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1.
Cell ; 135(7): 1251-62, 2008 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-19109896

RESUMO

Viral genomes are packaged into "procapsids" by powerful molecular motors. We report the crystal structure of the DNA packaging motor protein, gene product 17 (gp17), in bacteriophage T4. The structure consists of an N-terminal ATPase domain, which provides energy for compacting DNA, and a C-terminal nuclease domain, which terminates packaging. We show that another function of the C-terminal domain is to translocate the genome into the procapsid. The two domains are in close contact in the crystal structure, representing a "tensed state." A cryo-electron microscopy reconstruction of the T4 procapsid complexed with gp17 shows that the packaging motor is a pentamer and that the domains within each monomer are spatially separated, representing a "relaxed state." These structures suggest a mechanism, supported by mutational and other data, in which electrostatic forces drive the DNA packaging by alternating between tensed and relaxed states. Similar mechanisms may occur in other molecular motors.


Assuntos
Bacteriófago T4/metabolismo , Empacotamento do DNA , Proteínas Virais/química , Proteínas Virais/metabolismo , Montagem de Vírus , Cristalografia por Raios X , Modelos Moleculares , Eletricidade Estática
2.
J Virol ; 86(8): 4046-57, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22345478

RESUMO

DNA packaging by double-stranded DNA bacteriophages and herpesviruses is driven by a powerful molecular machine assembled at the portal vertex of the empty prohead. The phage T4 packaging machine consists of three components: dodecameric portal (gp20), pentameric large terminase motor (gp17), and 11- or 12-meric small terminase (gp16). These components dynamically interact and orchestrate a complex series of reactions to produce a DNA-filled head containing one viral genome per head. Here, we analyzed the interactions between the portal and motor proteins using a direct binding assay, mutagenesis, and structural analyses. Our results show that a portal binding site is located in the ATP hydrolysis-controlling subdomain II of gp17. Mutations at key residues of this site lead to temperature-sensitive or null phenotypes. A conserved helix-turn-helix (HLH) that is part of this site interacts with the portal. A recombinant HLH peptide competes with gp17 for portal binding and blocks DNA translocation. The helices apparently provide specificity to capture the cognate prohead, whereas the loop residues communicate the portal interaction to the ATPase center. These observations lead to a hypothesis in which a unique HLH-portal interaction in the symmetrically mismatched complex acts as a lever to position the arginine finger and trigger ATP hydrolysis. Transiently connecting the critical parts of the motor; subdomain I (ATP binding), subdomain II (controlling ATP hydrolysis), and C-domain (DNA movement), the portal-motor interactions might ensure tight coupling between ATP hydrolysis and DNA translocation.


Assuntos
Adenosina Trifosfatases/metabolismo , Bacteriófago T4/genética , Bacteriófago T4/metabolismo , Proteínas do Capsídeo/metabolismo , Empacotamento do DNA , DNA Viral/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Bacteriófago T4/química , Sítios de Ligação , Transporte Biológico , Endopeptidases/metabolismo , Ordem dos Genes , Sequências Hélice-Alça-Hélice , Hidrólise , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Alinhamento de Sequência , Proteínas do Core Viral/metabolismo , Proteínas Virais/química , Proteínas Virais/genética
3.
Nucleic Acids Res ; 39(7): 2742-55, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21109524

RESUMO

In genome packaging by tailed bacteriophages and herpesviruses, a concatemeric DNA is cut and inserted into an empty procapsid. A series of cuts follow the encapsidation of each unit-length 'headful' genome, but the mechanisms by which cutting is coupled to packaging are not understood. Here we report the first biochemical characterization of a headful nuclease from bacteriophage T4. Our results show that the T4 nuclease, which resides in the C-terminal domain of large 'terminase' gp17, is a weak endonuclease and regulated by a variety of factors; Mg, NaCl, ATP, small terminase gp16 and N-terminal ATPase domain. The small terminase, which stimulates gp17-ATPase, also stimulates nuclease in the presence of ATP but inhibits in the absence of ATP suggesting interdomain crosstalk. Comparison of the 'relaxed' and 'tensed' states of the motor show that a number of basic residues lining the nuclease groove are positioned to interact with DNA in the tensed state but change their positions in the relaxed state. These results suggest that conformational changes in the ATPase center remodel the nuclease center via an interdomain 'communication track'. This might be a common regulatory mechanism for coupling DNA cutting to DNA packaging among the headful packaging nucleases from dsDNA viruses.


Assuntos
Bacteriófago T4/enzimologia , Endodesoxirribonucleases/química , Proteínas Virais/química , Trifosfato de Adenosina/metabolismo , Bacteriófago T4/fisiologia , DNA/química , DNA/metabolismo , Clivagem do DNA , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Metais/química , Modelos Moleculares , Domínios e Motivos de Interação entre Proteínas , Cloreto de Sódio/química , Proteínas Virais/metabolismo , Montagem de Vírus
4.
J Biol Chem ; 285(31): 24282-9, 2010 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-20525695

RESUMO

Many double-stranded DNA viruses employ ATP-driven motors to translocate their genomes into small, preformed viral capsids against large forces resisting confinement. Here, we show via direct single-molecule measurements that a mutation T194M downstream of the Walker B motif in the phage lambda gpA packaging motor causes an 8-fold reduction in translocation velocity without substantially changing processivity or force dependence, whereas the mutation G212S in the putative C (coupling) motif causes a 3-fold reduction in velocity and a 6-fold reduction in processivity. Meanwhile a T194M pseudorevertant (T194V) showed a near restoration of the wild-type dynamics. Structural comparisons and modeling show that these mutations are in a loop-helix-loop region that positions the key residues of the catalytic motifs, Walker B and C, in the ATPase center and is structurally homologous with analogous regions in chromosome transporters and SF2 RNA helicases. Together with recently published studies of SpoIIIE chromosome transporter and Ded1 RNA helicase mutants, these findings suggest the presence of a structurally conserved region that may be a part of the mechanism that determines motor velocity and processivity in several different types of nucleic acid translocases.


Assuntos
DNA Viral/genética , DNA/genética , Mutação , Motivos de Aminoácidos , Sequência de Aminoácidos , Bacteriófago T4/metabolismo , Catálise , DNA Helicases/metabolismo , Microesferas , Dados de Sequência Molecular , Pinças Ópticas , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Proteínas Virais/metabolismo
5.
Mol Microbiol ; 69(5): 1180-90, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18627466

RESUMO

Most tailed bacteriophages and herpes viruses replicate genome as a concatemer which is cut by a 'headful' nuclease upon completion of genome packaging. Here, the catalytic centre of phage T4 headful nuclease, present in the C-terminal domain of 'large terminase' gp17, has been defined by mutational, biochemical and structural analyses. The crystal structure shows that this nuclease has an RNase-H fold, suggesting that it cuts DNA by a two-metal ion mechanism. The active centre has a Mg ion co-ordinated by three acidic residues, D401, E458 and D542. Mutations at any of these residues resulted in loss of nuclease activity, but the mutants can package linear DNA. The gp17's nuclease activity is modulated by the 'small terminase', gp16, by the N-terminal ATPase domain of gp17, and by the assembled packaging motor. These results lead to hypotheses concerning how phage headful nucleases cut the viral genomes before and after, but not during, DNA packaging.


Assuntos
Bacteriófago T4/enzimologia , Bacteriófago T4/fisiologia , Empacotamento do DNA , Desoxirribonucleases/metabolismo , Proteínas Virais/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Bacteriófago T4/química , Bacteriófago T4/genética , Bacteriófagos/genética , DNA Viral/genética , Desoxirribonucleases/química , Desoxirribonucleases/genética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Alinhamento de Sequência , Proteínas Virais/química , Proteínas Virais/genética , Montagem de Vírus
6.
J Mol Biol ; 369(1): 79-94, 2007 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-17428497

RESUMO

Tailed bacteriophages and large eukaryotic viruses employ powerful molecular motors to translocate dsDNA into a preassembled capsid shell. The phage T4 motor is composed of a dodecameric portal and small and large terminase subunits assembled at the special head-tail connector vertex of the prohead. The motor pumps DNA through the portal channel, utilizing ATP hydrolysis energy provided by an ATPase present in the large terminase subunit. We report that the ATPase motors of terminases, helicases, translocating restriction enzymes, and protein translocases possess a common coupling motif (C-motif). Mutations in the phage T4 terminase C-motif lead to loss of stimulated ATPase and DNA translocation activities. Surprisingly, the mutants can catalyze at least one ATP hydrolysis event but are unable to turn over and reset the motor. This is the first report of a catalytic block in translocating ATPase motor after ATP hydrolysis occurred. We suggest that the C-motif is an ATP hydrolysis sensor, linking product release to mechanical motion. A novel terminase-driven mechanism is proposed for translocation of dsDNA in viruses.


Assuntos
Trifosfato de Adenosina/metabolismo , Bacteriófago T4/genética , Bacteriófago T4/metabolismo , Empacotamento do DNA , DNA Viral/metabolismo , Adenosina Trifosfatases/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Bacteriófago T4/enzimologia , Transporte Biológico , Catálise , Proteínas de Ligação a DNA/metabolismo , Desoxirribonucleases/metabolismo , Endodesoxirribonucleases/química , Evolução Molecular , Hidrólise , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Ligação Proteica , Proteínas Virais/metabolismo
7.
Virology ; 468-470: 660-668, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25443668

RESUMO

The packaging motor of bacteriophage T4 translocates DNA into the capsid at a rate of up to 2000 bp/s. Such a high rate would require coordination of motor movements at millisecond timescale. Designing a cysteine-less gp17 is essential to generate fluorescently labeled motors and measure distance changes between motor domains by FRET analyses. Here, by using sequence alignments, structural modeling, combinatorial mutagenesis, and recombinational rescue, we replaced all nine cysteines of gp17 and introduced single cysteines at defined positions. These mutant motors retained in vitro DNA packaging activity. Single mutant motors translocated DNA molecules in real time as imaged by total internal reflection fluorescence microscopy. We discovered, unexpectedly, that a hydrophobic or nonpolar amino acid next to Walker B motif is essential for motor function, probably for efficient generation of OH(-) nucleophile. The ATPase Walker B motif, thus, may be redefined as "ß-strand (4-6 hydrophobic-rich amino acids)-DE-hydrophobic/nonpolar amino acid".


Assuntos
Adenosina Trifosfatases/metabolismo , Bacteriófago T4/metabolismo , Empacotamento do DNA/fisiologia , DNA Viral/genética , Escherichia coli/virologia , Adenosina Trifosfatases/genética , Bacteriófago T4/genética , Clonagem Molecular , Empacotamento do DNA/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação da Expressão Gênica , Biblioteca Gênica , Mutação , Conformação Proteica
8.
J Mol Biol ; 415(2): 329-42, 2012 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-22100308

RESUMO

ATP is the source of energy for numerous biochemical reactions in all organisms. Tailed bacteriophages use ATP to drive powerful packaging machines that translocate viral DNA into a procapsid and compact it to near-crystalline density. Here we report that a complex network of interactions dictates adenine recognition and ATP hydrolysis in the pentameric phage T4 large "terminase" (gp17) motor. The network includes residues that form hydrogen bonds at the edges of the adenine ring (Q138 and Q143), base-stacking interactions at the plane of the ring (I127 and R140), and cross-talking bonds between adenine, triphosphate, and Walker A P-loop (Y142, Q143, and R140). These interactions are conserved in other translocases such as type I/type III restriction enzymes and SF1/SF2 helicases. Perturbation of any of these interactions, even the loss of a single hydrogen bond, leads to multiple defects in motor functions. Adenine recognition is therefore a key checkpoint that ensures efficient ATP firing only when the fuel molecule is precisely engaged with the motor. This may be a common feature in the energy release mechanism of ATP-driven molecular machines that carry out numerous biomolecular reactions in biological systems.


Assuntos
Adenina/metabolismo , Bacteriófago T4/fisiologia , Empacotamento do DNA , Proteínas Virais/metabolismo , Montagem de Vírus , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Análise Mutacional de DNA , DNA Viral/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Homologia de Sequência de Aminoácidos , Proteínas Virais/química , Proteínas Virais/genética
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