Molecular Dynamics Simulation Analysis Of His226 Mutation On The Dynamics Of The Atpase Domain Of Dnak

Tez (Yüksek Lisans) -- İstanbul Teknik Üniversitesi, Fen Bilimleri Enstitüsü, 2017Thesis (M.Sc.) -- İstanbul Technical University, Institute of Science and Technology, 2017Proteinler, canlılar için yaşamsal açıdan önemli makromoleküllerdir. Sentezlendikten sonra fonksiyonel özelliklerini kazanmaları içinse doğru şekilde katlanmaları şarttır. Peptid zinciri sentezlenirken, üç boyutlu yapısını kazanması sürecinde görülen ilk etkileşimler hidrofobik etkileşimlerdir. Sonrasında ise iyonik etkileşimler,van der Waals etkileşimleri, dipol-dipol etkileşimleri, hidrojen bağları ile birlikte peptit zincirleri fonksiyonel hale geldiği üç boyutlu yapısına ulaşır. Bazı küçük proteinler bu şekilde tek başına katlanabilirken, pek çok protein katlanabilmek için şaperonlara ihtiyaç duyar. Proteinlerin düzgün katlanamaması ise, Alzheimer, Parkinson gibi çeşitli nörodejeneratif hastalıklara sebebiyet verebilir. Şaperonlar ilk kez 1978 yılında Ron Laskey tarafından bulundu. 1987 yılında R. John Ellis tarafından yapılan çalışmalarla birlikte, bu konudaki araştırmalar hız kazandı. Eksikliklerinde nörodejeneratif hastalıkların oluştuğunun keşfedilmesi ile beraber, şaperonlar ile ilgili çalışmalar oldukça sık gündeme gelmeye başlamıştır. Şaperonlar, normal şartlarda hücrede normal seviyelerde sentezlenir. Stres koşulları ise bu durumu değiştirebilir. Şaperonlar hücrenin aşırı ısı ve pH değişiminden olumsuz etkilenmemesini sağlamanın yanında oksidatif stres ve kimyasal stres gibi durumlara karşı da hücreyi korur. Bu gibi streslere karşı hücrenin bir savunma stratejisi olarak, şaperonların ekspresyonları artar ve tehdit altında olan hücre içi yaşamsal öneme sahip enzimlerin ve diğer proteinlerin yapısının bozularak fonksiyonel olarak zarar görmeleri engellenir. Hsp70, şaperon ailesinin en bilinen ve en yaygın üyesidir. Şaperonlar evrimsel süreçte korunmuş moleküler şaperonlar arasında yer almaktadır. Bakteri, arkea ve ökaryotlarda olmak üzere neredeyse tüm hücrelerde bulunurlar ve hayati açıdan önemli rollere sahiptirler. Örneğin, yeni eksprese edilmiş peptit zincirlerinin katlanması, agregat oluşumunun önlenmesi ya da proteinlerin belirli organellere translokasyonu Hsp70 tarafından gerçekleştirilir. Hsp70, katlanacak proteinin hidrofobik bölgelerine bağlanarak bu bölgelerin birbirleriyle doğru olmayan şekilde etkileşmesini önler, bu sayede proteinin yanlış katlanmasını engeller. Bu sebepten ötürü, Hsp70'in proteinlerin katlanmasında katalizör görevi görmediği, katlanması gereken proteinler için uygun ortam oluşturduğu düşünülmektedir. Çalışmamızda kullanılan E.coli Hsp70 homoloğu olan DnaK iki domenden oluşmaktadır. Bunlardan biri amino (N) ucunda bulunan nükleotit bağlayan ve ATPaz aktivitesi olan 44 kDa'lık Nükleotit Bağlanan Domen (NBD), diğeri ise karboksil (C) ucunda bulunan ve substrat bağlayan 25 kDa'lık Subsrat Bağlanan Domen (SBD)'dir. Bu iki domen ise oldukça korunmuş, domenler arasında bulunan xxiv hidrofobik bir bağlaç ile bağlanır. NBD ve SBD arasında allosterik bir etkileşim mevcuttur. Bu iki domen arasındaki iletişim ise domenler arasındaki bağlaç tarafından sağlanmaktadır. Her iki domende de gerçekleşen konformasyonel değişiklikler sonucunda, ATP bağlanması ve hidrolizi substrat affinitesini düzenlerken, ATP hidrolizi ise substrat bağlanması ile tetiklenmektedir. Yapılan kristalografi ve NMR çalışmaları sonucunda elde edilen yapılarda, ADP bağlı halde, bu iki domen ve bağlaç birbirinden ayrı şekilde gözlemlenmektedir. Bu durumda SBD'nin substrata olan afinitesi yüksektir ve dolayısıyla SBD, katlanacak protein üzerine kapanmış durumdadır. NBD'ye ATP bağlandığında ise, SBD açık bir konformasyonda ve bağlacı NBD'nin içlerine alacak şekilde NBD ile etkileşmektedir. Bu konformasyonda SBD'nin substrata afinitesi düşüktür ve katlanmış proteinin salınması ATP bağlı halde gerçekleştirilir. Sonrasında, SBD'ye bağlanan katlanması gerekli yeni bir protein, NBD'ye bağlı halde bululanan ATP'nin hidrolizini tetikler. ATP'nin ADP'ye hidrolizi sırasında SBD katlanması gereken proteinin üzerine kapanır, NBD ve SBD birbirinden tekrar ayrılır ve mekanizma bu döngü ile devam eder. Bu döngüye koşaperonlar eşlik eder. Hsp40 ailesi ve nükleotit değişim ailesi bu döngüde görevli koşaperonlar olarak bilinir. Hsp40 ailesi (DnaK için DnaJ), ATP hidrolizini hızlandırmakla görevliyken, nükleotit değişim faktörleri (DnaK için GrpE) ise hidroliz ile oluşan nükleotiti değiştirmek ve Hsp70'yi yeni döngüye hazırlama görevindedir. Mayer ve Bukau'nun 2015 yılında yaptığı son çalışmalar, ATP ile indüklenen substrat salınımının, substratın bağlanmasıyla uyarılan ATP hidrolizine göre şaperonun aktivitesinde daha önemli bir rol oynadığını ortaya koymaktadır. 2007 yılında, Swain grubu tarafından yapılan çalışmalarla, domenler arası bağlacın hidrofobik 389VLLL 392 sekansının NBD ve SBD arasındaki allosterik ilişkiden sorumlu olduğu bulunmuştur. Yine bu çalışmaya göre, bağlaç varlığında DnaK (1- 392), substrat tarafından uyarılmış yabanıl tip DnaK gibi davranmaktadır. Yabanıl tip, substrat ile uyarılmamış DnaK'nin aksine, DnaK (1-392)'nin pH bağımlı ve daha yüksek ATPase aktivitesi vardır. Diğer bir taraftan ise, bağlaç yokluğunda DnaK (1- 388), substrat tarafından uyarılmamış yabanıl tip DnaK'yi taklit etmektedir. Moleküler dinamik simulasyonları, 1950 yıllarının ikinci yarısına doğru ortaya çıkmıştır. 1960'larda daha da gelişmiş ve ilk kez 1977 yılında bovin pankreatik tripsin inibitörü ile birlikte proteinler üzerinde kullanılmaya başlanmıştır. In vivo ve in vitro olarak gözlemlemekte güç olabilecek bazı konularda fikir vermek için kullanılan önemli yollardan biri haline gelmiştir. Biyokimya ve biyofizikte oldukça geniş bir uygulama alanı bulmuştur. Bu çalışma DnaK (1-392) ve (1-388) kesik yapılarında, ATP bağlı durumda konformasyonel ve dinamiksel farklılıkları göstermeyi amaçlamanın yanısıra 226. konumdaki histidinin alanine mutasyonu halinde genel konformasyon ise de nasıl bir değişim olduğunu moleküler dinamik analizleriyle göstermeyi amaçlamaktadır. Kesik yapılar 4JN4 kodlu PDB (protein data bank) dosyasından modifiye edilip, NAMD/CHARMM-GUİ kullanılarak 200 ns boyunca yürütülmüştür. Bu süreçte ortalama karekökten sapma (RMSD) ve ortalama karekök değişimi (RMSF) gibi temel moleküler dinamik analizlerinin yanısıra, Başlıca Komponent Analizi (PCA), hedef amino asitler için zamana bağımlı uzaklık ölçümleri ve proteinin ne kadar kompakt olduğunu ölçmek amacıyla dönüş çapı (Rg) analizi yapılmıştır. Yapılan analizler sonucu bağlacın 389VLLL392 kısmının bulunmadığı DnaK (1-388) kesik yapısının, bağlacın bulunduğu DnaK (1-392) yapısına gore belirli bölgelerde xxv daha kapalı bir konformasyonda olduğu gözlemlenmiştir. Ayrıca bu iki yapı arasında farklı dinamikler olduğu da görülmektedir. Bir diğer önemli sonuç ise bağlaç varlığında His226'nın bu dinamikte önemli bir rolü olduğu yönündedir. 226 numaralı Histidinin alanine mutasyonu ile birlikte, konformasyonun daha da açıldığı, bazı amino asitlerin de bulunduğu konumdan oldukça saptığı görülmüştür. Bunlardan önemli olarak gördüğümüz katalitik bölgede bulunan ve ATP hidrolizinden sonra fosfatı kabul eden amino asit olduğu düşünülen 199. konumdaki Treonin yakından incelendiğinde simulasyon sürecinin sonlarına doğru ATP'den aniden ve önemli ölçüde uzaklaştığı görülmüştür. Bu durumun doğurabileceği sonuçlar, araştırma grubumuza ait diğer deneysel verileri desteklemekle birlikte Histidinin Hsp70 genel dinamiği için ne kadar önemli olduğunu da göstermektedir. Uzun vadede, yapılan bu moleküler dinamik simulasyon çalışmalarının Hsp70 çalışma mekanizmasını detaylı bir şekilde ortaya koyarak Hsp70 kaynaklı nörodejenaratif hastalıkların tedavisine ışık tutabileceği düşünülmektedir.Hsp70s are evolutionarily highly conserved ATP-dependent molecular chaperones which are ubiquitously expressed in the cell. They are found in three domains of life and have essential roles in cells, such as aiding proper folding of nascent polypeptides, prevention of polypeptide chains from misfolding and translocation of proteins across membranes. The diverse cellular functions of Hsp70s are based on the recognition of hydrophobic sequences of client protein. Hsp70s corporate with other co-chaperones like nucleotide exchange factors and J-domain proteins. In our study, we used DnaK which is an Escherichia coli homolog of Hsp70. DnaK have an N-terminal nucleotide-binding ATPase domain (NBD) and a C-terminal substrate-binding domain (SBD). These two domains are connected by a highly conserved hydrophobic interdomain linker. There is an allosteric communication between the domains via the hydrophobic linker. Substrate affinity is regulated by ATP binding and hydrolysis, which results in conformational changes in both domains, while ATP hydrolysis is stimulated by substrate binding. In 2007, Swain et al. revealed that the conserved hydrophobic 389VLLL392 sequence of the interdomain linker is responsible for the allosteric communication between NBD and SBD. According to this study, DnaK (1-392) behaves like the substrate-stimulated DnaK that is pH-dependent, and shows higher activity than that of the unstimulated fulllength protein. In contrast, DnaK (1-388) mimics the activity of the substrate-free form of the full-length DnaK. Which amino acids in the catalytic site are responsible in allosteric communication and pH-dependent ATPase activity in the presence of linker are not enlightened so far, however there are several research trying to find out the key residues and reveal the detailed mechanism of DnaK. In this study, the effect of linker 389VLLL392 on the ATP-bound protein conformation and H226A mutation on ATP-bound DnaK's (1-392) construct were investigated by using molecular dynamics simulations. MD simulation trajectories were analyzed by root mean square deviation (RMSD) and, root mean square fluctuation (RMSF) analysis, also by distance measurement in a time-dependent manner and, principle component analysis. From these analysis it was found that distance between the helices which contain His226 and its neighbour helix is closer to each other in DnaK (1-388) constructs. Moreover, this study reveals that His226 contributes the stabilization of residue Thr199 which is suspicious as a phosphate acceptor after the hydrolysis of ATP.Yüksek LisansM.Sc

Computational assessment of the effect of allosteric mutations on the dynamics of PDZ domains

PDZ domain-containing proteins are involved in intercellular interactions such as trafficking, signaling, cell to cell communication and organization of signaling complexes. PDZ domains are themselves small proteins which typically consist of 90 to 100 amino acids. However, the extra α helix structure at the carboxyl terminus introduces a selective structural feature to the third PDZ domain of PSD-95 which has a stabilizing effect and participates in allosteric communication. PDZ domains are the most commonly studied models to understand single domain allostery without resulting in significant structural changes. One change triggers another change at distal site, and the source of the ‘changes’ are localized perturbations such as a binding event, posttranslational modification, a mutation or light absorption. Mutations can alter the stabilization of the protein and result ON or OFF state for ligand binding. They can also cause a change in the active site and affect the ligand preference. Here we investigate the reasons leading to the allosteric regulation of mutations and their effect on the ligand preferences. By using third PDZ domain of postsynaptic density 95 (PSD-95) as a model system H372 directly connected to the binding site and G330 with a somewhat removed position were selected to assess the effect of allosteric mutations on the dynamics. In the literature, it was observed that the H372A and G330T/H372A mutations change ligand preferences from class I (T/S amino acid preference at position 2 of the ligand) to class II (hydrophobic amino acid preference at position 2 of the ligand). On the other hand, the G330T mutation leads to the recognition of both class I and class II types of ligands. Therefore, H372A is a ‘switching mutation’ while G330T mutation is ‘class bridging’. We have performed 200 ns molecular dynamics simulations for wild-type, H372A, G330T single mutants and a double mutant of third PDZ domain in the absence and presence of both types of ligands. The comparative study helps to identify the changes in the dynamics that are effective in the onset and prevention of allosteric communication. With the combination of free energy difference calculations and a detailed analysis of MD trajectories, the behavior of the PDZ domain under the mutations, which are ‘class bridging’(G330T) and ‘class changing’(H372A), and their effects on the ligand preferences and binding affinities are explained. We show that the ensemble view of allostery provides a better description of site-to-site coupling rather than a pathway view that assumes a direct connection between the effector and binding site

Protonien kuljetuskanavat hengitysketjun kompleksissa I molekyylidynamiikkasimulaatioilla tutkittuna

Energy production is one of the vital functions in living cells. In oxidative phosphorylation, the nutrients in foodstuff are converted to the energy currency of cells, adenosine triphosphate (ATP). Five respiratory complexes embedded in the inner mitochondrial membrane in eukaryotes, and in the inner cell membrane of bacteria, perform oxidative phosphorylation. The first enzyme in the respiratory chain, NADH(nicotinamide adenine dinucleotide)-ubiquinone oxidoreductase (complex I) is one of the largest known protein assemblies. Complex I is L-shaped and consists of two domains: the hydrophilic domain in the matrix and the hydrophobic domain in the membrane. The enzyme transfers two electrons from NADH to quinone, utilises the released free energy in pumping four protons across the membrane and thus participates in the generation of the proton electrochemical gradient. Dysfunctions and mutations in complex I have been connected to several incurable neurodegenerative diseases, for instance Alzheimer's disease. Understanding the molecular function of complex I is the key to unravel the dysfunctions and develop cures against these lethal diseases. The mechanism of complex I is elusive, and many questions concerning the coupling mechanism and proton pumping remain unsolved. The objective of this thesis is to study the possible proton translocation pathways in the antiporter-like subunits Nqo12-14 in the membrane arm of complex I. The research question is addressed using a computational method, known as atomistic molecular dynamics (MD) simulations. The results reveal a set of conserved hydrophilic residues coordinating the water wire formation. Additionally, a strong water mediated connection through the middle plane of the membrane arm in subunits Nqo12-14 is observed, giving rise to a tentative coupling element. Water dynamics along the long horizontal helix HL unique in subunit Nqo12 is also analysed, and a stabilising mechanism of subunits Nqo12-14 is proposed

The methyltransferase and helicase enzymes as therapeutic targets of Zika virus : a bio- computational analysis of interactions with potential inhibitors.

Doctoral of Philosophy in Pharmaceutical Sciences. University of KwaZulu-Natal, Westville, 2019.The rampant Zika virus has received worldwide attention after becoming a global crisis following the Brazilian epidemic in 2015. From an obscure and neglected pathogen, Zika virus is now a notorious virus associated with neurological disorders in infants and adults. Since 2016, the rapid research response from the global scientific community have led to the discovery of numerous potential small molecule inhibitors and vaccines against the Zika virus. Although, in spite of this massive research initiative, there is still no effective antiviral nor vaccine that has made it out of clinical trials. The design and development of new chemical entities demands excessive cost, time and resources. Therefore, this study applies computer-aided drug design techniques, which accelerates the rational drug design process. Computational approaches including molecular docking, virtual screening, molecular modeling and molecular dynamics facilitate the filtration of large databases of compounds to sift out potential lead compounds. Furthermore, research has dedicated several resources toward FDA-approved drug repurposing. Generally, drugs have similar effects on viruses of the same family; hence drugs that have previously been effective in treating other flaviviruses, such as Dengue virus and West Nile virus, are being tested for its potential inhibition of Zika virus. However, the ability of these drugs to pass the bloodbrain barrier to treat infected neurons poses a challenge to anti-Zika virus drug discovery. This study proposes innovative strategies to design drugs that are capable of passing the blood-brain barrier, and to be able to use drugs that are impermeable via drug delivery mechanisms. This study also assesses the bioavailability and blood-brain barrier permeability of screened drugs to scrutinize the list of potential Zika virus inhibitors. Apart from identifying potential inhibitors, understanding the structural dynamics of viral targets and molecular mechanisms underlying potential inhibition of the virus is imperative. This study explores the structural and molecular dynamics of key targets of the Zika virus, the NS3 helicase and the NS5 methyltransferase enzymes, using computational approaches mentioned above and several others elaborated in this thesis. These computational methods also allowed the identification of precise interactions, amino acid residues, inhibitory mechanisms and pharmacophoric features involved in binding of lead compounds to these enzymes. IX Chapter 4 represents the first study of this thesis, which presents a concise literature background of Zika virus and identifies blood-brain barrier permeability as a core challenge in anti-Zika virus drug development. This study also provides approaches that may enable researchers to create effective anti-Zika virus drugs. Chapter 5 is the subsequent study of this thesis, which applies molecular dynamics to comparatively investigate the mechanism of inhibition and binding mode of two potential inhibitors, sinefungin and compound 5, to the NS5 methyltransferase. The specific pharmacophoric moieties of the most stable inhibitor are also identified in this study. Chapter 6 is the final study of this thesis, which examines the structural dynamics of the Zika virus NS3 helicase enzyme upon binding of ATPase inhibitor and flavivirus lead compound, resveratrol, and reports the key interactions and amino acid residues of the NS3 helicase that contribute highly to binding of resveratrol. This thesis presents an all-inclusive in silico assessment to advance research in drug design and development of Zika virus inhibitors, thus providing a greater understanding of the structural dynamics that occur in unbound and inhibitor-bound Zika virus target enzymes. Therefore, the constituents of this thesis are considered an essential platform in the progression of research toward anti-ZIKV drug design, discovery and delivery against Zika virus

A computational perspective of influenza a virus targets : neuraminidase and endonuclease.

Ph. D. University of KwaZulu-Natal, Durban 2016.Through the ages the viruses have plagued mankind claiming the lives of millions, pre-dating any advancements in the medicinal sciences. One such pathogenic virus is influenza A, which has been implicated in the 1918-Spanish flu, the 2006-avian flu outbreak and the 2009-swine flu pandemic. It is a highly sophisticated species, alluding efforts to thwart the spread of disease and infection. One of the main reasons influenza has survived this long is simple evolution. Natural mutation within the genome of virions expressed in proteins, enzymes or molecular structure render us unable to predict or take preventative measures against possible infection. Thus, research efforts toward the competitive inhibition of biological pathways that lead to the spread of disease, have become attractive targets. The influenza A virus has a number of chemotherapeutic targets, such as: 1) The surface antigens, hemagglutinin and neuraminidase, 2) RNA-dependent RNA polymerase, and 3) The M2 proton channel. Influenza RNA polymerase is composed of three large segments encoding polymerase acidic protein (PA), polymerase basic protein 1 (PB1) and polymerase basic protein 2 (PB2). The PA protein is an N-terminal domain subunit which contains the endonuclease activity. The influenza virus is incapable of synthesizing a 5’-mRNA cap, so it has adapted a cap-snatching mechanism whereby the PB2 subunit binds to the 5’-end of host mRNA, after which 10-14 nucleotides downstream the PA-subunit (aka PAN) cleaves the strand forming a primer for viral mRNA synthesis which is catalysed by the PB1 subunit. Influenza target identification is based primarily on evidence suggesting sequence conservation of each entity and its selective expression in the virus and not the host. In this thesis two enzymatic targets were investigated, the PA protein of RNA polymerase and neuraminidase. The studies focussed on using computational tools to: 1) provide insight into the mechanism of drug-resistance, 2) describe the conformational structure of the protein in the presence of point mutations and in complex with an inhibitor, 3) determine the essential binding pharmacophoric features to aid the design of new drug therapies. An array of computational techniques were employed in the studies, such as: molecular dynamics (MD) simulation, structure-based and ligand-based in silico screening, principal component analysis, radius of gyration analysis, binding free energy calculations and solventaccessible surface area analysis. The first study (Chapter 5) determined the mechanism of drug-resistance in influenza A neuraminidase as a consequence of antigenic variations. Two distinct mutations in the enzyme sequence that were investigated are H274Y and I222K. The active site residues of neuraminidase are conserved among the subtypes of influenza A. However, it was discovered that the occurrence of resistance to the drug oseltamivir, in the H1N1 species was different to the H5N1 virus. Although both systems shared a loss in hydrophobicity of the active site, the conformational distortion of the active site pocket distinguished the enzyme of the two viral entities, from one another. The discoveries made in the first study laid the foundation for the second study (Chapter 6), which was based on the in silico design and screen of potential neuraminidase inhibitors. As a result 10 characteristic molecular scaffolds were suggested as potential inhibitors. The pharmacophore design was constructed with consideration to the new conformational structure of the active site pocket. Chapter 7 is the third study of this thesis. The active site pocket enclosing the endonuclease activity of the PA subunit was investigated. Using molecular dynamics simulations and postdynamic analyses, a description of the protein conformation was offered. Subsequently, a pharmacophore was proposed as a potential scaffold to which endonuclease inhibitors may be modelled upon. It is my belief that the impact of the results derived from the above mentioned studies would greatly contribute to the development of new and effective anti-influenza drugs

Relaxation Estimation of RMSD in Molecular Dynamics Immunosimulations

Molecular dynamics simulations have to be sufficiently long to draw reliable conclusions. However, no method exists to prove that a simulation has converged. We suggest the method of "lagged RMSD-analysis" as a tool to judge if an MD simulation has not yet run long enough. The analysis is based on RMSD values between pairs of configurations separated by variable time intervals Δt. Unless RMSD(Δt) has reached a stationary shape, the simulation has not yet converged

Relaxation Estimation of RMSD in Molecular Dynamics Immunosimulations

Molecular dynamics simulations have to be sufficiently long to draw reliable conclusions. However, no method exists to prove that a simulation has converged. We suggest the method of “lagged RMSD-analysis” as a tool to judge if an MD simulation has not yet run long enough. The analysis is based on RMSD values between pairs of configurations separated by variable time intervals Δt. Unless RMSD(Δt) has reached a stationary shape, the simulation has not yet converged