Abstract

Molecular level understanding of physiochemical interactions responsible for the structure and dynamics of functional RNA is a major challenge to the scientific community. RNA structural diversity is mainly due to the presence of non-canonical base-pairs, phosphate mediated interactions and interactions between two or more nucleotides involving water molecules and ions that stabilize folded RNA structures. BPFIND and INCAR are two complementary tools developed to analyse RNA 3D structure in terms of hydrogen-bonded secondary structural elements interacting through primarily non-canonical tertiary base pairing interactions. Identification of recurring combinations of such elements or motifs, important in terms of architecture enhances our understanding of the RNA tertiary structures. First tool, Base Pair Finder (BPFIND), detects and analyzes all canonical and non-canonical basepairs and base triples involving at least two direct hydrogen bonds formed between polar atoms of the bases or sugar O2’ only. Apart from detection, the program also gives a quantitative estimate of the conformational deformation of detected base pairs in comparison to an ideal planar base pair, which helps to understand their specific role towards structural and functional diversity. This approach is constrained by its dependence on identification of strongly hydrogen bonded base pairs only. The second tool, namely INCAR (Inter Nucleotide Contact Annotator in RNA), compensates this limitation by following a bottom-up approach towards identifying and characterizing structural features. At the lower end it detects, all inter-nucleotide hydrogen bonding interactions (both strong and weak), water and ion mediated interactions. At the other end it can capture the recurrent structural motifs made of several base-pairs and higher order interactions. This python based automated tool efficiently manages space and time complexity to capture, categorize and suitably annotate all significant non-covalent interactions, within a non-redundant dataset of RNA crystal structures. Along with this we have developed a visualization tool, VASTERN, which helps users to view the backbone structure as well as base pairs to analyze the structure interactively.

Abstract

Lipases are used in several industrial processes and thermostable lipases are very important in the detergent industry and in organic synthesis. B. subtilis lipase (Lip A) is a monomeric protein without any bound ligand. Lip A preferentially hydrolyses C8 fatty acid esters and it is a "lidless" lipase that does not show interfacial activation [1]. The mechanisms of thermostability of lipases are varied and depend on type of lipase; nevertheless, some common features that contribute to thermostability include additional non-covalent interactions, increase in packing density of the hydrophobic core, stabilization of alpha-helix and shortening of loops between secondary structural elements [3]. We have studied wild type Lip A along with its five thermostable mutants derived from wild type by introducing 2, 3, 4, 6 and 9 point mutations respectively through directed evolution. Experimentally, it was observed that the thermostabililty of the mutant lipases increased with the number of mutations introduced, without affecting their catalytic activity at mesophilic temperatures and the most thermostable mutant with nine mutations shows remarkable enhancement in melting temperature and temperature optima of activity compared to wild type [2]. Long time molecular dynamics (MD) simulations performed at both low and high temperatures, have provided us molecular-level insights into how point mutations can affect protein structure and dynamics. From our simulations we observed a reduced rmsd in several key regions, an increased overall flexibility with decrease in flexibility in stabilizing regions, an increase in the number of hydrogen bonds, decrease in SASA of hydrophobic regions for the most thermostable mutant lipase. Through PCA analysis we also show that the most thermostable lipase show a C-terminal stabilization through reduced fluctuation, which was not observed for the wild type and other less thermostable mutant lipases. However, thermostable lipases must retain their threedimensional structures and required flexibility at high temperatures in order to retain activity. The encouraging outcome suggests that the high-temperature MD simulations and the structure–thermostability relationships may be considered as a valuable tool for understanding the thermostability of lipases.

Abstract

Independent folding units which have the capability of carrying out biological functions have been classified as “protein domains”. These minimal structural units lead not only to considerable sequence changes of protein domains of similar folds and functions, but also gives rise to remarkable length variations under evolutionary pressure. Rapid and heuristic sequence search algorithms are generally sensitive and effective in recognizing protein domains that are distantly related within large sequence databases, but are not well-suited to identify remote homologues of varying lengths. An even more challenging aspect is introduced to distinguish reliable hits from a vast number of putative false positives that could have suboptimal sequence similarities. Here, the authors present a data-mining approach that provides stage-specific filters in sequence searches to reliably accumulate remote homologues, which encourages sampling of length variations albeit with a low false positive rate. Realization of such remote homologues with vivid length variations could contribute to better understanding of functional variety within protein domain superfamilies.

Abstract

In some bacteria, the intracellular concentration of several amino acids is controlled by riboswitches. One class of such riboswitches is Thermotoga maritima lysine-responsive riboswitch. It regulates the downstream expression of aspartate-semialdehyde dehydrogenase, which is involved in synthesis of precursor for methionine, threonine, lysine and diaminopimelate, depending on the presence or absence of small metabolite lysine. The GC-rich lysine riboswitch is among the longest and most complexly folded metabolite-sensing aptamers. Crystal structures of lysine sensing domain of riboswitch in presence and absence of lysine, feature a complex architecture, involving a modified four-way junction (J1-2, J2-3, J4-5 and J5-1) organized between coaxial bundles of three helices (P2, P3, P4) above and two helices (P1, P5) below. An intriguing structure to visualize is that adopted by P2, a long helix, which experiences two turns to have kissing-loop interactions between L2 and L3. The binding pocket of metabolite lysine is compact, bounded by the modified four-way junction, where lysine is specifically recognized through hydrogen bonds to its charged ends. In order to investigate the dynamics and conformational changes associated with the ligand binding, at the molecular level, we have carried out all-atom explicit solvent molecular dynamics simulations of aptamer domain of Thermotoga maritima asd lysine riboswitch in lysine-bound (HOLO) and lysine-free (APO) states using two widely accepted nucleic acids forcefields: CHARMM 27 and AMBER ff99parmbsc0. Detailed analysis, including principal component analysis and conformational clustering of the trajectories, reveal interesting insights into changes in conformational dynamics accompanying ligand binding. While confirming the existence of a preformed and ‘binding ready’ APO form and ligand binding induced stabilization of the expression platform linking P1 helix, our studies indicate importance of two tertiary interactions: loop-loop L2-L3 and helix-loop P2-L4 interactions in the functioning of lysine riboswitch, relating their role in stabilizing the aptamer domain. Our simulations also provide molecular insights into ligand binding mechanism by which lysine interacts with binding pocket to form a stable HOLO lysine riboswitch aptamer.

Abstract

Riboswitches are ligand dependent gene regulatory elements, found in mRNA transcripts. They are composed of two distinct domains: a ‘ligand-binding’ or aptamer domain and an expression platform. Communication between the two domains take place through ligand binding induced conformational changes which control downstream gene expression by turning it on or off at either the transcription and/or at the translation level. Till date, more than 20 different classes of riboswitches have been discovered and understanding their functional mechanisms constitutes an important area of research. Apart from crystal structure studies, experimental studies involving various biophysical and biochemical techniques are currently being carried out to understand the mechanism of discriminative ligand binding and the kinetics and thermodynamics of conformational changes affecting transcription and translation. These studies are also being complemented by computational studies involving the structure, dynamics and energetics of these riboswitches in their ligand-bound and ligand-free states. In continuation with our earlier QM and MD studies on the add Adenine riboswitch,1,2 we have carried out detailed studies on three different classes of riboswitches, PreQ1, Purine and Lysine riboswitches, which vary in their sizes as well as in their switching mechanisms. Providing an overview of the results of our investigations, the talk will highlight our insights gained on purine riboswitches, regarding the mechanisms of ligand binding (conformation selection versus induced fit) and its communication with the expression platform.

Abstract

BACKGROUND: Thermostable lipases are widely used in the detergent industry and in organic synthesis. B. subtilis lipase (Lip A) is a monomeric protein, which preferentially hydrolyses C8 fatty acid esters. Though the mechanism of thermostability depends on type of lipase, some common factors include additional non-covalent interactions, increase in packing density of the hydrophobic core, stabilization of alpha-helices and shortening of loops between secondary structural elements. RESULTS: We have studied wild type Lip A (WT) and six of its thermostable mutants containing 2, 3, 4, 6, 9 and 12 point mutations respectively, derived from the wild type protein by directed evolution. Experimental observations showed that the most thermostable variant, with twelve mutations, shows remarkable enhancement in melting temperature and temperature optima of activity compared to WT, in vitro. Our MD simulations show decrease in SASA for core regions and slow unfolding rate at several key secondary structural regions due to presence of persistent salt bridges, hydrogen bonds and native contacts for the most thermostable mutant lipase. The principal component (PC) and Free energy landscape (FEL) analyses at 450K suggest that the most thermostable mutant showed least fluctuation and stayed within a single large minimum. The other less thermostable mutants and WT showed higher fluctuation and exhibited two or more minima along the first two PCs. Analysis of representative structures corresponding to each sub-states showed that the most thermostable mutant sampled conformations close to the native state most of the time during the simulation. The other less thermostable mutants and WT lipase sampled non-native conformations more frequently than native conformations. CONCLUSIONS: Our results demonstrate that, the most thermostable lipase maintains its stability and activity at high temperatures by restricting conformational fluctuation through the stabilization of key salt bridges and main-chain H-bonds, which further helped them to maintain the stability of the core regions apart from stabilizing the overall 3D fold.

Abstract

t has been proposed that combining chemical and thermal denaturation along with mechanical unfolding enables better understanding of the thermodynamics of protein folding, and associated pathways. We have used molecular dynamics (MD) simulations, and umbrella sampling technique to study the effect of thermal and chemical perturbations on the unfolding of Trp-cage miniprotein (TC5b). Free energy profiles for the unfolding of the minicage protein were obtained at two different temperatures, and in presence and absence of urea. The distance between the penultimate terminal residues was used as the reaction coordinate for calculating the free energies. The unfolding involves opening of the Trp-cage, and separation of the secondary structure elements followed by the unfolding of the α-helix. Several energetic and geometric analyses including inter- and intramolecular interactions, and radial distribution functions and their relevance to the folding pathways will be discussed. Similar study on the titin I27 protein will also be presented.

Abstract

SAM-III riboswitch present in the 5’-UTR of mRNA that translates to SAM synthetase in certain bacteria is involved in regulating the biosynthesis of S-adenosylmethionine (SAM) by acting as an on-off switch for the translation process. We have used molecular dynamics (MD) and steered MD simulations to understand the ligand recognition and the switching mechanisms. Computations on model systems indicated that the riboswitch does not recognize SAM over a structurally related analog, S-adenosylhomocysteine (SAH). Nonetheless, the RNA uses nonspecific interactions to stabilize SAM in its binding pocket compared to SAH. Calculations were done in the absence of the ligand to examine the conformational changes responsible for the transition between on and off states. While the on- state is capable of sampling a large conformational space, formation of duplexlike structure comprising the adenine part of the ligand and sequentially distant nucleotides seems to be crucial for the on- to off- state transition.

Abstract

Vpu is an 81-residue protein encoded by human immunodeficiency virus type I (HIV-1) that facilitates viral release from host cells. The protein has a cytoplasmic domain and a helical transmembrane (TM) domain, of which the latter oligomerizes to form cation-specific ion channels. The number of TM domains that constitute the channel is still unclear, with experimental studies indicating the existence of a variety of oligomeric states. In this study, we have examined the possibility of Vpu to exist in tetra-, penta-, and hexameric states using comprehensive molecular dynamics (MD) simulations. By modeling the TM domain as an ideal α-helix, we carried out replica-exchange MD simulations in an implicit membrane environment for obtaining suitable starting structures, which were then subjected to extensive MD simulations in a fully hydrated lipid bilayer environment. The results show that the pentameric form is the most stable oligomeric state (the tetramer and hexamer models lose their initial channel-like structure), with helices in the pentamer being held together by strong van der Waals interactions. Hydrogen bonds between lipid headgroups and basic/hydrophilic residues on the protein are stronger in the pentamer than in the tetramer or the hexamer, indicating that these interactions might play a role in adhering the pentamer to the membrane. Free energy calculations using umbrella sampling technique have been performed to examine the potassium ion permeation through the pentameric pore. The results show a high free energy barrier corresponding to ion transport indicating weak ion channel activity in agreement with previous experimental biophysical studies.

Abstract

Chemical modifications on the backbone of the oligonucleotides have been shown to improve several factors such as increased cellular uptake, altered duplex conformation and stability. Modified oligonucleotides have also been shown to be potential antisense agents. Some of the common modifications include 2'-O-methyl, 2'-O,4'-C-methylene--D-ribofuranosyl (LNA) and phosphoroamidate. In general, the modifications at ribose sugar of the oligonucleotide attribute special features to the duplexes than other modifications. Molecular dynamics (MD) simulations are valuable tools to study their dynamics, conformational preferences, stabilities etc. Here, we have employed different simulation techniques such as classical MD, replica exchange MD to understand the impact of modification on the duplex properties. The simulations revealed several important aspects related to duplex stability and relation with their base content. Our results also suggest that the differential changes observed in nucleic acids depends on the position, nature of the modified site. This study also provides a molecular level picture for the origin of structural changes arise due to different types of modifications which is very important in designing the most fruitful antisense oligonucleotides.