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THEORETICAL STUDIES ON THE ENERGETICS OF SPECIFICITY IN SOME PROTEIN-DNA SYSTEMS

by

NIDHI ARORA

DEPARTMENT OF CHEMISTRY

SUBMITTED

IN FULFILMENT OF THE REQUIREMENTS OF THE DEGREE OF

DOCTOR OF PHILOSOPHY

tothe

INDIAN iNSTITUTE OF TECHNOLOGY, DELHI

INDIA March 1997

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OER ILFICATE

OEis is to certifs' that the thesis entit!叫、'THEORETICAL STUDIES ON THE ENERGETICS OF SPECIFICITY IN SOME PROTEII-DNA SYSTEMS", being sub面tted 句脇. NIDHI ARORA to the Indian Institute of千chno1ogy,氏面for the 醐紅d of the degree of U帆tor of Philosophy in Chemistry is a record of bonaffide

research work carried out 句 her,協. Ni面んora has worked under my guidance and supervision, and has fuiffihled the requirements for the sub面ssion of this thesis whi叱to 叫畑owledge, has辰ached the requisite standa川.

OEe results contained in this dissertation have not 1,<光n sub面tted in part or full to 如y other Univ郎ity or Institute for the aw肌-d of any degree or diploma.

Dat ・:晒: i ュ, n 了ト

B. Jayaram

Associate Professor 氏partment of Che面s町 Indian Institute of Technology, Delhi

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J was likeαchild on the crossroads, wiable lo decide which road was the right one. It i町ルB.血ルram, 婦ioりhis矛'sive enih硲一αid clear undeパ励威ng of the s呼cl別ided me in the right di,ぞcli伽in勝earch. He inoulded四涜価,clearing町 concepts, removing the cobwe加and in the p,りcess co盾tanlly encow召即ng ind習ndent 伽呼t andl如rk His mature criticism 柳one of the匂factors I厩trouゆrmed the vagi紹concepts in町"und into this p,質entableform. I wish toのo!郎四能叩se既of gratil泌α屈卿pect toルB.阿varamfor his四lienee, understau威ng auゼ功たgiぬ切ice

伽り咽切ut this切戒

It was P,可MN G叩ta who in加dz記edme to the戸eld of researcん built in nie the coがdence for i肌均'endent wo承 αid to accept no coinpronhises in research I am 即ゆi to him for his co摺tant encouragement 伽り呼out 町αca庇flic career at IlT Delhi.

抑sincere thai需 to the He屈ル即カnent of Chemisりfor providing the necessalプfacilities and to all声屈かmembersfor their val如bleαかice and cooperaiルfl.

乃is work co而not have been caクled ota without the he加and si似加rtof町 声enむes/柳加ケぬfl阿れJa加 A chin加D賀,加Isna Ri概助M知ぬ呼1, ぬdhna

防arma and Si万it B. Dixit who gルe meα戸伽Iんaring during叩stres.勇i向's and 1姫むed me with en/h硲厨m. It will be loo little to町1筑:in島リto them.

I am immenseケ伽1晒I to ぬtish BI加而'aj,凡ivi Kiran, Auiil 働nar, E.

珂町eねran,ルijni Madan, Remi Ba如晶町ん戒αep, B. K Pra'ガian,乃切nod SI励71V, Abhishek Uj肌拘り助il Garg and Ka励ikL加ta whoんルe been veグhe姉4'! to me in nu刀紹ro w乎呈

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I owe a great de'il to 町in-伽俄町 h盗加回四brothe,; i,> sister and四 戸irenlsfor being a stur'かs明加rl at all times andfor their mme盾e cooperation而ing

the course of this切rk Wo,濡are too little lo appreciate their

施砂 αthat応 are due to aft members of the staf/Jr their /dnd coopera如n throughout the course of四切虚

町stance加'n the Department ofScience and Technolo即の5刀 α万 theん威an>加tiMe of Tee)加。阿部 Delhi is gratefully

M

泌亥居み ノーーー

Vid/ii Arora

III

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Protein-DNA interactions oceng at al! !evels of DNA expression and lication are crucial determinants for the coordinated and controlled function ofcells, and are therefore extremely important in the life of a cefi. The remarkable specificity with which restriction endonucleases bind DNA and cut it at speciffic sites, the affnity of ressor to its cognate opeor, and the identiffication oflesions in DNA fol!owed by their repair are but a few instances of the bioche面ca! processes which are intriguing and are, of late, responsible for a surge in e即町imental and theoretica! studies di氏尤ted tow即ds obtaining a complete picture ofthe interactions函ng place at the mo!ecular level. In this regard, both theoiy as weil as experiment have beneffitted signifficantly with the availabi!ity of ato面C coordinates of a number of protein-DNA comp!exes.笥ese coup!ed with the avai!abi!ity of ever increasing computer power and with advances made in obtaining numerical solutions to biomo!ecular problems have enhanced our understanding considerably, but no ru!es for sp<ガffic reco師tion have hitherto b<如deffined. The cuirent status on protein-DNA specifficity is reviewed in Chapter I.正e present work takes a closer look at specifficity at the molecular level by forming an energetic perspective of these interactions.

Estimation of the thermodynC parameters of binding requires a potential of mean force between the two interacting macromolecules. Several forceffields perform well with proteins but not with DNA because of the dominance of electrostatic interactions in the latter and the indispensability ofa satisfactory account ofsolvent and counterions. For an expeditious and accurate estimation ofthe energetics ofnucleic acid systems, a distance dependent dielectricction,以r) in the electrostatic term of the forceffield,面th a proper

VII

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calibrati叫hasb<知recommended by several workers, in lieu ofexplicit molecular solvent 卿resenはion. Based on the collective experience in modeling small molecules and DNA with and without molecular solvent emer帥g from diverse Laboはones, we have put together a computationally simple forceffield exclusively for protein-DNA energetics. OEe methodology adopted is detailed in Chapter II・丑e面nimurn that is e即αted of such a dielectric価ction is to reproduce the base pa浦ng energies in solution. Base pairing thus provided a convenient testing ground.

liie base pairing energies h poly(的迦ly(dT) and poly (dG)ーpoly(dC) homopolymers have been (Chapter III) using parameters fflom recent versions of AMB眠 Cル駅MM, GROMOS and OPLS forceffields and employing different dielectric continuum models n印rely以r)=i .,4., 80., r and MI-ILF. An analysis of the base pa匠ng energetics in canonical B-DNA su留ests that a dielectric constant of面ty

{叫H}or a distance卿endent dielectric 鋤ction {D(加)面面C邸phase四ite well 議恒le a modiffied臣n四町ーいvery function Wil-ILF) is suitable for solution conditions.

Other 面釦如dielectric加ctions are not so satisfactory. ilie飼te difference Poisson- Boltzmann calculations and the MEILF邸ults indicate that the elαなostatic contribution to the base pairing energy in B-DNA in solution is in the range of -2.0 to -3.0 kcallmol per H-bond.

Afler characteriガng our approach with the latest forceffields 句estimating the base pa面g energetics, it was deemed impo比斌to gain the same level of co面dence in estimating in伽一protein energetics before proceed暁to the more complex protein-DNA systems. 'flic CO.. .1-IN interaction energies for topoloいcal neighbo邸involved in (i, 1 +4) H-bond interactions were estimated for a nonaひ叩tide of po1y (L-alanine) in 叫

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conformation (Chapter 1り. OEe total H-bond energetics is calculated as a sum of the contributions due to electrostatic and van der WぬIs interactions be氏V(児nthe閉比onyl of the ith residue and面de ofthe (i糾)tu residue empi叫iflg parameter sets 如m AMBER GROMOS, CHA肌皿OPLS and ECEPP force石elds using diLerent dielectric continuum models. All the forceffields capture the energetics in solutionトI .0 kca而0l) well within the experimentally predicted value of・0.5 to -2.0 koeL/mol.島ese are also sups刃ited by the FDPB values indicating the predo面nant role played by electrostatics in H-bonding. OEe modiffied Hngerty-山ely function estimates a H-bond strength of--P1.0 koe而olinOE- helices面le the same価ction leads to a value of -2.0 koe功如I in base pairing without any modiffications or'extra p雛ameteri風io氏 indicating the role of conte油ual e価加ts and g叩me町on the H-bond ene卿器

As the interactions in both DNA and protein in solution were captured in close correlation with eゆenmenち we applied our methodolo留to the well chamcterizガ入 卿ressor-OL1 system as a test case for studying protein-DNA interactions us暁OPLS p雛ameters (Chapterり. M宙e con宙Til血g most of the H-bond interactions noted in the X-ray studies, this investigation led to an identiffication of the tum-he!ix3-tum rather than the helix2-加rn-helix3 as being more impo比組t energetically for s叫iffic binding. I武k and key hypoth質is puts forth s加etu抱I complemen切rity as a hall mark of biomolecular recognition and this, as supportedりthe si師fleantly high van der WぬIs contribution su鉢町加sed on a hydrogen bond malガX, appears to be an essential mode of recognition in the入reprt溺。トope胤。野stem. A partitioning of the ener即tics into contributions due to phosphodiester group, sugar and bases reveals that the phosphate backbone plays a signifficant role in guiding the protein towards the DNA. In fact the m面mum number of H-bonds are with the phosphodiester group which contribute largely to the total

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electrostatic interaction. OEe sugars however show negligible contribution. Binding enけ師Cs impli叫te base atoms as being critical determinants of.specifficity with a van der Waals contribution of一58% to the total van der Wぬis interaction energy. A si面lar partitioning for the protein sub面ts reveals that the N-terminal amis make a large elαカostatic contribution(一 82% of total electros鳳ic interaction energy) to the total energetics besides si師fficant van der Wぬis interactions. OEe h-t-h motif interacts mainly 面ough van der Waalsト48% of the total van der Waal energy) with relatively面nor electrostatic and hydrophobic contributions.亜e t-h-t motif (containing the recognition helix and fl加tking加LI-ns) however emerges as the most impo比nt contributor to van der Waals interactions(,-58 殉 also aided in binding by the favourable electrostatic and hydrophobic interactions.

Intermolecular interaction energi昭of I i protein-DNA complexes have b和 eva!雌ed(Ch寧er VI) with a view to determining some common principles, if any,面ch may be responsible for附ciffic recognition ofαゴtain DNA sequen邸 in preference to others, fflom an energetic point of view. Results reveal that inespective of the number of residues or bases involved in compIe臓ion, the intei武tion energies in all the comp!exes fall in a very close range implicating the presence of邸tricting factors that de血e protein- DNA interactions. An i conclusion that e merg<認 is that packing e価cts at the inte由ce mani脇ted via the vander \\価Is interactions contribute門uallyα more strongly than elα力-o離而Cs.

Ap血itioning of the tota! ener留into contributions山e to direct and i而irect α河es reveals that more than the directα氏ゆase-residue interactions) that contributes si師fficantly but it is the 加iino acid side chain-DNA backbone interactions議拓ch are

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responsible for a maximum contribution to the tota! interaction energy. Over al!, these two components together contribute a signifficant 70-90% to the tota! energetics in diverse systems studied here with different force fleld OEe conclusion that the 仙il beyond the recognition he!ix is important for recognition is not only a feature of 入 repressor -operator comp!ex but a of al! the helix-turn-helix proteins considerガ here.胸drogen bonds are routinely implicated in processes involving molecu!ar recognition.丑e stren帥 of the 句drogen bonds at the protein-DNA

determined here to be of the order of--1.0 kcallmo囲-bond.

xl

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CERTI刃CATE

A

(次入り牙

Za

灰憂洗伍

w

パ 月

BS

ACT

LIST OF FIGURES

C

'VOER

INTRODUCHON

筋庇泌財巧如of恥叩ciかifl Prote加-DIロInteractions:

乃eCw,ぞntS勿t賀

1.1.1 Di.氏ctR<阻do叱 Rα刃師tion of the Exposed Edges of Bases h the Grooves

i . 1 .2 Indirect Readout: Role of Sugar Phosphate Backbone and Nucleic Acid Bendability

幼uctwal Motifsof Diロbinding乃りte加

1.2.1 Hel

泌恥

rn-Helix (hth) 1.2.2 Zinc Finger

i .2.3 Leucine Zipper 1.2.4 Hornα

omain

刀紹

oretical Stud

es on Pi

tein-DNA co

le

s

&窄

of the present Work References

lo J C1 つム J j

CHAPTER II 11且 つL つー つ一 っつ ォ什

2. 2. 2.

THEORY AND METHODOLOGY Energetics ofBase Pairing

The Sire呼hofH-bo及太 inαHelices

E肥卿ties of入R甲ぞssor-(加rator Co叩lex 取ergetics of Some Piりtei,i-D1ロ伽叩1e町 Finite切免rence Poisson-Boltzmann Coた油tio暦 References

一へ一 くJ OC I つム j IIJ j j j 月什 4. d. d.

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50 50 52 兇 め 66 CHAPTER 111. ENERGETICS OF BASE PAIRING [N艮DNA

Results and Discussion

CHAPTER IV. THE STRENGTH OF HYDROGEN BONDS IN a- HELICES 70

4.1 In加山ction 70

4.2 加ご如0陥1 71

4.3 Calculations 74

4.3 ResultsαガDiscussion 74

4.4 Cone厨励Is . 83

R予ルflees 84

CHAV正RV

「う一つ」

一 一一へ へ」

5.4

ENERGETIC BASIS FOR MOLECULAR ORIGINS OF 87 SPECWICITY I1 入 REPRESSOR-OPERATOR COMPLEX

Introduction . 87

Calculatio摺and Results 幻

Discussion 93

の励おio溜 %

References 97

CHAPTER 切

6.2

ヘノse LU AU うつ

6.4

ENERGY ANALYSIS OF SOME PROOEIN-DNA COMPLEXES

Introduction Bac惚切und

Caたulations and Set即 Results and Discussion Conchむio盾

References

1 一へLU CU 4. 99 99 10 12 12 15 15

L へつ J4 「つ j j

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I 59 CHAPTER VII SUMI"l隣RY, PERSPECFIVES AND SUGGESTIONS FOR

FUTURE晒TORK

引ひL:TA(加A(圧肌フR

AJ 「.1 AU I Ilt つ」

References

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