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1.
J Biol Chem ; 300(4): 107166, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38490435

RESUMO

Clamp loaders are pentameric ATPases that place circular sliding clamps onto DNA, where they function in DNA replication and genome integrity. The central activity of a clamp loader is the opening of the ring-shaped sliding clamp and the subsequent binding to primer-template (p/t)-junctions. The general architecture of clamp loaders is conserved across all life, suggesting that their mechanism is retained. Recent structural studies of the eukaryotic clamp loader replication factor C (RFC) revealed that it functions using a crab-claw mechanism, where clamp opening is coupled to a massive conformational change in the loader. Here we investigate the clamp loading mechanism of the Escherichia coli clamp loader at high resolution using cryo-electron microscopy. We find that the E. coli clamp loader opens the clamp using a crab-claw motion at a single pivot point, whereas the eukaryotic RFC loader uses motions distributed across the complex. Furthermore, we find clamp opening occurs in multiple steps, starting with a partly open state with a spiral conformation, and proceeding to a wide open clamp in a surprising planar geometry. Finally, our structures in the presence of p/t-junctions illustrate how the clamp closes around p/t-junctions and how the clamp loader initiates release from the loaded clamp. Our results reveal mechanistic distinctions in a macromolecular machine that is conserved across all domains of life.


Assuntos
Replicação do DNA , Escherichia coli , Microscopia Crioeletrônica , Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Conformação Proteica , Proteína de Replicação C/metabolismo , Proteína de Replicação C/química , Proteína de Replicação C/genética , Modelos Moleculares , Estrutura Quaternária de Proteína
2.
J Biol Chem ; 299(3): 103021, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36791911

RESUMO

Tail tube assembly is an essential step in the lifecycle of long-tailed bacteriophages. Limited structural and biophysical information has impeded an understanding of assembly and stability of their long, flexible tail tubes. The hyperthermophilic phage P74-26 is particularly intriguing as it has the longest tail of any known virus (nearly 1 µm) and is the most thermostable known phage. Here, we use structures of the P74-26 tail tube along with an in vitro system for studying tube assembly kinetics to propose the first molecular model for the tail tube assembly of long-tailed phages. Our high-resolution cryo-EM structure provides insight into how the P74-26 phage assembles through flexible loops that fit into neighboring rings through tight "ball-and-socket"-like interactions. Guided by this structure, and in combination with mutational, light scattering, and molecular dynamics simulations data, we propose a model for the assembly of conserved tube-like structures across phage and other entities possessing tail tube-like proteins. We propose that formation of a full ring promotes the adoption of a tube elongation-competent conformation among the flexible loops and their corresponding sockets, which is further stabilized by an adjacent ring. Tail assembly is controlled by the cooperative interaction of dynamic intraring and interring contacts. Given the structural conservation among tail tube proteins and tail-like structures, our model can explain the mechanism of high-fidelity assembly of long, stable tubes.


Assuntos
Bacteriófagos , Caudovirales , Bacteriófagos/metabolismo , Caudovirales/metabolismo , Conformação Molecular , Modelos Moleculares , Proteínas da Cauda Viral/química
3.
J Biol Chem ; 299(5): 104656, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36990216

RESUMO

Proliferating cell nuclear antigen (PCNA) is a sliding clamp protein that coordinates DNA replication with various DNA maintenance events that are critical for human health. Recently, a hypomorphic homozygous serine to isoleucine (S228I) substitution in PCNA was described to underlie a rare DNA repair disorder known as PCNA-associated DNA repair disorder (PARD). PARD symptoms range from UV sensitivity, neurodegeneration, telangiectasia, and premature aging. We, and others, previously showed that the S228I variant changes the protein-binding pocket of PCNA to a conformation that impairs interactions with specific partners. Here, we report a second PCNA substitution (C148S) that also causes PARD. Unlike PCNA-S228I, PCNA-C148S has WT-like structure and affinity toward partners. In contrast, both disease-associated variants possess a thermostability defect. Furthermore, patient-derived cells homozygous for the C148S allele exhibit low levels of chromatin-bound PCNA and display temperature-dependent phenotypes. The stability defect of both PARD variants indicates that PCNA levels are likely an important driver of PARD disease. These results significantly advance our understanding of PARD and will likely stimulate additional work focused on clinical, diagnostic, and therapeutic aspects of this severe disease.


Assuntos
Alelos , Ataxia Telangiectasia , Reparo do DNA , Antígeno Nuclear de Célula em Proliferação , Temperatura , Humanos , Ataxia Telangiectasia/genética , Ataxia Telangiectasia/metabolismo , Reparo do DNA/genética , Replicação do DNA , Antígeno Nuclear de Célula em Proliferação/química , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ligação Proteica/genética , Estabilidade Proteica , Cromatina/genética , Cromatina/metabolismo , Especificidade por Substrato
4.
Cell ; 137(4): 659-71, 2009 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-19450514

RESUMO

Clamp loaders load sliding clamps onto primer-template DNA. The structure of the E. coli clamp loader bound to DNA reveals the formation of an ATP-dependent spiral of ATPase domains that tracks only the template strand, allowing recognition of both RNA and DNA primers. Unlike hexameric helicases, in which DNA translocation requires distinct conformations of the ATPase domains, the clamp loader spiral is symmetric and is set up to trigger release upon DNA recognition. Specificity for primed DNA arises from blockage of the end of the primer and accommodation of the emerging template along a surface groove. A related structure reveals how the psi protein, essential for coupling the clamp loader to single-stranded DNA-binding protein (SSB), binds to the clamp loader. By stabilizing a conformation of the clamp loader that is consistent with the ATPase spiral observed upon DNA binding, psi binding promotes the clamp-loading activity of the complex.


Assuntos
Trifosfato de Adenosina/metabolismo , DNA Polimerase III/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/química , Catálise , Cristalografia por Raios X , DNA/metabolismo , DNA Polimerase III/química , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Modelos Moleculares , RNA/metabolismo
5.
Proc Natl Acad Sci U S A ; 118(17)2021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33888587

RESUMO

Many viruses utilize ringed packaging ATPases to translocate double-stranded DNA into procapsids during replication. A critical step in the mechanochemical cycle of such ATPases is ATP binding, which causes a subunit within the motor to grip DNA tightly. Here, we probe the underlying molecular mechanism by which ATP binding is coupled to DNA gripping and show that a glutamate-switch residue found in AAA+ enzymes is central to this coupling in viral packaging ATPases. Using free-energy landscapes computed through molecular dynamics simulations, we determined the stable conformational state of the ATPase active site in ATP- and ADP-bound states. Our results show that the catalytic glutamate residue transitions from an active to an inactive pose upon ATP hydrolysis and that a residue assigned as the glutamate switch is necessary for regulating this transition. Furthermore, we identified via mutual information analyses the intramolecular signaling pathway mediated by the glutamate switch that is responsible for coupling ATP binding to conformational transitions of DNA-gripping motifs. We corroborated these predictions with both structural and functional experimental measurements. Specifically, we showed that the crystal structure of the ADP-bound P74-26 packaging ATPase is consistent with the structural coupling predicted from simulations, and we further showed that disrupting the predicted signaling pathway indeed decouples ATPase activity from DNA translocation activity in the φ29 DNA packaging motor. Our work thus establishes a signaling pathway that couples chemical and mechanical events in viral DNA packaging motors.


Assuntos
Adenosina Trifosfatases/metabolismo , Ácido Glutâmico/metabolismo , Simulação de Dinâmica Molecular , Empacotamento do Genoma Viral , Transdução de Sinais
6.
Nucleic Acids Res ; 49(11): 6474-6488, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34050764

RESUMO

Double-stranded DNA viruses package their genomes into pre-assembled capsids using virally-encoded ASCE ATPase ring motors. We present the first atomic-resolution crystal structure of a multimeric ring form of a viral dsDNA packaging motor, the ATPase of the asccφ28 phage, and characterize its atomic-level dynamics via long timescale molecular dynamics simulations. Based on these results, and previous single-molecule data and cryo-EM reconstruction of the homologous φ29 motor, we propose an overall packaging model that is driven by helical-to-planar transitions of the ring motor. These transitions are coordinated by inter-subunit interactions that regulate catalytic and force-generating events. Stepwise ATP binding to individual subunits increase their affinity for the helical DNA phosphate backbone, resulting in distortion away from the planar ring towards a helical configuration, inducing mechanical strain. Subsequent sequential hydrolysis events alleviate the accumulated mechanical strain, allowing a stepwise return of the motor to the planar conformation, translocating DNA in the process. This type of helical-to-planar mechanism could serve as a general framework for ring ATPases.


Assuntos
Adenosina Trifosfatases/química , Empacotamento do Genoma Viral , Proteínas Virais/química , Adenosina/química , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/metabolismo , Arginina/química , Fagos Bacilares/enzimologia , Domínio Catalítico , Cristalografia por Raios X , Simulação de Dinâmica Molecular , Fosfatos/química , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Proteínas Virais/metabolismo
7.
Proc Natl Acad Sci U S A ; 117(38): 23571-23580, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32907938

RESUMO

DNA replication requires the sliding clamp, a ring-shaped protein complex that encircles DNA, where it acts as an essential cofactor for DNA polymerases and other proteins. The sliding clamp needs to be opened and installed onto DNA by a clamp loader ATPase of the AAA+ family. The human clamp loader replication factor C (RFC) and sliding clamp proliferating cell nuclear antigen (PCNA) are both essential and play critical roles in several diseases. Despite decades of study, no structure of human RFC has been resolved. Here, we report the structure of human RFC bound to PCNA by cryogenic electron microscopy to an overall resolution of ∼3.4 Å. The active sites of RFC are fully bound to adenosine 5'-triphosphate (ATP) analogs, which is expected to induce opening of the sliding clamp. However, we observe the complex in a conformation before PCNA opening, with the clamp loader ATPase modules forming an overtwisted spiral that is incapable of binding DNA or hydrolyzing ATP. The autoinhibited conformation observed here has many similarities to a previous yeast RFC:PCNA crystal structure, suggesting that eukaryotic clamp loaders adopt a similar autoinhibited state early on in clamp loading. Our results point to a "limited change/induced fit" mechanism in which the clamp first opens, followed by DNA binding, inducing opening of the loader to release autoinhibition. The proposed change from an overtwisted to an active conformation reveals an additional regulatory mechanism for AAA+ ATPases. Finally, our structural analysis of disease mutations leads to a mechanistic explanation for the role of RFC in human health.


Assuntos
ATPases Associadas a Diversas Atividades Celulares , Replicação do DNA/fisiologia , Antígeno Nuclear de Célula em Proliferação , Proteína de Replicação C , ATPases Associadas a Diversas Atividades Celulares/química , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Microscopia Crioeletrônica , Humanos , Antígeno Nuclear de Célula em Proliferação/química , Antígeno Nuclear de Célula em Proliferação/metabolismo , Antígeno Nuclear de Célula em Proliferação/ultraestrutura , Proteína de Replicação C/química , Proteína de Replicação C/metabolismo , Proteína de Replicação C/ultraestrutura
8.
Biochemistry ; 60(3): 170-181, 2021 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-33433210

RESUMO

In addition to encoding the tertiary fold and stability, the primary sequence of a protein encodes the folding trajectory and kinetic barriers that determine the speed of folding. How these kinetic barriers are encoded is not well understood. Here, we use evolutionary sequence variation in the α-lytic protease (αLP) protein family to probe the relationship between sequence and energy landscape. αLP has an unusual energy landscape: the native state of αLP is not the most thermodynamically favored conformation and, instead, remains folded due to a large kinetic barrier preventing unfolding. To fold, αLP utilizes an N-terminal pro region similar in size to the protease itself that functions as a folding catalyst. Once folded, the pro region is removed, and the native state does not unfold on a biologically relevant time scale. Without the pro region, αLP folds on the order of millennia. A phylogenetic search uncovers αLP homologs with a wide range of pro region sizes, including some with no pro region at all. In the resulting phylogenetic tree, these homologs cluster by pro region size. By studying homologs naturally lacking a pro region, we demonstrate they can be thermodynamically stable, fold much faster than αLP, yet retain the same fold as αLP. Key amino acids thought to contribute to αLP's extreme kinetic stability are lost in these homologs, supporting their role in kinetic stability. This study highlights how the entire energy landscape plays an important role in determining the evolutionary pressures on the protein sequence.


Assuntos
Proteínas de Bactérias/química , Evolução Molecular , Modelos Moleculares , Filogenia , Dobramento de Proteína , Serina Endopeptidases/química , Proteínas de Bactérias/genética , Estabilidade Enzimática , Cinética , Serina Endopeptidases/genética
9.
J Biol Chem ; 295(12): 3783-3793, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32014998

RESUMO

Tailed bacteriophages use a DNA-packaging motor to encapsulate their genome during viral particle assembly. The small terminase (TerS) component of this DNA-packaging machinery acts as a molecular matchmaker that recognizes both the viral genome and the main motor component, the large terminase (TerL). However, how TerS binds DNA and the TerL protein remains unclear. Here we identified gp83 of the thermophilic bacteriophage P74-26 as the TerS protein. We found that TerSP76-26 oligomerizes into a nonamer that binds DNA, stimulates TerL ATPase activity, and inhibits TerL nuclease activity. A cryo-EM structure of TerSP76-26 revealed that it forms a ring with a wide central pore and radially arrayed helix-turn-helix domains. The structure further showed that these helix-turn-helix domains, which are thought to bind DNA by wrapping the double helix around the ring, are rigidly held in an orientation distinct from that seen in other TerS proteins. This rigid arrangement of the putative DNA-binding domain imposed strong constraints on how TerSP76-26 can bind DNA. Finally, the TerSP76-26 structure lacked the conserved C-terminal ß-barrel domain used by other TerS proteins for binding TerL. This suggests that a well-ordered C-terminal ß-barrel domain is not required for TerSP76-26 to carry out its matchmaking function. Our work highlights a thermophilic system for studying the role of small terminase proteins in viral maturation and presents the structure of TerSP76-26, revealing key differences between this thermophilic phage and its mesophilic counterparts.


Assuntos
Adenosina Trifosfatases/metabolismo , Bacteriófagos/metabolismo , Endodesoxirribonucleases/metabolismo , Montagem de Vírus/fisiologia , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Microscopia Crioeletrônica , DNA Viral/química , DNA Viral/metabolismo , Endodesoxirribonucleases/química , Endodesoxirribonucleases/genética , Simulação de Dinâmica Molecular , Mutagênese , Ligação Proteica , Conformação Proteica em alfa-Hélice , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Eletricidade Estática
10.
Nucleic Acids Res ; 47(13): 6826-6841, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31114918

RESUMO

Proliferating cell nuclear antigen (PCNA) is a sliding clamp that acts as a central co-ordinator for mismatch repair (MMR) as well as DNA replication. Loss of Elg1, the major subunit of the PCNA unloader complex, causes over-accumulation of PCNA on DNA and also increases mutation rate, but it has been unclear if the two effects are linked. Here we show that timely removal of PCNA from DNA by the Elg1 complex is important to prevent mutations. Although premature unloading of PCNA generally increases mutation rate, the mutator phenotype of elg1Δ is attenuated by PCNA mutants PCNA-R14E and PCNA-D150E that spontaneously fall off DNA. In contrast, the elg1Δ mutator phenotype is exacerbated by PCNA mutants that accumulate on DNA due to enhanced electrostatic PCNA-DNA interactions. Epistasis analysis suggests that PCNA over-accumulation on DNA interferes with both MMR and MMR-independent process(es). In elg1Δ, over-retained PCNA hyper-recruits the Msh2-Msh6 mismatch recognition complex through its PCNA-interacting peptide motif, causing accumulation of MMR intermediates. Our results suggest that PCNA retention controlled by the Elg1 complex is critical for efficient MMR: PCNA needs to be on DNA long enough to enable MMR, but if it is retained too long it interferes with downstream repair steps.


Assuntos
Proteínas de Transporte/fisiologia , Reparo de Erro de Pareamento de DNA , DNA Fúngico/metabolismo , Mutação , Antígeno Nuclear de Célula em Proliferação/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Proteínas de Transporte/genética , Cristalografia por Raios X , Replicação do DNA , DNA Fúngico/genética , Proteínas de Ligação a DNA/metabolismo , Edição de Genes , Genes Fúngicos , Proteína 2 Homóloga a MutS/metabolismo , Proteína 3 Homóloga a MutS/metabolismo , Conformação de Ácido Nucleico , Mutação Puntual , Antígeno Nuclear de Célula em Proliferação/fisiologia , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/metabolismo , Fase S , Proteínas de Saccharomyces cerevisiae/metabolismo , Sumoilação
11.
Nucleic Acids Res ; 45(6): 3591-3605, 2017 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-28082398

RESUMO

Many viruses use a powerful terminase motor to pump their genome inside an empty procapsid shell during virus maturation. The large terminase (TerL) protein contains both enzymatic activities necessary for packaging in such viruses: the adenosine triphosphatase (ATPase) that powers DNA translocation and an endonuclease that cleaves the concatemeric genome at both initiation and completion of genome packaging. However, how TerL binds DNA during translocation and cleavage remains mysterious. Here we investigate DNA binding and cleavage using TerL from the thermophilic phage P74-26. We report the structure of the P74-26 TerL nuclease domain, which allows us to model DNA binding in the nuclease active site. We screened a large panel of TerL variants for defects in binding and DNA cleavage, revealing that the ATPase domain is the primary site for DNA binding, and is required for nuclease activity. The nuclease domain is dispensable for DNA binding but residues lining the active site guide DNA for cleavage. Kinetic analysis of DNA cleavage suggests flexible tethering of the nuclease domains during DNA cleavage. We propose that interactions with the procapsid during DNA translocation conformationally restrict the nuclease domain, inhibiting cleavage; TerL release from the capsid upon completion of packaging unlocks the nuclease domains to cleave DNA.


Assuntos
Adenosina Trifosfatases/química , DNA Viral/metabolismo , Endodesoxirribonucleases/química , Proteínas Virais/química , Adenosina Trifosfatases/metabolismo , Bacteriófagos/enzimologia , Bacteriófagos/genética , Sítios de Ligação , Clivagem do DNA , Endodesoxirribonucleases/metabolismo , Modelos Moleculares , Domínios Proteicos , Proteínas Virais/metabolismo , Montagem de Vírus
12.
Proc Natl Acad Sci U S A ; 112(29): E3792-9, 2015 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-26150523

RESUMO

Many viruses package their genomes into procapsids using an ATPase machine that is among the most powerful known biological motors. However, how this motor couples ATP hydrolysis to DNA translocation is still unknown. Here, we introduce a model system with unique properties for studying motor structure and mechanism. We describe crystal structures of the packaging motor ATPase domain that exhibit nucleotide-dependent conformational changes involving a large rotation of an entire subdomain. We also identify the arginine finger residue that catalyzes ATP hydrolysis in a neighboring motor subunit, illustrating that previous models for motor structure need revision. Our findings allow us to derive a structural model for the motor ring, which we validate using small-angle X-ray scattering and comparisons with previously published data. We illustrate the model's predictive power by identifying the motor's DNA-binding and assembly motifs. Finally, we integrate our results to propose a mechanistic model for DNA translocation by this molecular machine.


Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Bacteriófagos/enzimologia , Bacteriófagos/genética , Empacotamento do DNA , Genoma Viral , Montagem de Vírus , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Arginina/metabolismo , Fenômenos Biofísicos , Eletroforese em Gel de Poliacrilamida , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Reprodutibilidade dos Testes , Thermus thermophilus/virologia , Proteínas Virais/química , Proteínas Virais/metabolismo
13.
Biopolymers ; 105(8): 532-46, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26918303

RESUMO

Sliding clamps are ring-shaped polymerase processivity factors that act as master regulators of cellular replication by coordinating multiple functions on DNA to ensure faithful transmission of genetic and epigenetic information. Dedicated AAA+ ATPase machines called clamp loaders actively place clamps on DNA, thereby governing clamp function by controlling when and where clamps are used. Clamp loaders are also important model systems for understanding the basic principles of AAA+ mechanism and function. After nearly 30 years of study, the ATP-dependent mechanism of opening and loading of clamps is now becoming clear. Here I review the structural and mechanistic aspects of the clamp loading process, as well as comment on questions that will be addressed by future studies. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 532-546, 2016.


Assuntos
Adenosina Trifosfatases , Trifosfato de Adenosina , Proteínas de Ligação a DNA , DNA , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Animais , DNA/biossíntese , DNA/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos
14.
Proc Natl Acad Sci U S A ; 109(24): 9414-9, 2012 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-22635267

RESUMO

Ultrahigh-resolution (< 1.0 Å) structures have revealed unprecedented and unexpected details of molecular geometry, such as the deformation of aromatic rings from planarity. However, the functional utility of such energetically costly strain is unknown. The 0.83 Å structure of α-lytic protease (αLP) indicated that residues surrounding a conserved Phe side-chain dictate a rotamer which results in a ~6° distortion along the side-chain, estimated to cost 4 kcal/mol. By contrast, in the closely related protease Streptomyces griseus Protease B (SGPB), the equivalent Phe adopts a different rotamer and is undistorted. Here, we report that the αLP Phe side-chain distortion is both functional and conserved in proteases with large pro regions. Sequence analysis of the αLP serine protease family reveals a bifurcation separating those sequences expected to induce distortion and those that would not, which correlates with the extent of kinetic stability. Structural and folding kinetics analyses of family members suggest that distortion of this side-chain plays a role in increasing kinetic stability within the αLP family members that use a large Pro region. Additionally, structural and kinetic folding studies of mutants demonstrate that strain alters the folding free energy landscape by destabilizing the transition state (TS) relative to the native state (N). Although side-chain distortion comes at a cost of foldability, it suppresses the rate of unfolding, thereby enhancing kinetic stability and increasing protein longevity under harsh extracellular conditions. This ability of a structural distortion to enhance function is unlikely to be unique to αLP family members and may be relevant in other proteins exhibiting side-chain distortions.


Assuntos
Proteínas de Bactérias/química , Serina Endopeptidases/química , Cinética , Modelos Moleculares , Dobramento de Proteína , Streptomyces griseus/enzimologia , Termodinâmica
15.
BMC Biol ; 10: 34, 2012 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-22520345

RESUMO

Clamp loaders are pentameric ATPases of the AAA+ family that operate to ensure processive DNA replication. They do so by loading onto DNA the ring-shaped sliding clamps that tether the polymerase to the DNA. Structural and biochemical analysis of clamp loaders has shown how, despite differences in composition across different branches of life, all clamp loaders undergo the same concerted conformational transformations, which generate a binding surface for the open clamp and an internal spiral chamber into which the DNA at the replication fork can slide, triggering ATP hydrolysis, release of the clamp loader, and closure of the clamp round the DNA. We review here the current understanding of the clamp loader mechanism and discuss the implications of the differences between clamp loaders from the different branches of life.


Assuntos
Adenosina Trifosfatases/metabolismo , Replicação do DNA , Evolução Molecular , Adenosina Trifosfatases/química , Bactérias/química , Bactérias/classificação , Bactérias/enzimologia , Bactérias/genética , Bacteriófago T4/química , Bacteriófago T4/classificação , Bacteriófago T4/enzimologia , Bacteriófago T4/genética , Eucariotos/química , Eucariotos/classificação , Eucariotos/enzimologia , Eucariotos/genética , Hidrólise , Modelos Moleculares , Filogenia , Estrutura Terciária de Proteína
16.
bioRxiv ; 2023 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-38076975

RESUMO

Clamp loaders are pentameric ATPases that place circular sliding clamps onto DNA, where they function in DNA replication and genome integrity. The central activity of a clamp loader is the opening of the ring-shaped sliding clamp, and the subsequent binding to primer-template (p/t)-junctions. The general architecture of clamp loaders is conserved across all life, suggesting that their mechanism is retained. Recent structural studies of the eukaryotic clamp loader Replication Factor C (RFC) revealed that it functions using a crab-claw mechanism, where clamp opening is coupled to a massive conformational change in the loader. Here we investigate the clamp loading mechanism of the E. coli clamp loader at high resolution using cryo-electron microscopy (cryo-EM). We find that the E. coli clamp loader opens the clamp using a crab-claw motion at a single pivot point, whereas the eukaryotic RFC loader uses motions distributed across the complex. Furthermore, we find clamp opening occurs in multiple steps, starting with a partly open state with a spiral conformation, and proceeding to a wide open clamp in a surprising planar geometry. Finally, our structures in the presence of p/t-junctions illustrate how clamp closes around p/t-junctions and how the clamp loader initiates release from the loaded clamp. Our results reveal mechanistic distinctions in a macromolecular machine that is conserved across all domains of life.

17.
Elife ; 112022 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-35731107

RESUMO

Clamp loaders place circular sliding clamp proteins onto DNA so that clamp-binding partner proteins can synthesize, scan, and repair the genome. DNA with nicks or small single-stranded gaps are common clamp-loading targets in DNA repair, yet these substrates would be sterically blocked given the known mechanism for binding of primer-template DNA. Here, we report the discovery of a second DNA binding site in the yeast clamp loader replication factor C (RFC) that aids in binding to nicked or gapped DNA. This DNA binding site is on the external surface and is only accessible in the open conformation of RFC. Initial DNA binding at this site thus provides access to the primary DNA binding site in the central chamber. Furthermore, we identify that this site can partially unwind DNA to create an extended single-stranded gap for DNA binding in RFC's central chamber and subsequent ATPase activation. Finally, we show that deletion of the BRCT domain, a major component of the external DNA binding site, results in defective yeast growth in the presence of DNA damage where nicked or gapped DNA intermediates occur. We propose that RFC's external DNA binding site acts to enhance DNA binding and clamp loading, particularly at DNA architectures typically found in DNA repair.


Assuntos
Trifosfato de Adenosina , Saccharomyces cerevisiae , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , DNA/metabolismo , Replicação do DNA , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteína de Replicação C/química , Proteína de Replicação C/genética , Proteína de Replicação C/metabolismo , Saccharomyces cerevisiae/metabolismo
18.
Elife ; 112022 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-35179493

RESUMO

Sliding clamps are ring-shaped protein complexes that are integral to the DNA replication machinery of all life. Sliding clamps are opened and installed onto DNA by clamp loader AAA+ ATPase complexes. However, how a clamp loader opens and closes the sliding clamp around DNA is still unknown. Here, we describe structures of the Saccharomyces cerevisiae clamp loader Replication Factor C (RFC) bound to its cognate sliding clamp Proliferating Cell Nuclear Antigen (PCNA) en route to successful loading. RFC first binds to PCNA in a dynamic, closed conformation that blocks both ATPase activity and DNA binding. RFC then opens the PCNA ring through a large-scale 'crab-claw' expansion of both RFC and PCNA that explains how RFC prefers initial binding of PCNA over DNA. Next, the open RFC:PCNA complex binds DNA and interrogates the primer-template junction using a surprising base-flipping mechanism. Our structures indicate that initial PCNA opening and subsequent closure around DNA do not require ATP hydrolysis, but are driven by binding energy. ATP hydrolysis, which is necessary for RFC release, is triggered by interactions with both PCNA and DNA, explaining RFC's switch-like ATPase activity. Our work reveals how a AAA+ machine undergoes dramatic conformational changes for achieving binding preference and substrate remodeling.


Assuntos
Replicação do DNA , Saccharomyces cerevisiae , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Microscopia Crioeletrônica , DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteína de Replicação C/química , Proteína de Replicação C/genética , Proteína de Replicação C/metabolismo , Saccharomyces cerevisiae/genética
19.
Cell Rep ; 40(2): 111064, 2022 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-35830796

RESUMO

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a signaling protein required for long-term memory. When activated by Ca2+/CaM, it sustains activity even after the Ca2+ dissipates. In addition to the well-known autophosphorylation-mediated mechanism, interaction with specific binding partners also persistently activates CaMKII. A long-standing model invokes two distinct S and T sites. If an interactor binds at the T-site, then it will preclude autoinhibition and allow substrates to be phosphorylated at the S site. Here, we specifically test this model with X-ray crystallography, molecular dynamics simulations, and biochemistry. Our data are inconsistent with this model. Co-crystal structures of four different activators or substrates show that they all bind to a single continuous site across the kinase domain. We propose a mechanistic model where persistent CaMKII activity is facilitated by high-affinity binding partners that kinetically compete with autoinhibition by the regulatory segment to allow substrate phosphorylation.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Processamento de Proteína Pós-Traducional , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Domínio Catalítico , Fosforilação
20.
Mol Cancer Res ; 19(6): 1015-1025, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33619228

RESUMO

FANCJ (BRIP1/BACH1) is a hereditary breast and ovarian cancer (HBOC) gene encoding a DNA helicase. Similar to HBOC genes, BRCA1 and BRCA2, FANCJ is critical for processing DNA inter-strand crosslinks (ICL) induced by chemotherapeutics, such as cisplatin. Consequently, cells deficient in FANCJ or its catalytic activity are sensitive to ICL-inducing agents. Unfortunately, the majority of FANCJ clinical mutations remain uncharacterized, limiting therapeutic opportunities to effectively use cisplatin to treat tumors with mutated FANCJ. Here, we sought to perform a comprehensive screen to identify FANCJ loss-of-function (LOF) mutations. We developed a FANCJ lentivirus mutation library representing approximately 450 patient-derived FANCJ nonsense and missense mutations to introduce FANCJ mutants into FANCJ knockout (K/O) HeLa cells. We performed a high-throughput screen to identify FANCJ LOF mutants that, as compared with wild-type FANCJ, fail to robustly restore resistance to ICL-inducing agents, cisplatin or mitomycin C (MMC). On the basis of the failure to confer resistance to either cisplatin or MMC, we identified 26 missense and 25 nonsense LOF mutations. Nonsense mutations elucidated a relationship between location of truncation and ICL sensitivity, as the majority of nonsense mutations before amino acid 860 confer ICL sensitivity. Further validation of a subset of LOF mutations confirmed the ability of the screen to identify FANCJ mutations unable to confer ICL resistance. Finally, mapping the location of LOF mutations to a new homology model provides additional functional information. IMPLICATIONS: We identify 51 FANCJ LOF mutations, providing important classification of FANCJ mutations that will afford additional therapeutic strategies for affected patients.


Assuntos
Proteína BRCA1/genética , DNA Helicases/genética , Análise Mutacional de DNA/métodos , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Mutação/genética , Neoplasias/genética , RNA Helicases/genética , Linhagem Celular Tumoral , Cisplatino/farmacologia , Códon sem Sentido , Reagentes de Ligações Cruzadas/farmacologia , Técnicas de Inativação de Genes , Células HeLa , Humanos , Mutação com Perda de Função , Mitomicina/farmacologia , Mutação/efeitos dos fármacos , Mutação de Sentido Incorreto , Neoplasias/patologia
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