Nucleotide-Induced Conformational Changes in an Isolated Escherichia coli DNA Polymerase III Clamp Loader Subunit

Podobnik, Marjetka; Weitze, Tanya F.; O’Donnell, Michael; Kuriyan, John

doi:10.1016/s0969-2126(03)00027-3

Cited by 31 publications

(29 citation statements)

References 51 publications

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“…Adenosine 5Ј-[␤,␥-imido]triphosphate did not bind to the clamp loader, in agreement with previous results (15,26). ATP␥S was found to behave similarly to ATP and binds with a stoichiometry of 2 and K d of 0.78 M.…”

Section: Resultssupporting

confidence: 91%

“…3) with an independent-site (noncooperative) model yields dissociation constants (K d ) that are similar for ATP and ADP (3.7 and 3.0 M, respectively), with a stoichiometry of 2 for both nucleotides. These values for K d are comparable to those obtained for the binding of ATP and ADP to the isolated ␥-subunit (26) and suggest that ATP binding to the clamp-loader complex does not convert the complex to a different state, perhaps because of the absence of the clamp and DNA. Although it is possible that the apparent similarity in the binding energy of ATP and ADP reflects the conversion of part of the binding energy of ATP into a conformational change in the clamp loader, the crystal structure of the ATP␥S complex suggests instead that the interfacial interactions that recognize ATP may not occur after nucleotide addition in the absence of the clamp.…”

Section: Resultssupporting

confidence: 74%

“…A crystal structure of a truncated ␥-subunit with ATP␥S bound suggested that conformational changes in ␥ D after ATP binding would break this tight interface and allow a third ATP to bind to ␥ C (26). It is apparent from the crystal structure and ITC data presented here that a conformational change originating solely from ATP binding is not enough to break open this interface or convert the clamp loader to an active conformation.…”

Section: Simulations Suggest That the Open Conformation Of The Clampmentioning

confidence: 76%

“…ITC experiments used a MicroCal VP isothermal titration calorimeter (Microcal, Northampton, MA) as described (26). After degassing each sample under vacuum, nucleotide solution containing ATP, ATP␥S, ADP, or adenosine 5Ј-[␤,␥-imido]triphosphate (50-150 M) was titrated into protein solution (2.5-6.1 M) in 10-l injections 300 s apart.…”

Section: Methodsmentioning

confidence: 99%

“…Control titrations of nucleotide into ITC buffer demonstrated that there was no significant heat of dilution or injection for any of the tested nucleotides (data not shown). Data were analyzed as described (26).…”

Section: Methodsmentioning

confidence: 99%

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Structural analysis of the inactive state of the Escherichia coli DNA polymerase clamp-loader complex

Kazmirski

Podobnik

Weitze

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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Clamp-loader complexes are heteropentameric AAA ؉ ATPases that load sliding clamps onto DNA. The structure of the nucleotide-free Escherichia coli clamp loader had been determined previously and led to the proposal that the clamp-loader cycles between an inactive state, in which the ATPase domains form a closed ring, and an active state that opens up to form a ''C'' shape. The crystal structure was interpreted as being closer to the active state than the inactive state. The crystal structure of a nucleotide-bound eukaryotic clamp loader [replication factor C (RFC)] revealed a different and more tightly packed spiral organization of the ATPase domains, raising questions about the significance of the conformation seen earlier for the bacterial clamp loader. We describe crystal structures of the E. coli clamp-loader complex bound to the ATP analog ATP␥S (at a resolution of 3.5 Å) and ADP (at a resolution of 4.1 Å). These structures are similar to that of the nucleotide-free clamp-loader complex. Only two of the three functional ATP-binding sites are occupied by ATP␥S or ADP in these structures, and the bound nucleotides make no interfacial contacts in the complex. These results, along with data from isothermal titration calorimetry, molecular dynamics simulations, and comparison with the RFC structure, suggest that the more open form of the E. coli clamp loader described earlier and in the present work corresponds to a stable inactive state of the clamp loader in which the ATPase domains are prevented from engaging the clamp in the highly cooperative manner seen in the fully ATP-loaded RFC-clamp structure.AAA ϩ ATPase ͉ clamp loader ͉ DNA polymerase III ͉ DNA replication ͉ replication factor C C hromosomal replicases in all of the branches of life achieve high processivity by tethering their catalytic subunits to sliding DNA clamps (1-3). The closed circular sliding clamps are loaded onto DNA by ATP-dependent clamp-loader complexes (4), which are conserved heteropentameric ATPase assemblies. Most sliding clamps are stable closed rings in solution, although recent studies suggest that the clamp in T4 bacteriophage may be open in solution and that the role of the clamp loader might be to stabilize the open form and guide it to DNA (5). The subunits of the clamp-loader complexes consist of three conserved domains, with the first two being closely related to the nucleotide-binding domains of AAA ϩ ATPases (6-10). The AAA ϩ domains of the clamp loader are suspended loosely from a circular helical collar that is formed by the third (C-terminal) domains.ATP binding to the clamp loader triggers a conformational change that allows the complex to bind to and open the clamp and load it onto DNA (11, 12). In an intriguing control mechanism, the interaction with DNA stimulates the otherwise suppressed ATPase activity of the clamp loader, leading to ATP hydrolysis (11, 13) and separation of the clamp-loader complex from the DNA-loaded clamp. How ATP binding is coupled to loading of the clamp on DNA is poorly understood, and eluc...

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Section: Resultssupporting

confidence: 91%

Section: Resultssupporting

confidence: 74%

Section: Simulations Suggest That the Open Conformation Of The Clampmentioning

confidence: 76%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Structural analysis of the inactive state of the Escherichia coli DNA polymerase clamp-loader complex

Kazmirski

Podobnik

Weitze

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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Review: The lord of the rings: Structure and mechanism of the sliding clamp loader

Kelch

2016

Biopolymers

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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.

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What is the relationship between the global structures of apo and holo proteins?

Bryliński

Skolnick

2007

Proteins

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It is well known that ligand binding and release may induce a wide range of structural changes in a receptor protein, varying from small movements of loops or side chains in the binding pocket to large‐scale domain hinge‐bending and shear motions or even partial unfolding that facilitates the capture and release of a ligand. An interesting question is what in general are the conformational changes triggered by ligand binding? The aim of this work is analyze the magnitude of structural changes in a protein resulting from ligand binding to assess if the state of ligand binding needs to be included in template‐based protein structure prediction algorithms. To address this issue, a nonredundant dataset of 521 paired protein structures in the ligand‐free and ligand‐bound form was created and used to estimate the degree of both local and global structure similarity between the apo and holo forms. In most cases, the proteins undergo relatively small conformational rearrangements of their tertiary structure upon ligand binding/release (most root‐mean‐square‐deviations from native, RMSD, are <1 Å). However, a clear difference was observed between single‐ and multiple‐domain proteins. For the latter, RMSD changes greater than 1 Å and sometimes larger were found for almost 1/3 of the cases; these are mainly associated with large‐scale hinge‐bending movements of entire domains. The changes in the mutual orientation of individual domains in multiple‐domain proteins upon ligand binding were investigated using a mechanistic model based on mass‐weighted principal axes as well as interface buried surface calculations. Some preferences toward the anticipated mechanism of protein domain movements are predictable based on the examination of just the ligand‐free structural form. These results have applications to protein structure prediction, particularly in the context of protein domain assembly, if additional information concerning ligand binding is exploited. Proteins 2008. © 2007 Wiley‐Liss, Inc.

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Nucleotide-Induced Conformational Changes in an Isolated Escherichia coli DNA Polymerase III Clamp Loader Subunit

Cited by 31 publications

References 51 publications

Structural analysis of the inactive state of the Escherichia coli DNA polymerase clamp-loader complex

Structural analysis of the inactive state of the Escherichia coli DNA polymerase clamp-loader complex

Review: The lord of the rings: Structure and mechanism of the sliding clamp loader

What is the relationship between the global structures of apo and holo proteins?

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