Protonless NMR Experiments for Sequence-Specific Assignment of Backbone Nuclei in Unfolded Proteins

Bermel, Wolfgang; Bertini, Ivano; Felli, Isabella C.; Lee, Yong Min; Luchinat, Claudio; Pierattelli, Roberta

doi:10.1021/ja0582206

Cited by 177 publications

(193 citation statements)

References 33 publications

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“…The prediction that the WIP N fragment is disordered was confirmed by its CD curve and NMR fingerprint spectra. Nevertheless, in this study well established 13 C-detected experiments [22,62] [53] and are able as shown here to identify regions of transient structure. The interpretation of RDCs for IDPs under anisotropic conditions is challenging, since normally a folded structure is required for analysis of such data.…”

Section: Discussionsupporting

confidence: 66%

New insights into the role of the disordered WIP N‐terminal domain revealed by NMR structural characterization

et al. 2015

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WASp-interacting protein (WIP) is an intrinsically disordered 503-residue polypeptide with a key role in actin polymerization in activated T cells. Its interaction with actin is mediated by a pair of conserved actin binding motifs (ABMs) at the WIP N-terminus, a domain that has not been investigated in its unbound form. Here we use NMR to investigate the biophysical behavior of the N-terminal ABM in WIP using protonless 13 C 0 -detected spectroscopy. Secondary chemical shifts, residual dipolar couplings and temperature effects identify residual structure throughout the ABM, which exhibits transient helical and b-strand character for residues 30-42 and 44-62, respectively. These observed structural propensities echo the structure observed in the actin-bound state of the ABM. Furthermore, residues preceding the canonical ABM (17-25) and conserved among WIP-related proteins exhibit transient b-strand character, suggesting that the WIP N interaction epitope extends towards the N-terminal polyproline motif. This suggests a possible role for this region in mediating the WIP interaction with polyproline binders such as profilin. In revealing these features of the WIP ABM this study demonstrates the unique ability of NMR in characterizing unstructured domains and provides necessary information for further investigation of WIP-mediated protein-protein interactions.

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Section: Discussionsupporting

confidence: 66%

New insights into the role of the disordered WIP N‐terminal domain revealed by NMR structural characterization

et al. 2015

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“…The first step toward development of small molecule inhibitors targeting MLL-menin requires detailed characterization of this interaction. In this work we found that (i) menin interacts with MLL with a low nanomolar affinity (K d ϳ 10 nM); (ii) two short fragments of MLL, MBM1 (MLL 4 -15 ), and MBM2 (MLL [23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40] ), are involved in the interaction with menin; (iii) MBM1 represents the high affinity menin binding motif; (iv) in competition experiments, the MBM1 peptide is capable of causing efficient displacement of MLL comprising MBM1 and MBM2 from menin; (v) MBM1 binds to menin in an extended conformation, and (vi) a set of hydrophobic resi-dues (Phe 9 , Pro 10 , and Pro 13) has the most significant contribution to the binding affinity.…”

Section: Discussionmentioning

confidence: 99%

“…To examine whether MBM1 and MBM2 represent two independent binding motifs, we designed two fluorescein-labeled peptides: MLL 4 -15 (MBM1; MLL residues 1-3 are not involved in binding to menin as shown by ITC experiments, see below and Table 1) and MLL [23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40] (MBM2). Binding of these peptides to menin was determined by fluorescence polarization (FP).…”

Section: Menin Interacts With the N Terminus Of Mll With Highmentioning

confidence: 99%

Molecular Basis of the Mixed Lineage Leukemia-Menin Interaction

Grembecka

Belcher

Hartley

et al. 2010

Journal of Biological Chemistry

105

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Chromosomal translocations targeting the mixed lineage leukemia (MLL) gene result in MLL fusion proteins that are found in aggressive human acute leukemias. Disruption of MLL by such translocations leads to overexpression of Hox genes, resulting in a blockage of hematopoietic differentiation that ultimately leads to leukemia. Menin, which directly binds MLL, has been identified as an essential oncogenic co-factor required for the leukemogenic activity of MLL fusion proteins. Here, we characterize the molecular basis of the MLL-menin interaction. Using 13 C-detected NMR experiments, we have mapped the residues within the intrinsically unstructured fragment of MLL that are required for binding to menin. Interestingly, we found that MLL interacts with menin with a nanomolar affinity (K d ϳ 10 nM) through two motifs, MBM1 and MBM2 (menin binding motifs 1 and 2). These motifs are located within the N-terminal 43-amino acid fragment of MLL, and the MBM1 represents a high affinity binding motif. Using alanine scanning mutagenesis of MBM1, we found that the hydrophobic residues Phe 9 , Pro 10 , and Pro 13 are most critical for binding. Furthermore, based on exchange-transferred nuclear Overhauser effect measurements, we established that MBM1 binds to menin in an extended conformation. In a series of competition experiments we showed that a peptide corresponding to MBM1 efficiently dissociates the menin-MLL complex. Altogether, our work establishes the molecular basis of the menin interaction with MLL and MLL fusion proteins and provides the necessary foundation for development of small molecule inhibitors targeting this interaction in leukemias with MLL translocations. Chromosomal translocations involving the mixed lineage leukemia (MLL)3 gene result in human acute myeloid and lymphoid leukemias, affecting both children and adults (1, 2). Fusion of MLL with one of 60 partner genes forms chimeric oncogenes encoding MLL fusion proteins, which results in enhanced proliferation and blockage of blood cell differentiation ultimately leading to the development of acute leukemia (3). Translocations of MLL are particularly prevalent in infants with acute myeloid leukemia and acute lymphoblastic leukemia and constitute up to 80% of all infant acute leukemia cases (4). Patients with leukemias harboring MLL translocations have a very poor prognosis using available therapies (20% event-free survival at 3 years), and it is clear that novel targeted therapies are urgently needed to treat these leukemias (3, 5). MLL belongs to the evolutionary conserved family of TRX (Drosophila Trithorax) proteins that positively regulate gene expression during development (6 -8). MLL has been shown to associate with promoters of Ͼ5000 human genes, suggesting that it might play a global role in transcription (9). MLL is an important regulator of Hox gene expression, which is required for normal hematopoiesis (10). Disruption of MLL by chromosomal translocations up-regulates expression of Hox genes, including Hoxa7, Hoxa9, and the Hox cofactor Meis1, r...

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“…This motivated the use of HR-detection and direct carbon detection for the assignment of IDPs. 7,10,11 However, when we reduced the pH to 6.0 the 1 H/ 15 N signals of Tau were not attenuated at higher temperatures but rather increased by about 40% due to reduced amide proton exchange, more rapid tumbling, and improved relaxation properties. We thus asked if the favorable relaxation properties help to acquire very highdimensional NMR spectra.…”

Section: Introductionmentioning

confidence: 90%

Automatic Assignment of the Intrinsically Disordered Protein Tau with 441-Residues

et al. 2010

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Abstract:Intrinsically disordered proteins carry out many important functions in the cell. However, the lack of an ordered structure causes dramatic signal overlap and complicates the NMR-based characterization of their structure and dynamics. Here we demonstrate that the resonance assignment of 441-residue Tau and its smaller isoforms, htau24 (383 residues) and htau23 (352 residues), three prototypes of intrinsically disordered proteins, which bind to microtubules and play a key role in Alzheimer disease, can be obtained within 5 days by a combination of sevendimensional NMR spectra with optimized methods for automatic assignment. Chemical shift differences between the three isoforms provide evidence for the global folding of Tau in solution.In the past decade intrinsically disordered proteins (IDPs) obtained significant attention as more than 30% of eukaryotic proteins comprise unstructured regions larger than 50 consecutive residues. 1 A hallmark of IDPs is the lack of a well-structured threedimensional fold. 2 Despite the absence of a rigid secondary or tertiary structure, IDPs carry out many important functions in the cell, for example the regulation of transcription and translation, the storage of small molecules, and the regulation of the self-assembly of large multiprotein complexes. 3 Tau, a prototype of IDPs, binds to microtubules and plays a key role in Alzheimer disease. 4 Since disordered proteins tend to be highly flexible and have variable conformations, they have mostly not been amenable for structure analysis by crystallography. Thus NMR spectroscopy is the only method that allows a description of their conformations and dynamics with high resolution. 5 The lack of an ordered structure, however, causes dramatic signal overlap limiting previous NMR investigations of IDPs to around 200 residues (e.g., refs 6, 7). For the largest Tau isoform with 441 residues, htau40 ( Figure 1), the number of overlapping signals is 3.5-fold higher than that in the largest currently assigned globular protein. 8 In addition, the large number of proline residues, the strongly repetitive primary sequence in the repeat domains, and the complexity of the sequence further complicate the resonance assignment. 8,9 Only as part of an ongoing project taking several months to years and building on the assignment of smaller fragments of Tau in a "divide-andconquer" strategy, we were recently able to assign the backbone resonances of the full-length protein. 8 Here we demonstrate that the resonance assignment of 441-residue Tau and its smaller isoforms, htau24 (383 residues) and htau23 (352 residues) (Figure 1), can be obtained within 5 days by a combination of seven-dimensional NMR spectra with optimized methods for automatic assignment. Chemical shift differences between the three isoforms provide evidence for the global folding of Tau in solution.Due to the lack of stable hydrogen bonds, NMR spectra of IDPs are sensitive to solvent exchange. At pH 6.8 an increase in temperature from 5 to 35°C results in the broadening of ab...

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Protonless NMR Experiments for Sequence-Specific Assignment of Backbone Nuclei in Unfolded Proteins

Cited by 177 publications

References 33 publications

New insights into the role of the disordered WIP N‐terminal domain revealed by NMR structural characterization

New insights into the role of the disordered WIP N‐terminal domain revealed by NMR structural characterization

Molecular Basis of the Mixed Lineage Leukemia-Menin Interaction

Automatic Assignment of the Intrinsically Disordered Protein Tau with 441-Residues

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