HELICASES - PowerPoint Presentation

Slide1

HELICASES

Batlle Masó, Laura

Rosich

Sangrà

, Elena

Sumarroca

Bordas

, Marina

Torrecilas

Testa, TatianaSlide2

INDEX

Introduction

Materials and methods

Monomeric

helicase

:

PcrA

Hexameric

helicase

:

DnaB

Alignments and Superimpositions

ConclusionSlide3

INTRODUCTION

Definition

and

function

Superfamilies

Hexameric

helicases

RecA

like

and AAA+

domains

Evolution

Slide4

1. Definition and Function

Helicases are enzymes that unwind duplex DNA, RNA or DNA-RNA hybrids. They use energy derived from ATP hydrolisis to separate base-paired nucleic acids.

They

play roles in

cellular

processes

which

involve nucleic acids: DNA replication and repair Transcription Translation Ribosome synthesis RNA maduration and splicing Nuclear export processes

Eric J. On Helicases and other motor proeins. Cur Opin Stuct Biol. 2008 April; 18(2):243-257Slide5

1. Definition and Function

DNA

vs

RNA

helicases

Closely

related in structure and sequenceRNA – encoded

by organisms from all kingdoms of life and by many viruses.RNA helicases outnumber DNA helicasesNomenclature for subfamilies:Singleton MR, Dillngham MS. Structure and mechanism of helicases and nucleic acid

translocases. Annu. Rev. Biochem. 2007; 76:23-50.Slide6

2. Superfamilies (SF)

SF – 1

SF – 2

SF – 3

SF – 4

SF – 5

SF – 6

MONOMERIC

A & B helicasesHEXAMERICA helicasesB helicases

B helicases

Alfa

Helicases

Beta

Helicases

Classification based on the primary amino acid sequences of the

helicases

.

A & B

helicasesSlide7

2. Superfamilies (SF)

SF1 & SF2

SF3

– SF6

General

Very

prevalent

Monomeric

Form hexameric ringsFunctionSeveral diverse DNA and RNA manipulationsReplication forkDomainsTwo recA-likeRecA-like orAAA+ATP-binding siteAt the interface of these two domainsConsists of elements derived from monomers in the complexSlide8

2. Superfamilies (SF)

Fairman-Williams

ME,

Guenther

U,

Jankowsky

E. SF1 and SF2

helicases

: family matters. Curr Opin Struct Biol. 2010 June; 20(3):313-324Patel SS, Picha KM. Structure and function of hexameric helicases. Annu. Rev. Biochem. 2000; 69:651-97Slide9

Why a ring?

It

decrease

the

probability

of complete dissociation from the DNA3. Hexameric helicasesSlide10

3. Hexameric helicases

Types

DNA or RNA

Direction of unwinding

Examples

Bacteriophage

Helicases

ssDNA5’-3’- T7 gp4 ProteinsT4 gp41 Protein- SPP1 G40P ProteinPlasmid-Encoded HelicasessDNA5’-3’RSF1010 RepA ProteinBacterial HelicasesssDNA/ssRNA5’-3’- E.Coli DnaB ProteinE.coli RuvB Protein

E.coli rho ProteinArchaeal HelicasessDNA3’-5’Methanobacterium thermoautotrophicum MCM

Eukaryotic Viral

Helicases

dsDNA

3’-5’

SV40 and

Polyoma

Large

T Antigen Proteins

Papillomavirus

E1 Protein

Eukaryotic

Helicases

ssDNA

3’-5’

Human Bloom’s Syndrome protein

Mammalian MCM 4,6,7Slide11

4. Domains

AAA+

RecA

-like

Jiqing

Y,

Osborne

AR. RecA-like motor ATPases – lessons from structures. Biochemica et Biophysica Acta. 2004; 1-18Slide12

5. Evolution of DnaB

helicase

DnaB

originated

from a duplication of RecA-like ancestor after

the divergence of the bacteria from Archaea and eukaryotesThe replication fork helicases in Bacteria and Archaea/Eukaryota have evolved independently

Leipe DD, Aravind L. The bacterial replicative helicase DnaB evolved from a RecA duplication. Cold

Spring

Harbor

Laboratory

Press

ISSN

. 2000; 10:5-16Slide13

MATERIALS AND METHODS

Data bases

Sequence

alignment

Structural

aligmentSuperimpositions

DisplaySlide14

1. Databases

PDB

Uniprot

SCOP

PfamSlide15

2. Sequence aligment

T –

coffee

Clustalw

Clustal

format

T_

coffee

input.fa > output.faClustalw input.fa > output.fa Slide16

3. Structural aligment

HMMER

HMM fetch

HMM

align

HMM

scanSlide17

4. Superimpositions

Rough

STAMP



RMSD (Root

mean

Standard

deviation)

INPUT.out.pdb(output)

.pdb(input)Slide18

5. Display

ChimeraSlide19

PCR A

Structure

Motifs

Mechanism

Slide20

1.

Structure

4 structural domains:

2 α-β parallel domains (

dominis

1a and 2a)

2 additional domains (1b and 2b)

Velankar

SS, Soultanas P, Dillingham MS, Subramanya HS, Wigley DB. Crystal Structures of Complexes of PcrA DNA Helicase with DNA Substrate Indicate an Inchworm Mechanism. Cell 1999, 97 (75-84)Slide21

RecA-like

core

1.

Structure

Caruthers MJ, McKay D.

Helicase

structure and mechanism. Current opinion in Structural Biology 2002, 12: 123-133Slide22

1.

Structure

Caruthers MJ, McKay D.

Helicase

structure and mechanism. Current opinion in Structural Biology 2002, 12: 123-133Slide23

Walker A

Walker B

Motif I

(Walker A):

Amino group of lysine interacts with phosphates of

MgATP

/

MgADP

Hydroxyl of serine or threonine coordinates Mg2+ ionMotif II (Walker B):D227 forms salt bridge with K568 of motif V

2. MotifsSlide24

Walker A (I)

and

Walker B (II)

Walker A

Walker B

Motif I

(Walker A):

Amino group of lysine interacts with phosphates of

MgATP/MgADP

Lys37-ATP2. MotifsSlide25

Motif II (Walker B):

D227 forms salt bridge with K568 of motif V

Lys568

Asp227

2.

MotifsSlide26

Motif

Ia

:

Backbone carbonyl of F64 hydrogen bonds with ribose hydroxyl of

ssDNA

Motif IV

:

R359 binds DNA and forms a salt bridge to E600N361 interacts with ssDNAMotif TxGx:T91 and H93 interact with terminal nucleotide on ssDNA.2. MotifsSlide27

Ia

, IV

and

TxGx

Ia

IV

TxGx

2. MotifsSlide28

Motif Ia

:

Backbone carbonyl of F64 hydrogen bonds with ribose hydroxyl of

ssDNA

Hydrogen

bond

Phe64

2. MotifsSlide29

Motif IV:

N361

interacts with

ssDNA

Asn361

2.

MotifsSlide30

Motif IV

:

R359 binds DNA and forms a salt bridge to E600

Arg359

Glu600

2.

MotifsSlide31

Motif III

:

D251 and D253 form salt bridges with K309 and R206 respectively

Q254 interacts with

γ

phosphate of ATP

Y257, W259 and R260 interact with oligonucleotide

Motif V

:H565 interacts with ssDNAK568 forms salt bridge with E224 and D227 of motif IIE571 interacts with ribose of ATP2. MotifsSlide32

III

and

V

III

V

2.

MotifsSlide33

Motif III :

D251 and D253 form salt bridges with K309 and

R306

respectively

Asp251

Asp253

Lys309

Arg306

2. MotifsSlide34

Caruthers MJ, McKay D.

Helicase

structure and mechanism. Current opinion in Structural Biology 2002, 12: 123-133

2.

MotifsSlide35

Motif V:

K568

forms salt bridge with E224 and D227 of motif

II

2.

Motifs

Lys568

Glu224

Asp227Slide36

Caruthers MJ, McKay D.

Helicase

structure and mechanism. Current opinion in Structural Biology 2002, 12: 123-133

2.

MotifsSlide37

Motif VI

:

R610 interacts with

γ

phosphate of ATP

2.

MotifsSlide38

VI

2.

MotifsSlide39

2.

Motifs

Motif

VI

:

R610 interacts with

γ

phosphate of ATP Slide40

3. Mechanism

Active

Rolling

Inchworm

Requirement

for a dimeric protein Each subunit binds to ssDNA or dsDNA but not at the same time Large

step sizes Consistent with any oligomeric state of the protein

Binding

to

both

ssDNA

and

dsDNA

at

the

same

time

Smaller

step

sizes

Velanker

SS,

Soultnas P, Dillingham MS, Subramanya HS, Wigley DB. Crystal structures of complexes of PcrA

DNA helicase with a DNA substrate indicate an inchworm mechanism. Cell. 1999 Apr 2; 97 (1): 75-84.Slide41

3. Mechanism

Active

Rolling

Inchworm

Patel SS,

Donmex

I. Mechanism of

helicases. J Biol Chem. 2006 Jul 7; 281 (27).Slide42

DNA B

Introduction

Structure

Motifs

MechanismSlide43

HELICASES

SUPER FAMILY 4

DnaB family

RepA

T4 and T7

bacteriophages

DnaB

1.

IntroductionSlide44

Ring – shaped hexameric helicasa

6 identical monomers

2 structural domains:

1

α

-

β domain = CTD

1 α domain = NTD conected by a linker2. StructureSlide45

NTD

CTD

2.

StructureSlide46

Hall MC,

Matson

SW.

Helicase

motifs

:

the engine that powers DNA unwinding.

Molecular Microbiology. 1999; 34(5): 867-877.Bailey S, Eliason WK, Steitz TA. The crystal structure of the Thermus aquaticus DnaB helicase monomer. Nucleic Acids Research. 2007; 35(14): 4728-36.3. MotifsSlide47

Walker A

Walker B

3.

MotifsSlide48

Contacts with GDP

3.

Motifs

GLY 215

LYS 216

THR 217

Walker ASlide49

H1a

H2 (

Walker B

)

3.

MotifsSlide50

3.

Motifs

ASP 320

GLU 241

H1a

H2 (

Walker B

)Slide51

3.

Motifs

H3Slide52

3. Motifs

H3

GLN

362Slide53

4. Mechanism

Brownian

motor

Stepping

One

nucleic acid binding site Two conformational changes, tight state and weak state Power stroke motion + brownian

motion Two nucleic acid binding sites Six conformacional changes, for each subunit Power stroke motion Slide54

4. Mechanism

Brownian

motor

SteppingSlide55

ALIGNMENTS

PcrA

Sequence

alignment

Structural

alignment

Superimposition2. DnaBSequence alignmentStructural alignmentSuperimposition3. HelicasesSequence alignmentStructural alignmentSuperimpositionSlide56

1.

PcrA

Uniprot

ID

Organism

C3QZ11

Bacteriodes

sp.O34580Bacillus SubtilisP9WNP4Mycobacterium tuberculosisP56255Geobacillus stearothermophilusQ3DRY9Streptococcus agalactiaeQ8CRT9Straphylococcus epidermidisQ9S3Q0

Leuconostroc citreumQ53727Straphylococcus aureusProgram: T – coffeeTemplates:Slide57

1.

PcrA

Sequence

alignment

Walker A

Walker BSlide58

1.

PcrA

Structural

alignment

Conserved

N - terminal

Non

conserved C - terminalSlide59

1. PcrA

Superimposition

PcrA

vs

PcrA+ATPPcrA (1PJR)PcrA

+ ATP (3PJR)DNAATP RMSD: 2’33Sc: 4’27 Slide60

2.

DnaB

Uniprot

ID

Organism

A1AIN1

Escherichia

coli

O78411Guillardia thetaP59966Mycobacterium bovisQ55418Synechocystis sp.O30477Rhodothermus marinusP0A1Q4

Salmonella typhimuriumP47340Mycoplasma genitaliumP75539Mycoplasma pneumoniae

Q8YZA1

Nostoc

sp

.

P45256

Haemophilus

influenzae

P51333

Prophyra

purpurea

P9WMR2

Mycobacterium

tuberculosi

Q9X4C9

Geobacillus

stearothermophilus

Program

:

T –

coffee

Templates

:Slide61

2.

DnaB

Sequence

alignment

Walker A

Walker BSlide62

Structural alignment

Non

conserved

N

- terminal

Conserved

C - terminal

2.

DnaBSlide63

3.

Helicases

PDB

ID

Organism

Type

of

helicase

1E0KEnterobacteria phage T7Hexamer2REBEscherichia coliMonomeric1PJRGeobacillus stearothermophilusMonomeric4ESV

Geobacillus stearothermophilusHexameric1A1VHepatitis c virusMonomeric

1FUU

Saccharomyces

cerevisiae

Monomeric

1PV4

Escherichia

coli

Hexameric

1UAA

Escherichia

coli

Monomeric

1D2M

Thermus

thermophilus

Monomeric

Program

:

Clustalw

Templates

:Slide64

Sequence alignment

Walker A

Walker B

3.

HelicasesSlide65

Helicase

PDB ID

Family

Superfam

Domains

Organism

Pfam

Pfam’s

codeRecA2REBRecA protein-like (ATP-ase-domain)P-loop containing nucleoside triphosphate hydrolasesa: 3-268Escherichia coli (strain K12)RecAPF00154DnaB4ESV

a: 183-441Geobacillus stearothermophilus DnaBPF00772HCV

1A1V

RNA

helicase

P-

loop

containing

nucleoside

triphosphate

hydrolases

a:

190-325

b: 326-624

Hepatitis

C virus

genotype

1ª (

isolate

H)HCV corePF01542

IF4A1FUUTandem

AAA-

ATPase

domain

P-

loop

containing

nucleoside

triphosphate

hydrolases

a: 11-225

b: 226-394

Saccharomyces

cerevisiae

(

strain

ATCC 204508/S288c) (

Baker’s

yeast

)

Helicase

C-terminal

domain

PF00271

Rho

1PV4

RecA

protein-like

(

ATPase-domain

)

P-

loop

containing

nucleoside

triphosphate

hydrolases

a-f: 129-417

Escherichia

coli

(

strain

K12)

Rho N-terminal

domain

PF07498

3.

HelicasesSlide66

Helicase

PDB ID

Family

Superfam

Domains

Organism

Pfam

Pfam’s

codeRep1UAATandem AAA- ATPase domainP-loop containing nucleoside triphosphate hydrolasesa: 2-307b: 308-640Escherichia coli (strain K12)UvrD helicasePF00580

UvrB1D2MTandem AAA- ATPase domainP-loop containing nucleoside

triphosphate

hydrolases

a: 2-409

b: 410-583

Thermus

thermophilus

(

strain

HBB/ATCC 27634/DSM 579)

UvrB

PF12344

T7

1E0K

RecA

protein-like

(

ATPase

domain)

P-loop containing nucleoside

triphosphate

hydrolases

chains

a-f

Bacteriophage

T7

DnaB_C

PF03796

PcrA

1PJR

Tandem

AAA-

ATPase

domain

P-

loop

containing

nucleoside

triphosphate

hydrolases

a: 1-318

b: 319-651

Bacillus

stearothermophilus

UvrD-helicase

PF00580

3.

HelicasesSlide67

Structural alignment

: PDB ID

N –

terminal

conserved

C – terminal

conserved1PJR

1E0K1FUU4ESV1UAA2REB1D2M1PV4 3. HelicasesSlide68

Structural

alignment

: N – terminal

domain

Conserved

Non-conserved

Non-conserved

3. HelicasesSlide69

Structural alignment

: C – terminal

domain

Conserved

Non-conserved

Non-conserved

3.

HelicasesSlide70

Superimposition PcrA

- Rep

PcrA

(1PJR)

Rep

(1UAA)

RMSD: 1’39

Sc: 4’53

3. HelicasesSlide71

CONCLUSIONS

Helicases

are

essential

proteins

for every living organism.The ATP-binding

motifs (Walker A and Walker B) are highly conserved.The important structures are mantained along evolution in order to preserve the function of the enzymes.Slide72

BIBLIOGRAPHY

Enemark

EJ, Tor LJ. On

helicases

and

other

motor proteins. Curr Opin

Struct Biol. 2008 April; 18(2): 243-257. Singleton MR, Dillingham MS, Wigley DB. Structure and mechanism of helicases and nucleic acid translocases. Annu Rev Biochem. 2007; 76: 23-50. Hall MC, Matson SW. Helicase motifs: the engine that powers DNA unwinding. Molecular Microbiology

. 1999; 34(5): 867-877. Patel SS, Picha KM. Structure and function of hexameric helicases. Annu Rev Biochem. 2000; 69: 651-97.

Fairman-Williams

ME,

Guenther

UP,

Jankowsky

E. SF1 and SF2

helicases

:

family

matters

.

Curr

Opin

Struct

Biol

. 2010

June; 20(3): 313-324. Leipe DD, Aravind L,Grishin NV, Koonin EV. The Bacterial replicative helicase DnaB evolved

from a RecA duplication. Genome

Res

. 2000 Jan; 10(1): 5-16.

Neuwald

AF,

Aravind

L,

Spouge

JL,

Koonin

EV. AAA+: A

class

of

chaperone-like

ATPases

associated

with

the

assembly

,

operation

, and

disassembly

of

protein

complexes

.

Genome

Res

. 1999 Jan; 9(1): 27-43.

Ye

J,

Osborne

AR,

Groll

M,

Rapoport

TA.

RecA-like

motor

ATPases

–

lessons

from

structures

.

Biochim

Biophys

Acta

. 2004

Nov

4; 1659(1): 1-18.

Soultanas

P,

Wigley

DB.

Site-directed

mutagenesis

reveals

roles for

conserved

amino

acid

residues

in

the

hexameric

DNA

helicase

DnaB

from

Bacillus

stearothermophilus

.

Nucleic

Acids

Research

. 2002; 30: 4051-4060.

Bailey

S,

Eliason

WK,

Steitz

TA. The

crystal

structure

of

the

Thermus

aquaticus

DnaB

helicase

monomer

.

Nucleic

Acids

Research

. 2007; 35(14): 4728-36.

Soultanas

P.

Loading

mechanisms

of ring

helicases

at

replication

origins

.

Mol

Microbiol

. 2012

Apr

; 84(1): 6-16.

Bailey

S,

Eliason

WK,

Steitz

TA.

Structure

of

hexameric

DnaB

helicase

and

its

complex

with

a

domain

of

DnaG

primase

.

Science

. 2007

Oct

; 318(5849): 459-63.

Itsathitphaisarn

O,

Wing

RA,

Eliason

WK,

Wang

J. The

non-planar

structure

of

DnaB

hexamer

with

its

substrates

suggests

a

different

mechanisms

of

translocation

.

Cell

. 2012

Oct

12; 151(2): 267-277.Slide73

QUESTIONS

1. In

which

processes

are

helicases

involved? a. DNA replication b. Ribosome

synthesis c. Nuclear export processes d. DNA repair e. All are correct2. Helicases are classified in six superfamilies: a. Hexameric helicases are SF3, SF4, SF5 and SF6 b. Monomeric helicases are SF3, SF4, SF5 and SF6 c. SF1 and SF2 are hexameric helicases

d. All superfamilies are hexameric helicases e. All superfamilies are monomeric helicasesSlide74

QUESTIONS

3.

PcrA

:

a. The

organisms

that have it are gram-negative

bacteria b. Belongs to SF1 c. A and B are correct d. Hexameric helicase e. All are correct4. Motifs of PcrA: a. Walker A is motif I and Walker B is motif II b. Walker A is the only motif c. Walker A interacts with DNA d. Walker B interacts with DNA e. All are

correctSlide75

QUESTIONS

5.

DnaB

:

a.

Hexameric

helicase b. Bacterial helicase c. A and B are

correct d. It doesn’t have B – sheet folds e. All are correct6. Motifs of DnaB: a. Are located at the C-terminal part of DnaB b. DnaB has 5 conserved motifs c. A and B are correct d. The N-terminal part of DnaB is

conserved e. All are correct Slide76

QUESTIONS

7.

About

PcrA

and

DnaB

helicases: a. PcrA belongs to SF1 b.

DnaB belongs to SF4 c. PcrA participates in the replication of some plasmids d. DnaB is the main replicative helicase of eubacteria kingdom e. All are correct8. When aligning PcrA helicases from different organisms

: a. Walker A motif is conserved b. Walker A motif is not conserved c. The N-terminal domain is not structurally

conserved

d. The

C-terminal

domain

is

structurally

conserved

e. Any

motif

is

conservedSlide77

QUESTIONS

9.

When

aligning

DnaB

helicases from different organisms:

a. Walker A and Walker B motifs are conserved b. N-terminal domain is structurally conserved c. C-terminal domain is structurally conserved d. A and B are correct e. A and C are correct10. When aligning different types of helicases: a. The N-terminal domain always

contains the Walker A motif b. The C-terminal domain always contains the Walker B motif c. A and B are correct

d. DNA and

ATP-binding

motifs

are

conserved

among

helicases

e. All

are

correct

Slide78

Thank

you

!