Quantitative Conformational Analysis of Functionally Important Electrostatic Interactions in the Intrinsically Disordered Region of Delta Subunit of Bacterial RNA Polymerase

Kubáň, Vojtěch; Srb, Pavel; Štégnerová, Hana; Padrta, Petr; Zachrdla, Milan; Jaseňáková, Zuzana; Šanderová, Hana; Vítovská, Dragana; Krásný, Libor; Kovaĺ, T.; Dohnálek, Jan; Ziemska-Legięcka, Joanna; Grynberg, Marcin; Jarnot, Patryk; Gruca, Aleksandra; Jensen, Malene Ringkjøbing; Blackledge, Martin; Žı́dek, Lukáš

doi:10.1021/jacs.9b07837

Cited by 16 publications

(11 citation statements)

References 52 publications

(83 reference statements)

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“…8). PREs over the entire ANP32A molecule (folded and unfolded domains) were calculated for each of the conformers calculated for each complex, and these conformations were used as a basis set from which ensembles were selected using the ASTEROIDS approach 36,58 . Ensemble size was estimated on the basis of direct and cross-validated PRE profiles (60 conformers were used for both h and av complexes).…”

Section: Methodsmentioning

confidence: 99%

Molecular basis of host-adaptation interactions between influenza virus polymerase PB2 subunit and ANP32A

et al. 2020

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Avian influenza polymerase undergoes host adaptation in order to efficiently replicate in human cells. Adaptive mutants are localised on the C-terminal (627-NLS) domains of the PB2 subunit. In particular, mutation of PB2 residue 627 from E to K rescues polymerase activity in mammalian cells. A host transcription regulator ANP32A, comprising a long Cterminal intrinsically disordered domain (IDD), is responsible for this adaptation. Human ANP32A IDD lacks a 33 residue insertion compared to avian ANP32A, and this deletion restricts avian influenza polymerase activity. We used NMR to determine conformational ensembles of E627 and K627 forms of 627-NLS of PB2 in complex with avian and human ANP32A. Human ANP32A IDD transiently binds to the 627 domain, exploiting multivalency to maximise affinity. E627 interrupts the polyvalency of the interaction, an effect compensated by an avian-unique motif in the IDD. The observed binding mode is maintained in the context of heterotrimeric influenza polymerase, placing ANP32A in the immediate vicinity of known host-adaptive PB2 mutants.

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Section: Methodsmentioning

confidence: 99%

Molecular basis of host-adaptation interactions between influenza virus polymerase PB2 subunit and ANP32A

et al. 2020

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“…The combined analysis thus results in an accurate, integrated description of the ensemble of states sampled by both wild-type and mutant protein in solution, providing insight into the impact of the electrostatic charge distribution on local and long-range conformational behavior. 156 Interestingly, the loss of longrange contacts induced by mutagenesis influences cell fitness and transcription efficiency in vitro. While the complete knockout of the delta subunit makes transcription too fast and insensitive to regulation by initiating nucleoside triphosphates, the mutation disrupting long-range contacts has the opposite effect: it inhibits transcription from promoters that form unstable complexes with RNA polymerase.…”

Section: Accurate Mapping Of the Conformational Landscape Of Idpsmentioning

confidence: 99%

NMR Provides Unique Insight into the Functional Dynamics and Interactions of Intrinsically Disordered Proteins

et al. 2022

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Intrinsically disordered proteins are ubiquitous throughout all known proteomes, playing essential roles in all aspects of cellular and extracellular biochemistry. To understand their function, it is necessary to determine their structural and dynamic behavior and to describe the physical chemistry of their interaction trajectories. Nuclear magnetic resonance is perfectly adapted to this task, providing ensemble averaged structural and dynamic parameters that report on each assigned resonance in the molecule, unveiling otherwise inaccessible insight into the reaction kinetics and thermodynamics that are essential for function. In this review, we describe recent applications of NMR-based approaches to understanding the conformational energy landscape, the nature and time scales of local and long-range dynamics and how they depend on the environment, even in the cell. Finally, we illustrate the ability of NMR to uncover the mechanistic basis of functional disordered molecular assemblies that are important for human health.

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“…NMR chemical shifts (CS), residual dipolar couplings (RDCs), scalar couplings as well as paramagnetic relaxation enhancements (PREs) have, in the past, been used to derive conformational ensembles describing IDPs (reviewed in [ 11 ]). Those parameters have often been used in conjunction with small angle scattering techniques, providing additional information on the overall extension of the disordered protein ensemble [ 42 , 46 , 88 , 121 , 122 , 123 ]. Single molecule fluorescence, on the other hand, and in particular smFRET, providing access to specific distances in the protein chain due to site specific attachment of the fluorophores, has mainly obtained distances assuming the behavior of the protein chain according to a polymer chain model [ 15 , 18 , 98 , 124 ].…”

Section: Towards a Quantitative Combination Of Smfret And Nmrmentioning

confidence: 99%

Synergies of Single Molecule Fluorescence and NMR for the Study of Intrinsically Disordered Proteins

2021

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Single molecule fluorescence and nuclear magnetic resonance spectroscopy (NMR) are two very powerful techniques for the analysis of intrinsically disordered proteins (IDPs). Both techniques have individually made major contributions to deciphering the complex properties of IDPs and their interactions, and it has become evident that they can provide very complementary views on the distance-dynamics relationships of IDP systems. We now review the first approaches using both NMR and single molecule fluorescence to decipher the molecular properties of IDPs and their interactions. We shed light on how these two techniques were employed synergistically for multidomain proteins harboring intrinsically disordered linkers, for veritable IDPs, but also for liquid–liquid phase separated systems. Additionally, we provide insights into the first approaches to use single molecule Förster resonance energy transfer (FRET) and NMR for the description of multiconformational models of IDPs.

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Quantitative Conformational Analysis of Functionally Important Electrostatic Interactions in the Intrinsically Disordered Region of Delta Subunit of Bacterial RNA Polymerase

Cited by 16 publications

References 52 publications

Molecular basis of host-adaptation interactions between influenza virus polymerase PB2 subunit and ANP32A

Molecular basis of host-adaptation interactions between influenza virus polymerase PB2 subunit and ANP32A

NMR Provides Unique Insight into the Functional Dynamics and Interactions of Intrinsically Disordered Proteins

Synergies of Single Molecule Fluorescence and NMR for the Study of Intrinsically Disordered Proteins

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