Flexible Tethering of ASPP Proteins Facilitates PP-1c Catalysis

Zhou, Yeyun; Millott, Robyn; Kim, Hyeong-Jin; Peng, Shiyun; Edwards, Ross A.; Skene-Arnold, Tamara D.; Hammel, Michal; Lees‐Miller, Susan P.; Tainer, John A.; Holmes, Charles F.B.; Glover, J. N. Mark

doi:10.1016/j.str.2019.07.012

Cited by 25 publications

(25 citation statements)

References 80 publications

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“…However, SAXS data provides information on the structure adopted in arguably more physiologically and functionally relevant conditions. For example, recent comparisons of SEC‐SAXS data taken across the peak unveils functional DNA repair complex conformations in solution can sample the compact crystal structure conformations, but these interconvert with more extended conformations that enable the functional release of contacts …”

Section: Discussionmentioning

confidence: 99%

“…For example, recent comparisons of SEC-SAXS data taken across the peak unveils functional DNA repair complex conformations in solution can sample the compact crystal structure conformations, but these interconvert with more extended conformations that enable the functional release of contacts. 38 In the short term, moving away from crystallography as the gold standard, which has formed the backbone of CASP, is likely unwise.…”

Section: Solution Structure Vs Crystal Structurementioning

confidence: 99%

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Small angle X‐ray scattering‐assisted protein structure prediction in CASP13 and emergence of solution structure differences

et al. 2019

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Small angle X‐ray scattering (SAXS) measures comprehensive distance information on a protein's structure, which can constrain and guide computational structure prediction algorithms. Here, we evaluate structure predictions of 11 monomeric and oligomeric proteins for which SAXS data were collected and provided to predictors in the 13th round of the Critical Assessment of protein Structure Prediction (CASP13). The category for SAXS‐assisted predictions made gains in certain areas for CASP13 compared to CASP12. Improvements included higher quality data with size exclusion chromatography‐SAXS (SEC‐SAXS) and better selection of targets and communication of results by CASP organizers. In several cases, we can track improvements in model accuracy with use of SAXS data. For hard multimeric targets where regular folding algorithms were unsuccessful, SAXS data helped predictors to build models better resembling the global shape of the target. For most models, however, no significant improvement in model accuracy at the domain level was registered from use of SAXS data, when rigorously comparing SAXS‐assisted models to the best regular server predictions. To promote future progress in this category, we identify successes, challenges, and opportunities for improved strategies in prediction, assessment, and communication of SAXS data to predictors. An important observation is that, for many targets, SAXS data were inconsistent with crystal structures, suggesting that these proteins adopt different conformation(s) in solution. This CASP13 result, if representative of PDB structures and future CASP targets, may have substantive implications for the structure training databases used for machine learning, CASP, and use of prediction models for biology.

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Section: Discussionmentioning

confidence: 99%

Section: Solution Structure Vs Crystal Structurementioning

confidence: 99%

Small angle X‐ray scattering‐assisted protein structure prediction in CASP13 and emergence of solution structure differences

et al. 2019

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“…The interaction was lost when the SH3 domain was absent or the RARL motif mutated. Conversely, the C-terminal region (Cter, region 600-828) of iASPP which included the RARL and SILK motifs 23 , the Ank repeats and the SH3 domain was sufficient for robust association of PP1 with iASPP ( Supplementary Fig. 1a).…”

Section: Results Iaspp Engages In Multiple Intramolecular and Intermomentioning

confidence: 99%

iASPP contributes to cortex rigidity, astral microtubule capture and mitotic spindle positioning

Mangon

Salaün

Bouali

et al. 2019

Preprint

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The microtubule plus-end binding protein EB1 is the core of a complex protein network which regulates microtubule dynamics during important biological processes such as cell motility and mitosis. We found that iASPP, an inhibitor of p53 and predicted regulatory subunit of the PP1 phosphatase, associates with EB1 at microtubule plus-ends via a SxIP motif. iASPP silencing or mutation of the SxIP motif led to defective microtubule capture at the leading edge of migrating cells, and at the cortex of mitotic cells leading to abnormal positioning of the mitotic spindle. These effects were recapitulated by the knockdown of Myosin-Ic (Myo1c), identified as a novel partner of iASPP. Moreover, iASPP or Myo1c knockdown cells failed to round up during mitosis because of defective cortical rigidity. We propose that iASPP, together with EB1 and Myo1c, contributes to mitotic cell cortex rigidity, allowing astral microtubule capture and appropriate positioning of the mitotic spindle.

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“…For Rad51, which acts in HDR, the functionally flexible polymerization motif lies in the linker region between domains; this made it so challenging to see correct assemblies that a thermophile was employed to define the first intact Rad51 structure and assembly 22 . Fortunately, SAXS provides an accurate measure of the solution ensemble plus the means to examine unstructured regions and to assess conformational changes and assembly states critical to DNA repair activities; this is invaluable for complementing many X‐ray, cryo‐EM, and NMR structures 1,2,16,23–26 …”

Section: Introductionmentioning

confidence: 99%

“… 22 Fortunately, SAXS provides an accurate measure of the solution ensemble plus the means to examine unstructured regions and to assess conformational changes and assembly states critical to DNA repair activities; this is invaluable for complementing many X‐ray, cryo‐EM, and NMR structures. 1 , 2 , 16 , 23 , 24 , 25 , 26 …”

Section: Introductionmentioning

confidence: 99%

X‐ray scattering reveals disordered linkers and dynamic interfaces in complexes and mechanisms for DNA double‐strand break repair impacting cell and cancer biology

Hammel

Tainer

2021

Protein Science

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Evolutionary selection ensures specificity and efficiency in dynamic metastable macromolecular machines that repair DNA damage without releasing toxic and mutagenic intermediates. Here we examine non‐homologous end joining (NHEJ) as the primary conserved DNA double‐strand break (DSB) repair process in human cells. NHEJ has exemplary key roles in networks determining the development, outcome of cancer treatments by DSB‐inducing agents, generation of antibody and T‐cell receptor diversity, and innate immune response for RNA viruses. We determine mechanistic insights into NHEJ structural biochemistry focusing upon advanced small angle X‐ray scattering (SAXS) results combined with X‐ray crystallography (MX) and cryo‐electron microscopy (cryo‐EM). SAXS coupled to atomic structures enables integrated structural biology for objective quantitative assessment of conformational ensembles and assemblies in solution, intra‐molecular distances, structural similarity, functional disorder, conformational switching, and flexibility. Importantly, NHEJ complexes in solution undergo larger allosteric transitions than seen in their cryo‐EM or MX structures. In the long‐range synaptic complex, X‐ray repair cross‐complementing 4 (XRCC4) plus XRCC4‐like‐factor (XLF) form a flexible bridge and linchpin for DNA ends bound to KU heterodimer (Ku70/80) and DNA‐PKcs (DNA‐dependent protein kinase catalytic subunit). Upon binding two DNA ends, auto‐phosphorylation opens DNA‐PKcs dimer licensing NHEJ via concerted conformational transformations of XLF‐XRCC4, XLF–Ku80, and LigIVBRCT–Ku70 interfaces. Integrated structures reveal multifunctional roles for disordered linkers and modular dynamic interfaces promoting DSB end processing and alignment into the short‐range complex for ligation by LigIV. Integrated findings define dynamic assemblies fundamental to designing separation‐of‐function mutants and allosteric inhibitors targeting conformational transitions in multifunctional complexes.

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Flexible Tethering of ASPP Proteins Facilitates PP-1c Catalysis

Cited by 25 publications

References 80 publications

Small angle X‐ray scattering‐assisted protein structure prediction in CASP13 and emergence of solution structure differences

Small angle X‐ray scattering‐assisted protein structure prediction in CASP13 and emergence of solution structure differences

iASPP contributes to cortex rigidity, astral microtubule capture and mitotic spindle positioning

X‐ray scattering reveals disordered linkers and dynamic interfaces in complexes and mechanisms for DNA double‐strand break repair impacting cell and cancer biology

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