HELICASES Batlle Masó, Laura Rosich Sangrà, Elena Sumarroca Bordas, Marina Torrecilas Testa, Tatiana INDEX A. B. C. D. E. F. Introduction Materials and methods Monomeric helicase: PcrA Hexameric helicase: DnaB Alignments and Superimpositions Conclusion INTRODUCTION 1. 2. 3. 4. 5. Definition and function Superfamilies Hexameric helicases RecA like and AAA+ domains Evolution 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):243-257 1. Definition and Function DNA vs RNA helicases Closely related in structure and sequence RNA – encoded by organisms from all kingdoms of life and by many viruses. RNA helicases outnumber DNA helicases Nomenclature for subfamilies: Singleton MR, Dillngham MS. Structure and mechanism of helicases and nucleic acid translocases. Annu. Rev. Biochem. 2007; 76:23-50. 2. Superfamilies (SF) Classification based on the primary amino acid sequences of the helicases. Alfa Helicases Beta Helicases SF – 1 SF – 2 SF – 3 SF – 4 SF – 5 SF – 6 MONOMERIC A & B helicases A helicases B helicases B helicases A & B helicases HEXAMERIC 2. Superfamilies (SF) SF1 & SF2 SF3 – SF6 General Very prevalent Monomeric Form hexameric rings Function Several diverse DNA and RNA manipulations Replication fork Domains Two recA-like RecA-like or AAA+ ATP-binding site At the interface of these two domains Consists of elements derived from monomers in the complex 2. Superfamilies (SF) Fairman-Williams ME, Guenther U, Jankowsky E. SF1 and SF2 helicases: family matters. Curr Opin Struct Biol. 2010 June; 20(3):313-324 Patel SS, Picha KM. Structure and function of hexameric helicases. Annu. Rev. Biochem. 2000; 69:651-97 3. Hexameric helicases •Why a ring? Increases the processivity of their respective DNA polymerases • High processivity is needed to catalyze genome replication and recombination processes. By encircling the DNA, helicases are topologically linked to the DNA • It allows helicases to stay on the DNA longer. It decrease the probability of complete dissociation from the DNA 3. Hexameric helicases Types DNA or RNA Direction of unwinding Examples Bacteriophage Helicases ssDNA 5’-3’ - T7 gp4 Proteins -T4 gp41 Protein - SPP1 G40P Protein Plasmid-Encoded Helicase ssDNA 5’-3’ RSF1010 RepA Protein Bacterial Helicases ssDNA/ssRNA 5’-3’ - E.Coli DnaB Protein -E.coli RuvB Protein - E.coli rho Protein Archaeal Helicase ssDNA 3’-5’ Methanobacterium thermoautotrophicum MCM 3’-5’ -SV40 and Polyoma Large T Antigen Proteins - Papillomavirus E1 Protein 3’-5’ -Human Bloom’s Syndrome protein - Mammalian MCM 4,6,7 Eukaryotic Viral Helicases Eukaryotic Helicases dsDNA ssDNA 4. Domains • RecA-like Jiqing Y, Osborne AR. RecA-like motor ATPases – lessons from structures. Biochemica et Biophysica Acta. 2004; 1-18 • AAA+ 5. Evolution of DnaB helicase DnaB originated from a duplication of RecA-like ancestor after the divergence of the bacteria from Archaea and eukaryotes The 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; 10:516 MATERIALS AND METHODS 1. 2. 3. 4. 5. Data bases Sequence alignment Structural aligment Superimpositions Display 1. Databases PDB Uniprot SCOP Pfam 2. Sequence aligment T – coffee Clustalw T_coffee input.fa > output.fa Clustalw input.fa > output.fa Clustal format 3. Structural aligment HMMER HMM fetch Target protein HMM scan PFAM family of the target HMM from PFAM family alignment HMM align Sequence – based alignment 4. Superimpositions Rough STAMP .out INPUT .domains .pdb (input) Rough STAMP Transform Chimera .pdb (output) RMSD (Root mean Standard deviation) Proteins superimposed 5. Display Chimera PCR A 1. Structure 2. Motifs 3. Mechanism 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) 1. Structure RecA-like core Caruthers MJ, McKay D. Helicase structure and mechanism. Current opinion in Structural Biology 2002, 12: 123-133 1. Structure Caruthers MJ, McKay D. Helicase structure and mechanism. Current opinion in Structural Biology 2002, 12: 123-133 2. Motifs Walker A Walker B • Motif I (Walker A): o Amino group of lysine interacts with phosphates of MgATP/MgADP o Hydroxyl of serine or threonine coordinates Mg2+ ion • Motif II (Walker B): o D227 forms salt bridge with K568 of motif V 2. Motifs • Walker A Walker A (I) and Walker B (II) Lys37-ATP • Walker B • Motif I (Walker A): o Amino group of lysine interacts with phosphates of MgATP/MgADP 2. Motifs • Motif II (Walker B): o D227 forms salt bridge with K568 of motif V Asp227 Lys568 2. Motifs • Motif Ia : o Backbone carbonyl of F64 hydrogen bonds with ribose hydroxyl of ssDNA • Motif IV: o R359 binds DNA and forms a salt bridge to E600 o N361 interacts with ssDNA • Motif TxGx: o T91 and H93 interact with terminal nucleotide on ssDNA. 2. Motifs Ia, IV and TxGx • Ia • IV • TxGx 2. Motifs • Motif Ia : o Backbone carbonyl of F64 hydrogen bonds with ribose hydroxyl of ssDNA Hydrogen bond Phe64 2. Motifs • Motif IV: o N361 interacts with ssDNA Asn361 2. Motifs • Motif IV: o R359 binds DNA and forms a salt bridge to E600 Arg359 Glu600 2. Motifs • Motif III : o D251 and D253 form salt bridges with K309 and R206 respectively o Q254 interacts with γ phosphate of ATP o Y257, W259 and R260 interact with oligonucleotide • Motif V: o H565 interacts with ssDNA o K568 forms salt bridge with E224 and D227 of motif II o E571 interacts with ribose of ATP 2. Motifs III and V • III • V 2. Motifs • Motif III : o D251 and D253 form salt bridges with K309 and R306 respectively Asp251 Arg306 Asp253 Lys309 2. Motifs Caruthers MJ, McKay D. Helicase structure and mechanism. Current opinion in Structural Biology 2002, 12: 123-133 2. Motifs • Motif V: o K568 forms salt bridge with E224 and D227 of motif II Glu224 Lys568 Asp227 2. Motifs Caruthers MJ, McKay D. Helicase structure and mechanism. Current opinion in Structural Biology 2002, 12: 123-133 2. Motifs • Motif VI : o R610 interacts with γ phosphate of ATP 2. Motifs VI 2. Motifs • Motif VI: o R610 interacts with γ phosphate of ATP 3. Mechanism Active Rolling Inchworm • Requirement for a dimeric protein • Consistent with any oligomeric • Each subunit binds to ssDNA or dsDNA but not at the same time • Binding to both ssDNA and dsDNA at the same time • Large step sizes • Smaller step sizes state of the protein 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. 3. Mechanism Active Rolling Inchworm Patel SS, Donmex I. Mechanism of helicases. J Biol Chem. 2006 Jul 7; 281 (27). DNA B 1. 2. 3. 4. Introduction Structure Motifs Mechanism 1. Introduction SUPER FAMILY 4 DnaB family DnaB RepA T4 and T7 bacteriophages 2. Structure Ring – shaped hexameric helicasa 6 identical monomers 2 structural domains: • 1 α-β domain = CTD • 1 α domain = NTD • conected by a linker 2. Structure CTD NTD 3. Motifs 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. Motifs Walker A Walker B 3. Motifs Contacts with GDP Walker A GLY 215 LYS 216 THR 217 3. Motifs H1a H2 (Walker B) 3. Motifs H1a H2 (Walker B) ASP 320 GLU 241 3. Motifs H3 3. Motifs H3 GLN 362 4. Mechanism Brownian motor Stepping • One nucleic acid binding site • Two nucleic acid binding sites • Two conformational changes, tight state and weak state • Six conformacional changes, for each subunit • Power stroke motion + brownian motion • Power stroke motion 4. Mechanism Brownian motor Stepping ALIGNMENTS 1. PcrA a. Sequence alignment b. Structural alignment c. Superimposition 2. DnaB a. Sequence alignment b. Structural alignment c. Superimposition 3. Helicases a. Sequence alignment b. Structural alignment c. Superimposition 1. PcrA Program: T – coffee Templates: Uniprot ID Organism C3QZ11 Bacteriodes sp. O34580 Bacillus Subtilis P9WNP4 Mycobacterium tuberculosis P56255 Geobacillus stearothermophilus Q3DRY9 Streptococcus agalactiae Q8CRT9 Straphylococcus epidermidis Q9S3Q0 Leuconostroc citreum Q53727 Straphylococcus aureus 1. PcrA Sequence alignment Walker B Walker A 1. PcrA Structural alignment Conserved N - terminal Non conserved C - terminal 1. PcrA Superimposition PcrA vs PcrA+ATP - - PcrA (1PJR) PcrA + ATP (3PJR) DNA ATP RMSD: 2’33 Sc: 4’27 2. DnaB Program: T – coffee Templates: Uniprot ID Organism A1AIN1 Escherichia coli O78411 Guillardia theta P59966 Mycobacterium bovis Q55418 Synechocystis sp. O30477 Rhodothermus marinus P0A1Q4 Salmonella typhimurium P47340 Mycoplasma genitalium P75539 Mycoplasma pneumoniae Q8YZA1 Nostoc sp. P45256 Haemophilus influenzae P51333 Prophyra purpurea P9WMR2 Mycobacterium tuberculosi Q9X4C9 Geobacillus stearothermophilus 2. DnaB Sequence alignment Walker A Walker B 2. DnaB Structural alignment Non conserved N - terminal Conserved C - terminal 3. Helicases Program: Clustalw Templates: PDB ID Organism Type of helicase 1E0K Enterobacteria phage T7 Hexamer 2REB Escherichia coli Monomeric 1PJR Geobacillus stearothermophilus Monomeric 4ESV Geobacillus stearothermophilus Hexameric 1A1V Hepatitis c virus Monomeric 1FUU Saccharomyces cerevisiae Monomeric 1PV4 Escherichia coli Hexameric 1UAA Escherichia coli Monomeric 1D2M Thermus thermophilus Monomeric 3. Helicases Sequence alignment Walker A Walker B 3. Helicases Helicase PDB ID Family Superfam Domains Organism Pfam Pfam’s code RecA 2REB RecA proteinlike (ATPase-domain) P-loop containing nucleoside triphosphate hydrolases a: 3-268 Escherichia coli (strain K12) RecA PF00154 DnaB 4ESV a: 183-441 Geobacillus stearothermophil us DnaB PF00772 HCV 1A1V P-loop containing nucleoside triphosphate hydrolases a: 190-325 Hepatitis C virus genotype 1ª (isolate H) HCV core PF01542 Tandem AAA-ATPase domain P-loop containing nucleoside triphosphate hydrolases a: 11-225 Saccharomyces cerevisiae (strain ATCC 204508/S288c ) (Baker’s yeast) Helicas e Ctermina l domain PF00271 RecA proteinlike (ATPasedomain) P-loop containing nucleoside triphosphate hydrolases Escherichia coli (strain K12) Rho Ntermina l domain PF07498 IF4A Rho 1FUU 1PV4 RNA helicase b: 326-624 b: 226-394 a-f: 129-417 3. Helicases Helicase PDB ID Family Superfam Domains Organism Pfam Pfam’s code Rep 1UAA Tandem AAA- ATPase domain P-loop containing nucleoside triphosphate hydrolases a: 2-307 Escherichia coli (strain K12) UvrD helicas e PF00580 Tandem AAA- ATPase domain P-loop containing nucleoside triphosphate hydrolases Thermus thermophilus (strain HBB/ATCC 27634/DSM 579) UvrB PF12344 UvrB 1D2M b: 308-640 a: 2-409 b: 410-583 T7 1E0K RecA proteinlike (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 Bacillus stearothermophi lus UvrDhelicas e PF00580 b: 319-651 3. Helicases Structural alignment: PDB ID N – terminal conserved C – terminal conserved 1PJR 1E0K 1FUU 4ESV 1UAA 2REB 1D2M 1PV4 3. Helicases Structural alignment: N – terminal domain Non-conserved Conserved Non-conserved 3. Helicases Structural alignment: C – terminal domain Non-conserved Conserved Non-conserved 3. Helicases Superimposition PcrA - Rep - PcrA (1PJR) Rep (1UAA) RMSD: 1’39 Sc: 4’53 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. 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. QUESTIONS 1. In which processes are helicases involved? a. DNA replication b. Ribosome synthesis c. Nuclear export processes d. DNA repair e. All are correct 2. 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 helicases 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 correct 4. 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 correct 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 correct 6. 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 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 correct 8. 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 conserved 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 correct 10. 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 Thank you!