Taking a molecular motor for a spin: helicase mechanism studied by spin labeling and PELDOR

Constantinescu-Aruxandei, Diana; Petrovic-Stojanovska, Biljana; Schiemann, Olav; Naismith, James H.; White, Malcolm F.

doi:10.1093/nar/gkv1373

Cited by 16 publications

(19 citation statements)

References 60 publications

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“…Remarkably the structure of SaXPD with HD2 missing adopts the same closed state as the full-length protein, consistent with its stability, recently highlighted by spin labelling and mutational analysis of residues at this interface in TaXPD ( 48 ). Moreover, it was previously shown ( 12 , 13 ) that the conformation of the Arch domain was not affected by the 4FeS domain being disordered and thus by the disrupted interface between the two (PDB IDs: 3CRW and 2VL7).…”

Section: Discussionsupporting

confidence: 73%

“…Yet all structures show that these domains make a number of polar and hydrophobic interactions that hold together the domains. Moreover, a spin labelling/PELDOR study of TaXPD yielded no evidence for opening at the interface between the Arch and 4FeS domain ( 48 ). Since XPD binds ssDNA within a repair bubble without a free DNA end, either the protein has to undergo a large conformational change separating the two domains or the DNA does not pass through the pore.…”

Section: Discussionmentioning

confidence: 99%

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Mechanism of DNA loading by the DNA repair helicase XPD

Constantinescu-Aruxandei¹,

Petrovic-Stojanovska²,

Penedo³

et al. 2016

Nucleic Acids Res

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The xeroderma pigmentosum group D (XPD) helicase is a component of the transcription factor IIH complex in eukaryotes and plays an essential role in DNA repair in the nucleotide excision repair pathway. XPD is a 5′ to 3′ helicase with an essential iron–sulfur cluster. Structural and biochemical studies of the monomeric archaeal XPD homologues have aided a mechanistic understanding of this important class of helicase, but several important questions remain open. In particular, the mechanism for DNA loading, which is assumed to require large protein conformational change, is not fully understood. Here, DNA binding by the archaeal XPD helicase from Thermoplasma acidophilum has been investigated using a combination of crystallography, cross-linking, modified substrates and biochemical assays. The data are consistent with an initial tight binding of ssDNA to helicase domain 2, followed by transient opening of the interface between the Arch and 4FeS domains, allowing access to a second binding site on helicase domain 1 that directs DNA through the pore. A crystal structure of XPD from Sulfolobus acidocaldiarius that lacks helicase domain 2 has an otherwise unperturbed structure, emphasizing the stability of the interface between the Arch and 4FeS domains in XPD.

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Section: Discussionsupporting

confidence: 73%

Section: Discussionmentioning

confidence: 99%

Mechanism of DNA loading by the DNA repair helicase XPD

Constantinescu-Aruxandei¹,

Petrovic-Stojanovska²,

Penedo³

et al. 2016

Nucleic Acids Res

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“…Based on the Anfinsen dogma, it became customary to explain function in terms of a single conformation or of well-defined transitions between a few conformations defined at atomic resolution. While this is certainly a reasonable approximation in some cases [75][76][77], availability of distance distributions demonstrates that rather often conformation transitions are coupled to order-disorder transitions or are shifts in disorder equilibria [39,[78][79][80][81][82][83][84][85][86][87][88][89][90][91][92][93][94]. Among the systems addressed by PDS to date, the fraction where at least one state is genuinely disordered is surprisingly large.…”

Section: A Fuzzy Relation Of Structure To Functionmentioning

confidence: 99%

“…However, in the Toc34 GTPase homodimer, the GDP-bound state is tight and ordered and the GTB-bound state disordered [85]. In helicase PcrA from superfamily 1, the two motor domains are flexible with respect to each other in the apo-and ADP-bound states, while ATP-analogue binding brings them closer together and tightens the conformational bundle [94]. DNA binding further rigidifies the motor domains.…”

Section: A Fuzzy Relation Of Structure To Functionmentioning

confidence: 99%

The contribution of modern EPR to structural biology

Jeschke

2018

Emerging Topics in Life Sciences

112

133

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Electron paramagnetic resonance (EPR) spectroscopy combined with site-directed spin labelling is applicable to biomolecules and their complexes irrespective of system size and in a broad range of environments. Neither short-range nor long-range order is required to obtain structural restraints on accessibility of sites to water or oxygen, on secondary structure, and on distances between sites. Many of the experiments characterize a static ensemble obtained by shock-freezing. Compared with characterizing the dynamic ensemble at ambient temperature, analysis is simplified and information loss due to overlapping timescales of measurement and system dynamics is avoided. The necessity for labelling leads to sparse restraint sets that require integration with data from other methodologies for building models. The double electron-electron resonance experiment provides distance distributions in the nanometre range that carry information not only on the mean conformation but also on the width of the native ensemble. The distribution widths are often inconsistent with Anfinsen's concept that a sequence encodes a single native conformation defined at atomic resolution under physiological conditions.

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“…1,2 This technique is most widely applied to investigate proteins and protein complexes labelled at different positions with nitroxide spin labels introduced by site-directed mutagenesis. 3–7 …”

Section: Introductionmentioning

confidence: 99%

Coherent pump pulses in Double Electron Electron Resonance spectroscopy

Tait

Stoll

2016

Phys. Chem. Chem. Phys.

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The recent introduction of shaped pulses to Double Electron Electron Resonance (DEER) spectroscopy has led to significant enhancements in sensitivity through increased excitation bandwidths and improved control over spin dynamics. The application of DEER has so far relied on the presence of an incoherent pump channel to average out most undesired coherent effects of the pump pulse(s) on the observer spins. However, in fully coherent EPR spectrometers that are increasingly used to generate shaped pulses, the presence of coherent pump pulses means that these effects need to be explicitly considered. In this paper, we examine the effects of coherent rectangular and sech/tanh pump pulses in DEER experiments with up to three pump pulses. We show that, even in the absence of significant overlap of the observer and pump pulse excitation bandwidths, coherence transfer pathways involving both types of pulses generate spin echoes of considerable intensity. These echoes introduce artefacts, which, if not identified and removed, can easily lead to misinterpretation. We demonstrate that the observed echoes can be quantitatively modelled using a simple spin quantum dynamics approach that includes instrumental transfer functions. Based on an analysis of the echo crossing artefacts, we propose efficient phase cycling schemes for their suppression. This enables the use of advanced DEER experiments, characterized by high sensitivity and increased accuracy for long-distance measurements, on novel fully coherent EPR spectrometers.

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Taking a molecular motor for a spin: helicase mechanism studied by spin labeling and PELDOR

Cited by 16 publications

References 60 publications

Mechanism of DNA loading by the DNA repair helicase XPD

Mechanism of DNA loading by the DNA repair helicase XPD

The contribution of modern EPR to structural biology

Coherent pump pulses in Double Electron Electron Resonance spectroscopy

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