaining the diversity of cell wall lipids of Mycobacterium tuberculosis (Mtb). Here, we report how unique functional interfaces have been evolved in common folds of these enzymes that have helped sustain the lipid diversity in Mtb.

The characterization of Mtb FadB suggests that it cannot assimilate cis-unsaturated fatty acids due to a constricted pocket volume (256 Å3 ) as compared to its orthologs (>317 Å3 ). As a consequence, it becomes dependent on isomerization enzymes, a canonical function of ECH. Structure-based sequence alignment of ECH helped in classifying the 21 paralogs of Mtb ECH into two sub-classes, namely mono-functional (isomerase-only) and bi-functional (isomerase and hydratase) enzymes. A single negatively charged residue in the active site acts as an isomerase (mono-functional) while two or more negatively charged residues function as hydratase and isomerase (bi-functional). We also demonstrate that a functional complex of ECH with FadAB enables recycling of cis-unsaturated fatty acids depending on their bioavailability during different stages of pathogenesis.

We also present structural studies of the R-domain from Mtb that is involved in the termination step of biosynthesis by NRPS. The first crystal structure of R-domain solved to a resolution of 2.3 Å showed that these enzymes belong to a common short-chain dehydrogenase family of enzymes with an additional C-terminal domain and carry out NADPH-dependent reduction. A new crystal form of apo R-domain solved to a resolution of 1.81 Å helped in the identification of conformational changes in the loop regions near the canonical NADPH-binding site, viz., Gating and Catalytic loop. These loops prevent NADPH binding until carrier protein domains are loaded with fully mature products. Such a unique regulation system allows R- domains to exercise precise coordination with other domains to avoid wasteful expenditure of energy.
 

Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai, India

Understanding the Effect of Post-translational Acetylation of Lys-40 on α-Tubulin Structure using Molecular Dynamics Simulation

Post-translational modifications (PTMs) play an essential role in the structure, functions and dynamics of microtubules. PTMs generally occur at the C-terminal end of αβ-tubulin. The acetylation of α-tubulin at the Lys-40 position is unique as it occurs on the luminal surface of tubulin. This acetylation induces significant conformational changes in the H1 containing loop, which could have functional role(s) in microtubule stability during growth and intracellular transport. Post-translational acetylation at Lys-40 replaces ɛ-NH³⁺ group with ɛ-NHCOCH₃ group. However, the role of acetylation is not well understood at the atomic level due to lack of structural information. Here, we investigate the structural effect of acetylation of Lys40 on α-tubulin protein using molecular dynamics simulation. Analysis of dynamics simulation results shows that acetylation induces significant conformational changes in α-tubulin structure. Hence, to get a detailed understanding of the effect of acetylation, we further analyzed averaged structures after molecular dynamics simulation at different intervals. The analysis of averaged structures reveals that un-acetylated α-tubulin prefers H1 helix, H1 loop, H2 helix, S2 and S3 sheet conformations, which are similar to the conformations in the α-tubulin crystal structures. Whereas, the averaged structures of acetylated α-tubulin prefer only H1 helix, H1 loop, S2 sheet and S3 sheet conformation and do not prefer the H2 helix conformation. This is because acetylation increases hydrophobicity in the H1 loop containing region, which may affect the formation of H2 helix conformation. This may consequently lead to having a flexible H1 loop conformation which is freely available in the lumen of the microtubule. We propose that this flexible H1 loop region could be helpful in providing elasticity to the individual tubulin heterodimers, which may lead to a consortium effect in the polymeric microtubule structure. This may help to increase the microtubule stability under mechanical stress. Hence, our computational study provides an insight into the effect of acetylation of Lys40 on α-tubulin structure, as well as polymeric microtubule structure.
 

Indian Institute of Technology Kanpur

Fluctuations and noise in a cellular system:large deviation principle

Actins are cytoskeletal proteins whose polymerization can generate significant force and propel the cell forward. We study force generation by a set of parallel actin filaments growing against a non­rigid barrier or membrane whose shape undergoes thermal fluctuations. We consider two different types of barrier: one which is acted upon by an external load that pushes the membrane in a direction opposite to that of polymerization, and another in which the elastic tension prevents formation of local distortion in the membrane shape. Our main interest is to study the dependence of the velocity of the membrane on the external load or elastic tension, since this dependence holds the key to understand the force generation mechanism of the filaments. We find the shape fluctuations of the barrier strongly affects force­velocity relationship, particularly the relative time­scale between the filament polymerization and membrane fluctuations is crucial. In the first type of barrier, the velocity decays monotonically with the external load, but the force­velocity curve can be convex or concave depending on the time­scale. In the second case, the velocity shows non­monotonic dependence on the membrane tension, which is a result of the interplay of elastic force and polymerization force. In this case also, the above relative time­scale plays an important role in determining the shape of the curve.
 

1. Department of Physics, Indian Institute of Technology Madras, Chennai, India
2. Department of Physics, Indian Institute of Technology Palakkad, Palakkad, India
3. Institute of Mathematical Sciences, Chennai, India
4. DAMTP, Centre for Mathematical Sciences, University of Cambridge, Cambridge, UK

Collective dynamics of active polymers

Polymers in solutions show a wealth of topological phenomena such as entanglement, reptation, and defect production, which can be fully explained from a balance of conser- vative, viscous and thermal forces and torques. In the presence of activity, the latter must be augmented by forces and torques mediated by active flow in the ambient fluid. Here we study active polymers in solution using Brownian microhydrodynamics [1, 2] where forces and torques mediated by active flow are explicitly taken into account by adding active hydrodynamic contributions to Langevin equations describing the dynamics of in- dividual polymers [3–5]. Spontaneous and persistent collective motion in volumetric confinement leads, at moderate polymer densities, to chain entanglement which cannot be released through active reptation. Spontaneous motion in surface confinement leads to athermal production and annihilation of hairpins and disclinations. Our results suggest new active systems that combine aspects of polymeric, colloidal, and liquid crystalline soft matter.
 

1. Laskar, Abhrajit, and R. Adhikari. ”Brownian microhydrodynamics of active filaments.” Soft matter 11.47 (2015): 9073-9085.
2. R. Singh and R. Adhikari. Universal hydrodynamic mechanisms for crystallization in active colloidal suspensions. Phys. Rev. Lett., 117:228002, 2016.
3. Jayaraman, Gayathri, et al. ”Autonomous motility of active filaments due to sponta-neous flow-symmetry breaking.” Physical review letters 109.15 (2012): 158302.
4. Laskar, Abhrajit, et al. ”Hydrodynamic instabilities provide a generic route to spon-taneous biomimetic oscillations in chemomechanically active filaments.” Scientific re-ports 3 (2013).
5. Manna, Raj Kumar, PB Sunil Kumar, and R. Adhikari. ”Colloidal transport by active filaments.” The Journal of Chemical Physics 146.2 (2017): 024901.
   
    

Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai, India

Computational study of interactions of Epothilone-A with cancer drug resistant tubulin isotypes

­Epothilones are metabolites, produced by the soil-dwelling myxobacterium Sorangium cellulosum. They are microtubule stabilizing antimitotic agents (MSAA) and work on same principle as taxanes, they perturb microtubule dynamics and prevent proliferation of cancer cells by preventing cell division. They have more efficacy and milder adverse effects than taxane family members, and they have good solubility in water. Epothilones work against MDR cell lines, because of all these qualities, this may replace taxanes in few years. It already has been observed that tubulin isotypes show altered expression in a range of cancers. Analysis of clinical specimens has shown that high expression in tubulin isotypes results in chemotherapy drug resistance and poor patient outcome in many cancers. Interactions of potent drug Epothilone-A (EpoA) with tubulin isotypes has not been very well understood. In this work computation study has been accomplished to understand the binding mode of drug resistant tubulin isotypes with potential drug EpoA using sequence analysis, homology modeling, molecular docking, molecular dynamic simulations, binding free energy calculation and hydrogen bonding analysis.

* - These authors contributed equally to the work.
 

SNBNCBS, Kolkata

Actin filaments growing against a fluctuating barrier with elastic properties

Actins are cytoskeletal proteins whose polymerization can generate significant force and propel the cell forward. We study force generation by a set of parallel actin filaments growing against a non­rigid barrier or membrane whose shape undergoes thermal fluctuations. We consider two different types of barrier: one which is acted upon by an external load that pushes the membrane in a direction opposite to that of polymerization, and another in which the elastic tension prevents formation of local distortion in the membrane shape. Our main interest is to study the dependence of the velocity of the membrane on the external load or elastic tension, since this dependence holds the key to understand the force generation mechanism of the filaments. We find the shape fluctuations of the barrier strongly affects force­velocity relationship, particularly the relative time­scale between the filament polymerization and membrane fluctuations is crucial. In the first type of barrier, the velocity decays monotonically with the external load, but the force­velocity curve can be convex or concave depending on the time­scale. In the second case, the velocity shows non­monotonic dependence on the membrane tension, which is a result of the interplay of elastic force and polymerization force. In this case also, the above relative time­scale plays an important role in determining the shape of the curve.
 

1. Department of Mathematics, Indian Institute of Technology Ropar, India
2. Department of Chemistry and Center for Theoretical Biological Physics, Rice University, USA

Collective Dynamics of Interacting Molecular Motors along Parallel Filaments

Molecular motors or motor proteins perform various vital functions within a cell to keep it alive. During intracellular transport, they generally move in a group and carry cargoes along microtubules by converting chemical energy typically derived from hydrolysis of ATP into mechanical energy. Experimental evidences suggest that they interact with the nearest molecules of their filament. Single molecular experiments have been well explored in the literature, however the collective behavior of motors need yet to be explored.

In the presentation, the collective dynamics of interacting molecular motors along linear filaments are analyzed by discussing an open two-lane symmetrically coupled interactive TASEP model that incorporates the intra-lane interaction in the thermodynamically consistent fashion. The effect of repulsive as well as attractive interactions on the system's dynamical properties has been studied using vertical cluster mean field analysis and Monte Carlo simulations. The presence of inter-lane transitions into the system lessened the correlations. The role of different coupling rates and interaction splittings is measured on the maximal particle current flow. The analysis indicate that for strong coupling and large splittings, the maximal flow of the motors arises at a weak attractive interaction strength, which is experimentally known to exist among kinesin motor protein.
 

Department of Physics, Indian Institute of Technology, Kanpur

"Cut and Run" - A Neurofilament’s Trip to the Tip

Axonal transport is a perfect example to study the trip to the tip mediated by molecular motors on microtubule tracks decorated with MAPS (microtubule associated proteins). Neurofilaments (NFs), a kind of neuronal cytoskeleton needed for a healthy axon, is an indispensable component of neuron. Frequent switching of motion between anterograde and retrograde direction, and on and off track movements make them a suitable candidate for studying slow axonal transport. In addition, they are also amenable to "end to end annealing" (fusion) and "severing " (fission). Fusion results in longer NFs which prefer being stationary whereas fission generate a motile population of NFs. Through our biophysical model, intended to understand the collective phenomena of axonal transport, we have tried to capture the consequences of this “Fusion-Fission” interaction on NF transport dynamics mediated by motors on microtubule tracks with MAPs.
 

Indian Institute of Science, Bangalore

Investigating Mitochondrial Dynamics in Fission Yeast using Chemical Kinetics

The mitochondrial network in eukaryotes generates almost the entire gamut of ATP required to drive myriad active cellular processes. Mitochondrial network morphology is a reflection of cellular state. For example, aberrations in the network morphology might signify diseased states. The morphology is ultimately regulated by the balance between mitochondrial fission and fusion dynamics, the latter two processes being at the forefront of a multiscale regulatory process. In interphase fission yeast cells, cytoplasmic microtubule dynamicity plays an important role in establishing the set point of the balance between mitochondrial fission and fusion dynamics, with mitochondria bound to microtubules possibly not undergoing any fission (Mehta et al., 2017). We test this conjecture using kinetic models. The first step, in this regard, is to understand mitochondrial dynamics in the absence of cytoplasmic microtubules. For this, we analyze time series images of mitochondria in fission yeast cells having short microtubules due to treatment with the microtubule de-polymerizing drug MBC. We employ a deterministic kinetic model in which mitochondria are modeled as “polymers”, with the “monomer” being the smallest possible mitochondrial unit containing only a single nucleoid. The spectrum of possible fission and fusion events is constrained by the principle of conservation of mass and also by invoking a closure of sizes i.e. by considering a certain maximum mitochondrial size, as obtained from experimental data. Further, fission and fusion events are assumed to obey the law of mass action. We find that while several fission kinetic rate constants are significant, only a few rate constants for fusion assume substantial values. Also, most fusion rate constants are about two orders of magnitude lower than most fission rate constants. In the future, we intend to extend the model to the case where cytoplasmic microtubules exist with the aim of gaining insights into mitochondrial dynamics in interphase fission yeast cells, as mentioned previously.
 

References:

1. K. Mehta, M. K. Chug, S. Jhunjhunwala and V. Ananthanarayanan, Microtubule dynamics regulates mitochondrial fission, BioRxiv, 2017.

CSIR- Centre for cellular and Molecular biology, Hyderabad

Animalia specific paralog of DTD with relaxed substrate chiral specificity

D-aminoacyl-tRNA deacylase (DTD), a trans editing factor present in bacteria and eukaryotes, deacylate D-amino acids mischarged on tRNAs and maintain homochirality in protein synthesis. Presence of invariant Gly-cisPro is the mechanistic basis of strict rejection of L-amino acids. Thus, DTD acts on achiral glycine, an activity which is important to eliminate mischarged Gly-tRNAAla species. Here, we reports DTD variant named ATD (Animalia-specific tRNA deacylase), that harbours a Gly-transPro motif. The cis-to- trans switch of Gly-Pro motif creating an “additional” space in ATD’s active site pocket as compared to DTD’s Consequently, ATD achieves a “gain of function” through L-chiral selectivity as its active site pocket can accommodate smaller L-amino acids, in addition to glycine and D-amino acids. ATD is therefore able to rectify a unique tRNA miss selection by preferentially clearing L-alanine mischarged on tRNAThr (G4•U69) by eukaryotic AlaRS. The biochemical proofreading activity of ATD is conserved across diverse classes of phylum Chordata. Animalia genomes enriched in tRNAThr (G4•U69) are in strict association with the presence of ATD, underlining the necessity for a dedicated factor to proofread tRNA misaminoacylation. The study highlights the emergence of ATD during genome expansion as a key event associated with the evolution of Animalia.