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    Journal of Biomolecular Structure and Dynamics

    ISSN: 0739-1102 (Print) 1538-0254 (Online) Journal homepage:

    Comparative molecular dynamics simulationstudies for determining factors contributing to thethermostability of chemotaxis protein CheY

    Manish Paul, Mousumi Hazra, Arghya Barman & Saugata Hazra

    To cite this article: Manish Paul, Mousumi Hazra, Arghya Barman & Saugata Hazra (2014)Comparative molecular dynamics simulation studies for determining factors contributingto the thermostability of chemotaxis protein CheY, Journal of Biomolecular Structure andDynamics, 32:6, 928-949, DOI: 10.1080/07391102.2013.799438

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  • Comparative molecular dynamics simulation studies for determining factors contributing tothe thermostability of chemotaxis protein CheY

    Manish Paula, Mousumi Hazrab, Arghya Barmanc and Saugata Hazrad*aDepartment of Zoology, Naihati Rishi Bankim Chandra College, West Bengal State University, Naihati, North 24 Parganas,West Bengal, India; bDepartment of Microbiology, University of Kalyani, Kalyani, West Bengal, India; cDepartment of Chemistry,University of Miami, 1301 Memorial Drive, Coral Gables, FL 33146, USA; dDepartment of Biochemistry, Albert EinsteinCollege of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA

    Communicated by Ramaswamy H. Sarma

    (Received 6 November 2012; nal version received 22 April 2013)

    Comparative molecular dynamics simulations of chemotaxis protein CheY from thermophilic origin Thermotogamaritima and its mesophilic counterpart Salmonella enterica have been performed for 10 ns each at 300 and 350K, and20 ns each at 400 and 450K. The trajectories were analyzed in terms of different factors like root-mean-square deviation,root-mean-square uctuation, radius of gyration, solvent accessible surface area, H-bonds, salt bridge content, and pro-teinsolvent interactions which indicate distinct differences between the two of them. The two proteins also follow dis-similar unfolding pathways. The overall exibility calculated by the trace of the diagonalized covariance matrix displayssimilar exibility of both the proteins near their optimum growth temperatures. However, at higher temperatures meso-philic protein shows increased overall exibility than its thermophilic counterpart. Principal component analysis alsoindicates that the essential subspaces explored by the simulations of two proteins at different temperatures are nonover-lapping and they show signicantly different directions of motion. However, there are signicant overlaps within thetrajectories and similar direction of motions are observed for both proteins at 300K. Overall, the mesophilic proteinleads to increased conformational sampling of the phase space than its thermophilic counterpart. This is the rst everstudy of thermostability of CheY protein homologs by using protein dynamism as a main impact. Our study might beused as a model for studying the molecular basis of thermostability of two homologous proteins from two organismsliving at different temperatures with less visible differences.

    Keywords: CheY; signal transduction; MD simulation; GROMACS; thermostability; salt bridge; PCA; proteinengineering


    Chemotaxis is a biochemical signaling process in whichbacterial agellar rotation is controlled by several envi-ronmental cues such as pH, chemicals, temperature, etc.Motile bacteria are able to change the direction of theirmigration through solution in response to the concentra-tion gradients of attractants and repellents (Spohn &Scarlato, 2001). Chemotactic behavior requires detectionof the chemical signal, signal transduction, and subse-quent motor response. Chemotaxis helps bacteria tosearch for food and also escape hostile environments(Eisenbach & Caplan, 1998). There are a number ofproteins involved in the bacterial chemotactic signalcascade. Through several studies using molecular biol-ogy, genetics, biochemical, and biophysical methods it

    has been established that Che proteins play importantroles in the whole process. Che proteins are involved insignal transduction and form a two component system.Two component signal transduction pathways are denedby the conservation of a histidine kinase autophosphory-lating at a conserved histidine residue and a response reg-ulator with a conserved aspartate phosphorylation site(Allweiss, Dostal, Carey, Edwards, & Freter, 1977). Themembrane receptor proteins initially receive the environ-mental signal and transmit it to the cell interior. Afterreceiving the signal, the cytoplasmic proteins get stimu-lated and amplify the signal, (1) by working as a DNAbinding protein to enhance expression of particular genes,(2) by binding to transcription inducers to perform thesimilar job in an indirect way, and (3) by binding to

    *Corresponding author. Email: saugata.hazra@einstein.yu.eduManish Paul and Mousumi Hazra contributed equally to this work

    Journal of Biomolecular Structure and Dynamics, 2014Vol. 32, No. 6, 928949,

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  • downstream proteins to change their conformationscritical to some biological processes (Hoch, 2000).

    The two component system formed by Che proteinsis relatively complicated than the models, involving morethan two proteins. During chemotaxis, the sensor CheAreceives signals from transmembrane chemoreceptors withthe help of CheW (Bourret, Davagnino, & Simon, 1993;Gegner, Graham, Roth, & Dahlquist, 1992; Hazelbauer,Berg, & Matsumura, 1993) and then transfers thesignal to the response regulator, CheY, by the process ofphosphorylation. Phosphorylated CheY (CheYP) isdephosphorylated by its autophosphatase activity, a reac-tion enhanced by CheZ (Kuo & Koshland, 1989). A seriesof conformational changes and proteinprotein interac-tions occur in between those phosphotransfer eventsinvolving several other proteins (e.g. FliM, FliN, andFliG), which help switch motor to reverse the direction ofagellar rotation from counterclockwise to clockwise.

    The main focus of our current study is the proteinCheY. The length of this protein ranges from 120 to130 residues (Stock, Koshland, & Stock, 1985). CheY iscomposed of central patches of sheets surrounded by anumber of helices. The number of helix and strands varies in different bacterial species. CheY proteincontains different exible loop regions in its structure.Each CheY protein contains one receiver domain andone response regulatory domain, respectively. It plays akey role in the control of the bacterial movements inresponse to environmental chemotactic stimuli (Volz &Matsumura, 1991). The phosphoryl group is received byCheY from a conserved histidine residue of histidinekinase CheA (Djordjevic & Stock, 1998). This leads to aconformational change in the regulatory domain andhelps it to bind with the protein FliM. Binding to FliMleads to a complex series of interactions involving motorproteins and in this way, CheY plays a major role intransmitting chemical stimuli to the bacterial agella viaa signal transduction cascade (Matsumura, Rydel, Lina-meir, & Vacante, 1984; McEvoy, Hausrath, Randolph,Remington, & Dahlquist, 1998).

    The primary target of our study is to understand thefactors contributing towards the thermostability of CheY.We have selected two representatives of the CheY pro-tein, one is thermophilic and the other is mesophilic(Dasgupta & Dattagupta, 2008; Knaggs, Salsbury,Edgell, & Fetrow, 2007; Liang et al., 2009). Thermosta-bility is a property of a protein which could be utilizedto measure the potential of the protein to retain its sec-ondary structure content at its maximum tolerabletemperature. A thermophilic protein can retain its sec-ondary structure more compactly at higher temperaturescompared to its mesophilic counterpart. Proteins that arestable at high temperatures have attracted much interestbecause they have potential industrial applications(Bruins, Janssen, & Boom, 2001; Sagi, Khan, &

    Eisenbach, 2003). Thus, it is important to understandhow these thermophilic proteins remain stable at elevatedtemperatures. Such an understanding may help to eluci-date the critical principles of protein engineering andhelp in constructing the design of thermostable proteinsfor industrial applications. Thermostability is dependenton several factors like hydrogen bonding, hydrophobicpacking, helix dipole stabilization, etc. Protein thermosta-bility can also be increased by improving electrostaticinteraction and removing residues that are sensitive tooxidation or deamination. With a proper understandingof those factors, it is possible to perform knowledge-based protein engineering to generate proteins withhigher thermostability for industrial applications (Turner,Mamo, & Karlsson, 2007).

    Beside the applicative features, we also haveanother interesting motive for our study. It has beenreported that Thermotoga maritima CheY is muchmore thermostable than mesophilic proteinsEscherichia coli or Salmonella enterica CheY. Thedifference between the melting temperature (Tm) ofT. maritima CheY (TmCh