Structure of the BamC Two-Domain Protein Obtained by Rosetta with a Limited NMR Data Set

Warner, Lisa; Varga, Krisztina; Lange, Oliver; Baker, Susan L.; Baker, David C.; Sousa, Marcelo C.; Pardi, Arthur

doi:10.1016/j.jmb.2011.05.022

Cited by 45 publications

(40 citation statements)

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“…For the Rosetta methodology, a very limited set of NMR data, which are too sparse for conventional methods, is sufficient to accurately determine solution structures of proteins up to 40 kDa (25)(26)(27)(28). In fact, the NMR data serve only to guide conformational search toward the lowest-energy conformations in the folding landscape, whereas the details of the solution structures are determined by the physical chemistry implicit in the Rosetta all-atom energy function (25)(26)(27)(28). We calculated 2,000 Rosetta structures and selected the five lowest-energy structures for further analysis (Fig.…”

Section: Resultsmentioning

confidence: 99%

TDP-43 N terminus encodes a novel ubiquitin-like fold and its unfolded form in equilibrium that can be shifted by binding to ssDNA

Qin¹,

Lim²,

Wei

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

133

213

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Transactivation response element (TAR) DNA-binding protein 43 (TDP-43) is the principal component of ubiquitinated inclusions characteristic of most forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia-frontotemporal lobar degeneration with TDP-43-positive inclusions (FTLD-TDP), as well as an increasing spectrum of other neurodegenerative diseases. Previous structural and functional studies on TDP-43 have been mostly focused on its recognized domains. Very recently, however, its extreme N terminus was identified to be a double-edged sword indispensable for both physiology and proteinopathy, but thus far its structure remains unknown due to the severe aggregation. Here as facilitated by our previous discovery that protein aggregation can be significantly minimized by reducing salt concentrations, by circular dichroism and NMR spectroscopy we revealed that the TDP-43 N terminus encodes a well-folded structure in concentration-dependent equilibrium with its unfolded form. Despite previous failure in detecting any sequence homology to ubiquitin, the folded state was determined to adopt a novel ubiquitin-like fold by the CS-Rosetta program with NMR chemical shifts and 78 unambiguous longrange nuclear Overhauser effect (NOE) constraints. Remarkably, this ubiquitin-like fold could bind ssDNA, and the binding shifted the conformational equilibrium toward reducing the unfolded population. To the best of our knowledge, the TDP-43 N terminus represents the first ubiquitin-like fold capable of directly binding nucleic acid. Our results provide a molecular mechanism rationalizing the functional dichotomy of TDP-43 and might also shed light on the formation and dynamics of cellular ribonucleoprotein granules, which have been recently linked to ALS pathogenesis. As a consequence, one therapeutic strategy for TDP-43-causing diseases might be to stabilize its ubiquitin-like fold by ssDNA or designed molecules. 1, 2). Since then, numerous studies have confirmed that TDP43 protein is mechanistically linked to neurodegeneration (3, 4). TDP43 is a 414-residue protein that has been previously recognized to be composed of a nuclear localization signal (NLS), two RNA recognition motifs (RRM1 and RRM2) hosting a nuclear export signal (NES), and C-terminal glycine-rich prion-like domain (Fig. 1A). The NLS and NES regulate the shuttling of TDP-43 between the nucleus and the cytoplasm (5), whereas the RRM1 and RRM2 are responsible for binding to nucleic acids including single-or double-stranded DNA/RNA (5-8). The prion-like domain mediates protein-protein interactions between TDP-43 and other hnRNP members (9), which also hosts most known ALS-associated TDP-43 mutations.TDP43 is an aggregation-prone protein (1-4, 10-14), and its abnormal aggregation has been found in ∼97% ALS and ∼45% frontotemporal dementia (FTD) patients. Additionally, TDP-43 immunoreactive inclusions have also been observed in an increasing spectrum of other neurodegenerative disorders, which include ALS/parkinsonism-dementia complex of Guam, Alzhei...

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Section: Resultsmentioning

confidence: 99%

TDP-43 N terminus encodes a novel ubiquitin-like fold and its unfolded form in equilibrium that can be shifted by binding to ssDNA

Qin¹,

Lim²,

Wei

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

133

213

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“…For two targets (the N-and C-terminal domains of BamC), collaborators shared their data (25), and for two additional targets [sensory rhodopsin (26) and MBP (4)], the data were previously published. The latter two restraint sets were filtered to include only those restraints that would be obtained using ILV-methyl protonated samples.…”

Section: Resultsmentioning

confidence: 99%

“…The C α -rmsd to the corresponding reference structures range from 1.1-3.9 Å (Table 2 and SI Appendix, Table S5 for restriction to converged regions). The RASREC CS-Rosetta NMR structures of both domains of BamC were published previously (25). Subsequently, the X-ray structures of both domains became available (31).…”

Section: Determination Of Previously Unsolved Structures By Rasrec Csmentioning

confidence: 99%

Determination of solution structures of proteins up to 40 kDa using CS-Rosetta with sparse NMR data from deuterated samples

Lange

Rossi

Sgourakis

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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202

232

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We have developed an approach for determining NMR structures of proteins over 20 kDa that utilizes sparse distance restraints obtained using transverse relaxation optimized spectroscopy experiments on perdeuterated samples to guide RASREC Rosetta NMR structure calculations. The method was tested on 11 proteins ranging from 15 to 40 kDa, seven of which were previously unsolved. The RASREC Rosetta models were in good agreement with models obtained using traditional NMR methods with larger restraint sets. In five cases X-ray structures were determined or were available, allowing comparison of the accuracy of the Rosetta models and conventional NMR models. In all five cases, the Rosetta models were more similar to the X-ray structures over both the backbone and side-chain conformations than the "best effort" structures determined by conventional methods. The incorporation of sparse distance restraints into RASREC Rosetta allows routine determination of high-quality solution NMR structures for proteins up to 40 kDa, and should be broadly useful in structural biology.nuclear magnetic resonance | sparse data | maltose binding protein | structural genomics | genetic algorithms A dvances in hardware, sample preparation, pulse sequence development, and refinement techniques have expanded the size and complexity of proteins accessible to structure determination by solution-state NMR to include proteins that, until recently, were exclusively the realm of X-ray crystallography (1-3). However, despite a number of landmark studies (4-7), only a small percentage of structures solved by NMR and deposited in the Protein Data Bank exceed 20 kDa in molecular weight. Larger structures need to be assembled by combining structural information from individual domains, and require additional techniques to elucidate the spatial arrangement, such as shape fitting (5) and/or paramagnetic restraints (8).The 20-kDa general limit coincides with the two fundamental problems in solution-state NMR: resonance overlap and progressive increase in the transverse relaxation rate (1∕T 2 ). As the size of a molecule increases, so does the rotational correlation time and, consequently, the efficiency of 1 H-1 H relaxation mechanisms. One way to suppress these effects is to incorporate deuterium into the protein sample, diluting the 1 H-1 H relaxation networks and increasing 13 C and 15 N relaxation times, resulting in sharper line widths and dramatic improvement of the signalto-noise ratios (2, 9, 10). Perdeuteration is generally required for studies of larger proteins (11-14), particularly membrane proteins (15, 16).Unfortunately, deuteration also eliminates the majority of 1 H-1 H NOEs, the main source of long-range distance information in solution-state NMR. Several methods have emerged for reintroducing protons at selected sites to function as distance probes in the structure (11,17). Methyl groups of isoleucine δ1, leucine, and valine side chains are straightforward to label with 13 C and 1 H isotopes in an otherwise deuterated protein sample (12, ...

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“…BamC can be divided into three domains: the unstructured region at the far N terminus of the protein (BamC U ), the N-terminal domain (BamC N ), and the C-terminal domain (BamC C ) (13,(21)(22)(23). The truncated forms of BamC created for this study are as follows: 1) BamC N (residues 99 -217), 2) BamC C (residues 220 -344), 3) BamC NC (both the N-and C-terminal domains, residues 94 -344), and 4) BamC UN (the N-terminal unstructured region followed by the N-terminal domain, residues 26 -217).…”

Section: Resultsmentioning

confidence: 99%

Crystal Structure of β-Barrel Assembly Machinery BamCD Protein Complex

Kim

Aulakh

Paetzel

2011

Journal of Biological Chemistry

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Background:The Escherichia coli ␤-barrel assembly machinery complex (BamABCDE) facilitates outer membrane protein assembly. Results:The unstructured N terminus of BamC binds and blocks the proposed substrate-binding pocket on BamD. Conclusion:The unstructured N terminus of BamC is essential for BamCD interaction. Significance: The first Bam lipoprotein complex structure reveals how BamC and BamD interact.

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Structure of the BamC Two-Domain Protein Obtained by Rosetta with a Limited NMR Data Set

Cited by 45 publications

References 47 publications

TDP-43 N terminus encodes a novel ubiquitin-like fold and its unfolded form in equilibrium that can be shifted by binding to ssDNA

TDP-43 N terminus encodes a novel ubiquitin-like fold and its unfolded form in equilibrium that can be shifted by binding to ssDNA

Determination of solution structures of proteins up to 40 kDa using CS-Rosetta with sparse NMR data from deuterated samples

Crystal Structure of β-Barrel Assembly Machinery BamCD Protein Complex

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