Dataset Information

Utilization of paramagnetic relaxation enhancements for structural analysis of actin-binding proteins in complex with actin.

ABSTRACT: Actin cytoskeleton dynamics are controlled by various actin binding proteins (ABPs) that modulate the polymerization of the monomeric G-actin and the depolymerization of filamentous F-actin. Although revealing the structures of the actin/ABP complexes is crucial to understand how the ABPs regulate actin dynamics, the X-ray crystallography and cryoEM methods are inadequate to apply for the ABPs that interact with G- or F-actin with lower affinity or multiple binding modes. In this study, we aimed to establish the alternative method to build a structural model of G-actin/ABP complexes, utilizing the paramagnetic relaxation enhancement (PRE) experiments. Thymosin ?4 (T?4) was used as a test case for validation, since its structure in complex with G-actin was reported recently. Recombinantly expressed G-actin, containing a cysteine mutation, was conjugated with a nitroxyl spin label at the specific site. Based on the intensity ratio of the (1)H-(15)N HSQC spectra of T?4 in the complex with G-actin in the paramagnetic and diamagnetic states, the distances between the amide groups of T?4 and the spin label of G-actin were estimated. Using the PRE-derived distance constraints, we were able to compute a well-converged docking structure of the G-actin/T?4 complex that shows great accordance with the reference structure.

SUBMITTER: Huang S

PROVIDER: S-EPMC5031973 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Utilization of paramagnetic relaxation enhancements for structural analysis of actin-binding proteins in complex with actin.

Huang Shuxian S Umemoto Ryo R Tamura Yuki Y Kofuku Yutaka Y Uyeda Taro Q P TQ Nishida Noritaka N Shimada Ichio I

Scientific reports 20160922

Actin cytoskeleton dynamics are controlled by various actin binding proteins (ABPs) that modulate the polymerization of the monomeric G-actin and the depolymerization of filamentous F-actin. Although revealing the structures of the actin/ABP complexes is crucial to understand how the ABPs regulate actin dynamics, the X-ray crystallography and cryoEM methods are inadequate to apply for the ABPs that interact with G- or F-actin with lower affinity or multiple binding modes. In this study, we aimed ...[more]

PMID: 27654858

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Project description:The human DNA repair enzyme uracil DNA glycosylase (hUNG) locates and excises rare uracil bases that arise in DNA from cytosine deamination or through dUTP incorporation by DNA polymerases. Previous NMR studies of hUNG have revealed millisecond time scale dynamic transitions in the enzyme-nonspecific DNA complex, but not the free enzyme, that were ascribed to a reversible clamping motion of the enzyme as it scans along short regions of duplex DNA in its search for uracil. Here we further probe the properties of the nonspecific DNA binding surface of {(2)H(12)C}{(15)N}-labeled hUNG using a neutral chelate of a paramagnetic Gd(3+) cosolute (Gd(HP-DO3A)). Overall, the measured paramagnetic relaxation enhancements (PREs) on R(2) of the backbone amide protons for free hUNG and its DNA complex were in good agreement with those calculated based on their relative exposure observed in the crystal structures of both enzyme forms. However, the calculated PREs systematically underestimated the experimental PREs by large amounts in discrete regions implicated in DNA recognition and catalysis: active site loops involved in DNA recognition (268-274, 246-250), the uracil binding pocket (143-148, 169-170), a transient extrahelical base binding site (214-216), and a remote hinge region (129-132) implicated in dynamic clamping. These reactive hot spots were not correlated with structural, hydrophobic, or solvent exchange properties that might be common to these regions, leaving the possibility that the effects arise from dynamic sampling of exposed conformations that are distinct from the static structures. Consistent with this suggestion, the above regions have been previously shown to be flexible based on relaxation dispersion measurements and course-grained normal-mode analysis. A model is suggested where the intrinsic dynamic properties of these regions allows sampling of transient conformations where the backbone amide groups have greater average exposure to the cosolute as compared to the static structures. We conclude that PREs derived from the paramagnetic cosolute reveal dynamic hot spots in hUNG and that these regions are highly correlated with substrate binding and recognition.

Dataset Information

Utilization of paramagnetic relaxation enhancements for structural analysis of actin-binding proteins in complex with actin.

Publications

Utilization of paramagnetic relaxation enhancements for structural analysis of actin-binding proteins in complex with actin.

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