Methyl group reorientation under ligand binding probed by pseudocontact shifts

Lescanne, Mathilde; Ahuja, Puneet; Blok, Anneloes; Timmer, Monika; Åkerud, Tomas; Ubbink, Marcellus

doi:10.1007/s10858-018-0190-5

Cited by 17 publications

(23 citation statements)

References 37 publications

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“…The STD method can also provide useful information regarding clients bound to large chaperone systems . The PCS and RDC techniques have been applied in characterizations of chaperones either in their apo‐states or in complex with ligands . Furthermore, solid‐state NMR methods have also been recently successfully applied in the study of amyloids interactions with the chaperonin GroEL system …”

Section: Discussionmentioning

confidence: 99%

Mechanisms of Chaperones as Active Assistant/Protector for Proteins: Insights from NMR Studies

et al. 2020

Chin. J. Chem.

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Molecular chaperones are diverse families of proteins that play key roles in protein homeostasis. They assist the folding of client proteins or prevent them from irreversible aggregation under stress conditions. Diverse chaperone families contribute to different aspects of protein homeostasis by interacting with a wide range of client proteins. Despite the vital roles of chaperones in cell survival, the molecular mechanisms underlying chaperone functions remain elusive, due to the non‐specificity of chaperone‐client interactions and the intrinsic flexibility of the clients. Our understanding of the chaperone functional mechanisms, especially regarding chaperone‐client interactions, has greatly expanded in recent years, thanks to the significant contribution from various NMR studies. Solution NMR methods have unique advantages in characterizing disordered protein structures, detecting weak and non‐specific interactions, and probing conformational dynamics of proteins and protein complexes, etc., and therefore are especially powerful in the studies of chaperone structure‐function relationships. In this review, we summarize some of the current knowledge of molecular chaperones, with emphasis on common features of chaperone‐client interactions and examples on a number of specific systems in which solution NMR methods were used to provide essential insights into their functional mechanisms.

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Section: Discussionmentioning

confidence: 99%

Mechanisms of Chaperones as Active Assistant/Protector for Proteins: Insights from NMR Studies

et al. 2020

Chin. J. Chem.

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“…The utility of rapid, accurate methyl assignments is highlighted by recent studies that used NOEs between an unlabeled ligand and a methyl-labeled protein as restraints to generate models of the docked complex 32,41,53,54 and PCSs to measure reorientation of methyl groups upon ligand binding. 55 In the future, MethylFLYA could be extended to incorporate paramagnetic restraints, such as PREs or PCSs, or be combined with existing software packages that predominantly rely on these restraints. 26,27 Furthermore, MethylFLYA can straightforwardly be used to assign methyl resonances in solid-state NMR spectra.…”

Section: Discussionmentioning

confidence: 99%

Structure-based methyl resonance assignment with MethylFLYA

Pritišanac

Würz

Alderson

et al. 2019

Preprint

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Isotope-labeled methyl groups provide NMR probes that can be observed in very large, otherwise deuterated systems and enable investigations of protein structure, dynamics and mechanisms.However, the assignment of resonances to specific methyls in the protein is expensive and timeconsuming, which limits the use of methyl-based NMR for large proteins. To resolve this bottleneck, methyl assignment methods have been developed. However, these remain limited regarding complete automation, computational feasibility, and/or the extent and accuracy of the assignments. Here, we present the automated MethylFLYA method for the assignment of methyl groups that is based on methyl-methyl nuclear Overhauser effect spectroscopy (NOESY) peak lists. MethylFLYA was applied to five proteins (28-358 kDa) comprising a total of 708 isotopelabeled methyl groups, of which 674 had manually determined 1 H/ 13 C reference assignments and 614 showed cross peaks in the available NOESY peak lists. MethylFLYA confidently assigned 488 methyl groups, i.e. 79% of those with NOESY data. Of these, 460 agreed with the reference, 5 were different, and 23 concerned methyls without reference assignment. For high-quality NOESY spectra, automatic NOESY peak picking followed by resonance assignment with MethylFLYA can yield results comparable to those obtained from manually prepared peak lists, indicating the feasibility of unbiased, fully automatic methyl resonance assignment starting directly from the NMR spectra. Overall, MethylFLYA assigns significantly more methyl groups than other algorithms, has an average error rate of 1%, modest runtimes of 0.4-1.2 h, and flexibility to handle arbitrary isotope labeling patterns and include data from other types of NMR spectra.

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“…The location of the Ln 3+ ion and ∆χ-tensor were calculated with Numbat [22] using the crystal structure of the MPS1 TPR domain (PDB entry, 4H7Y) [21] and the PCSs for residues 59-199. The quality (Qa) scores of PCSs were calculated as previously described [23], using the following formula.…”

Section: Nmr Measurements Assignment and Data Analysismentioning

confidence: 99%

“…We have previously speculated that the interaction of the NTE module with the TPR exerts an auto-inhibitory function to the NTE, moderating the interaction with the NDC80C complex in the outer kinetochore [6]. Subsequently, an NMR analysis including the assignment of most amide resonances (87%) of the NTE-TPR module, showed that the NTE does not adopt a defined structure, except for a short segment (residues, [14][15][16][17][18][19][20][21][22][23], which has a propensity to form a helical turn [7]. This helical fragment is required for interactions with kinetochores and forms intermolecular interactions with the TPR domain.…”

Section: Introductionmentioning

confidence: 99%

The MPS1 kinase NTE region has helical propensity and preferred conformations towards the TPR domain

Hiruma

Matsoukas

Touw

et al. 2020

Preprint

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The mitotic spindle assembly checkpoint (SAC) ensures accurate segregation of chromosomes by preventing onset of anaphase until all chromosomes are properly attached to spindle microtubules. The Monopolar spindle 1 (MPS1) kinase is one of the SAC components, localizing at unattached kinetochores by an N-terminal localization module. This module comprises a flexible NTE module and the TPR domain, which we previously characterized for their contribution to kinetochore binding. Here we discuss the conformations of the highly flexible NTE with respect to the TPR domain, using paramagnetic NMR. The distance restraints derived from paramagnetic relaxation enhancements (PREs) show that the mobile NTE can be found in proximity of a large but specific part of the surface area of the TPR domain. To sample the conformational space of the NTE in the context of the NTE-TPR module, we used the ab initio Rosetta approach supplemented by paramagnetic NMR restraints. We find that many NTE residues have a propensity to form helical structures and that the module localizes at the convex surface of the TPR domain. This work demonstrates the highly dynamic nature of the interactions between the NTE and TPR domains and it shows that the convex rather than the canonical concave TPR surface mediates interactions, leading to the auto-inhibition that the TPR exerts upon the NTE region in the context of SAC signaling. 144Total 7 132 170 309

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Methyl group reorientation under ligand binding probed by pseudocontact shifts

Cited by 17 publications

References 37 publications

Mechanisms of Chaperones as Active Assistant/Protector for Proteins: Insights from NMR Studies

Mechanisms of Chaperones as Active Assistant/Protector for Proteins: Insights from NMR Studies

Structure-based methyl resonance assignment with MethylFLYA

The MPS1 kinase NTE region has helical propensity and preferred conformations towards the TPR domain

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