The NMR Exchange Format (NEF) is a community-driven standard for representing NMR experimental data in a consistent, interoperable, and machine-readable form. Built on the STAR syntax, NEF provides a structured framework for storing and exchanging chemical shifts, peak lists, various types of structural restraints, and related metadata, thus allowing for data exchange across software platforms. By enabling direct, lossless transfer of information, NEF simplifies multi-software workflows, improves reproducibility, and supports FAIR (Findable, Accessible, Interoperable, Reusable) data principles. We describe the NEF specification, its current implementation across commonly used NMR software packages, and its application in areas including biomolecular structure determination, metabolomics, and ligand screening. Testing demonstrates that NEF can be used to exchange complete datasets between programs without loss of information or functionality. We also outline recent developments and future directions, such as inclusion of NMR relaxation data and support for non-standard residue topologies. NEFs growing adoption highlights its potential as a unifying standard for NMR data, enabling more efficient, transparent and collaborative research.

Protein kinases often rely on docking site motifs to enhance substrate interactions and facilitate phosphorylation. For example, mitogen-activated protein kinases (MAPKs) utilize D- and F-motifs, which frequently act in concert to enable bipartite substrate binding. While these motifs are known to modulate phosphorylation efficiency, their quantitative impact on target site phosphorylation within long intrinsically disordered substrates remains largely unexplored. Using NMR spectroscopy, JNK1-dependent phosphorylation of JIP1, a 450-amino acid disordered substrate, is investigated, identifying eleven phosphosites with distinct phosphorylation efficiencies. By selectively disrupting JNK1 binding to the D- and F-motifs of JIP1, the determinants of phosphorylation efficiency are uncovered. Specifically, it is found that the D-motif selectively enhances phosphorylation in the C-terminal direction in a length-dependent manner, impressively increasing the phosphorylation efficiencies of sites located at sequence distances exceeding 120 amino acids, while the F-motif primarily promotes phosphorylation of a site located immediately N-terminal to the F-motif. Additionally, docking-site-independent phosphorylation is observed, whose efficiency is dictated by the intrinsic sequence preference of JNK1, as inferred from motif scores derived from positional scanning peptide arrays. The work highlights how docking site motifs and sequence context synergistically regulate phosphorylation efficiency, emphasizing the critical role of substrate architecture in determining MAPK-mediated signaling outcomes.

The nucleocapsid protein of severe acute respiratory syndrome coronavirus 2 encapsidates the viral genome and is essential for viral function. The central disordered domain comprises a serine-arginine-rich (SR) region that is hyperphosphorylated in infected cells. This modification regulates function, although mechanistic details remain unknown. We use nuclear magnetic resonance to follow structural changes occurring during hyperphosphorylation by serine arginine protein kinase 1, glycogen synthase kinase 3, and casein kinase 1, that abolishes interaction with RNA. When eight approximately uniformly distributed sites have been phosphorylated, the SR domain binds the same interface as single-stranded RNA, resulting in complete inhibition of RNA binding. Phosphorylation by protein kinase A does not prevent RNA binding, indicating that the pattern resulting from physiologically relevant kinases is specific for inhibition. Long-range contacts between the RNA binding, linker, and dimerization domains are abrogated, phenomena possibly related to genome packaging and unpackaging. This study provides insight into the recruitment of specific host kinases to regulate viral function.

Temperate bacteriophages can enter one of two life cycles following infection of a sensitive host: the lysogenic or the lytic life cycle. The choice between the two alternative life cycles is dependent upon a tight regulation of promoters and their cognate regulatory proteins within the phage genome. We investigated the genetic switch of TP901-1, a bacteriophage of Lactococcus lactis, controlled by the CI repressor and the modulator of repression (MOR) antirepressor and their interactions with DNA. We determined the solution structure of MOR, and we solved the crystal structure of MOR in complex with the N-terminal domain of CI, revealing the structural basis of MOR inhibition of CI binding to the DNA operator sites. 15N NMR Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion and rotating frame R1ρ measurements demonstrate that MOR displays molecular recognition dynamics on two different time scales involving a repacking of aromatic residues at the interface with CI. Mutations in the CI:MOR binding interface impair complex formation in vitro, and when introduced in vivo, the bacteriophage switch is unable to choose the lytic life cycle showing that the CI:MOR complex is essential for proper functioning of the genetic switch. On the basis of sequence alignments, we show that the structural features of the MOR:CI complex are likely conserved among a larger family of bacteriophages from human pathogens implicated in transfer of antibiotic resistance.

(Developmental Cell 50, 494–508.e1–e11; August 19, 2019) As a result of an author oversight in the originally published version of this article, the author Susanne Salomon was omitted from the author list. This error has now been corrected in the article online. The authors apologize for the error and any inconvenience that may have resulted. Temporal Ordering in Endocytic Clathrin-Coated Vesicle Formation via AP2 PhosphorylationWrobel et al.Developmental CellAugust 19, 2019In BriefWrobel et al. show that phosphorylation of the mammalian endocytic AP2 adaptor causes a conformational change that allows it to efficiently bind the protein NECAP. This, in turn, recruits membrane-remodeling proteins into clathrin-coated pits, which drive their formation toward final scission from the parent membrane. Full-Text PDF Open Access

Clathrin-mediated endocytosis (CME) is key to maintaining the transmembrane protein composition of cells' limiting membranes. During mammalian CME, a reversible phosphorylation event occurs on Thr156 of the μ2 subunit of the main endocytic clathrin adaptor, AP2. We show that this phosphorylation event starts during clathrin-coated pit (CCP) initiation and increases throughout CCP lifetime. μ2Thr156 phosphorylation favors a new, cargo-bound conformation of AP2 and simultaneously creates a binding platform for the endocytic NECAP proteins but without significantly altering AP2's cargo affinity in vitro. We describe the structural bases of both. NECAP arrival at CCPs parallels that of clathrin and increases with μ2Thr156 phosphorylation. In turn, NECAP recruits drivers of late stages of CCP formation, including SNX9, via a site distinct from where NECAP binds AP2. Disruption of the different modules of this phosphorylation-based temporal regulatory system results in CCP maturation being delayed and/or stalled, hence impairing global rates of CME.

In bacteria, nucleoid associated proteins (NAPs) take part in active chromosome organization by supercoil management, three-dimensional DNA looping and direct transcriptional control. Mycobacterial integration host factor (mIHF, rv1388) is a NAP restricted to Actinobacteria and essential for survival of the human pathogen Mycobacterium tuberculosis. We show in vitro that DNA binding by mIHF strongly stabilizes the protein and increases its melting temperature. The structure obtained by Nuclear Magnetic Resonance (NMR) spectroscopy characterizes mIHF as a globular protein with a protruding alpha helix and a disordered N-terminus, similar to Streptomyces coelicolor IHF (sIHF). NMR revealed no residues of high flexibility, suggesting that mIHF is a rigid protein overall that does not undergo structural rearrangements. We show that mIHF only binds to double stranded DNA in solution, through two DNA binding sites (DBSs) similar to those identified in the X-ray structure of sIHF. According to Atomic Force Microscopy, mIHF is able to introduce left-handed loops of ca. 100 nm size (similar to 300 bp) in supercoiled cosmids, thereby unwinding and relaxing the DNA.

Combination of solution and solid state NMR yields a molecular level view of the interactions between antibiotic teixobactin and bacterial cell wall building block lipid II.

Arkadia (Rnf111) is an E3 ubiquitin ligase that plays a central role in the amplification of transforming growth factor beta (TGF-β) signaling responses by targeting for degradation the negative regulators of the pathway, Smad6 and Smad7, and the nuclear co-repressors Ski and Skil (SnoN). Arkadia's function in vivo depends on the really interesting new gene (RING)–H2 interaction with the E2 enzyme UbcH5b in order to ligate ubiquitin chains on its substrates. A conserved tryptophan (W972) in the C-terminal α-helix is widely accepted as essential for E2 recruitment and interaction and thus also for E3 enzymatic activity. The present NMR-driven study provides an atomic-level investigation of the structural and dynamical properties of two W972 Arkadia RING mutants, attempting to illuminate for the first time the differences between a functional and a nonfunctional mutant W972A and W972R, respectively. A TGF-β-responsive promoter driving luciferase was used to assay for Arkadia function in vivo. These experiments showed that the Arkadia W972A mutant has the same activity as wild-type (WT) Arkadia in enhancing TGF-β signaling responses, while W972R does not. Only minor structural differences exist between the W972A RING domain and WT-RING. In contrast, the W972R mutant hardly interacts with E2. The loss of function correlates with structural changes in the C-terminal α-helix and an increase in the distance between the Zn(II) ions. Our data show that the position occupied by W972 within WT Arkadia is critical for the function of RING and that it depends on the nature of the residue at this position.

Reference EPFL-CONF-225200View record in Web of Science Record created on 2017-01-24, modified on 2017-05-12

Significance Protein structure determination is key to the detailed description of many biological processes. The critical factor that would allow general application of magic-angle spinning (MAS) solid-state NMR to this end is improvement in sensitivity and resolution for as many nuclear spins as possible. This is achieved here with detection of resolved 1 H resonances in protonated proteins by increasing MAS rates to frequencies of 100 kHz and above. For large proteins and assemblies, ultrafast spinning narrows spectral resonances better than Brownian motion on which solution NMR relies, removing a fundamental barrier to the NMR study of large systems. This is exploited here to determine the de novo structure of a 28-kDa protein dimer in a 2.5-MDa viral capsid assembly.

UNIO is a comprehensive software suite for protein NMR structure determination that enables full automation of all NMR data analysis steps involved—including signal identification in NMR spectra, sequence-specific backbone and side-chain resonance assignment, NOE assignment and structure calculation. Within the framework of the second round of the community-wide stringent blind NMR structure determination challenge (CASD-NMR 2), we participated in two categories of CASD-NMR 2, namely using either raw NMR spectra or unrefined NOE peak lists as input. A total of 15 resulting NMR structure bundles were submitted for 9 out of 10 blind protein targets. All submitted UNIO structures accurately coincided with the corresponding blind targets as documented by an average backbone root mean-square deviation to the reference proteins of only 1.2 Å. Also, the precision of the UNIO structure bundles was virtually identical to the ensemble of reference structures. By assessing the quality of all UNIO structures submitted to the two categories, we find throughout that only the UNIO–ATNOS/CANDID approach using raw NMR spectra consistently yielded structure bundles of high quality for direct deposition in the Protein Data Bank. In conclusion, the results obtained in CASD-NMR 2 are another vital proof for robust, accurate and unsupervised NMR data analysis by UNIO for real-world applications.

Here we introduce a new pulse sequence for resonance assignment that halves the number of data sets required for sequential linking by directly correlating sequential amide resonances in a single diagonal-free spectrum. The method is demonstrated with both microcrystalline and sedimented deuterated proteins spinning at 60 and 111 kHz, and a fully protonated microcrystalline protein spinning at 111 kHz, with as little as 0.5 mg protein sample. We find that amide signals have a low chance of ambiguous linkage, which is further improved by linking in both forward and backward directions. The spectra obtained are amenable to automated resonance assignment using general-purpose software such as UNIO-MATCH.

Nuclear magnetic resonance spectroscopy (NMR) can provide a great deal of information about structure and dynamics of biomolecules. The quality of an NMR structure strongly depends on the number of experimental observables and on their accurate conversion into geometric restraints. When distance restraints are derived from nuclear Overhauser effect spectroscopy (NOESY), stereo-specific assignments of prochiral atoms can contribute significantly to the accuracy of NMR structures of proteins and nucleic acids. Here we introduce a series of NOESY-based pulse sequences that can assist in the assignment of chiral CHD methylene protons in random fractionally deuterated proteins. Partial deuteration suppresses spin-diffusion between the two protons of CH2 groups that normally impedes the distinction of cross-relaxation networks for these two protons in NOESY spectra. Three and four-dimensional spectra allow one to distinguish cross-relaxation pathways involving either of the two methylene protons so that one can obtain stereospecific assignments. In addition, the analysis provides a large number of stereospecific distance restraints. Non-uniform sampling was used to ensure optimal signal resolution in 4D spectra and reduce ambiguities of the assignments. Automatic assignment procedures were modified for efficient and accurate stereospecific assignments during automated structure calculations based on 3D spectra. The protocol was applied to calcium-loaded calbindin D9k. A large number of stereospecific assignments lead to a significant improvement of the accuracy of the structure.

Cerebral dopamine neurotrophic factor (CDNF) is a promising therapeutic agent for Parkinson disease. As such, there has been great interest in studying its mode of action, which remains unknown. The three-dimensional crystal structure of the N terminus (residues 9-107) of CDNF has been determined, but there have been no published structural studies on the full-length protein due to proteolysis of its C-terminal domain, which is considered intrinsically disordered. An improved purification protocol enabled us to obtain active full-length CDNF and to determine its three-dimensional structure in solution. CDNF contains two well folded domains (residues 10-100 and 111-157) that are linked by a loop of intermediate flexibility. We identified two surface patches on the N-terminal domain that were characterized by increased conformational dynamics that should allow them to embrace active sites. One of these patches is formed by residues Ser-33, Leu-34, Ala-66, Lys-68, Ile-69, Leu-70, Ser-71, and Glu-72. The other includes a flexibly disordered N-terminal tail (residues 1-9), followed by the N-terminal portion of alpha-helix 1 (residues Cys-11, Glu-12, Val-13, Lys-15, and Glu-16) and residue Glu-88. The surface of the C-terminal domain contains two conserved active sites, which have previously been identified in mesencephalic astrocyte-derived neurotrophic factor, a CDNF paralog, which corresponds to its intracellular mode of action. We also showed that CDNF was able to protect dopaminergic neurons against injury caused by alpha-synuclein oligomers. This advises its use against physiological damages caused by alpha-synuclein oligomers, as observed in Parkinson disease and several other neurodegenerative diseases.

We present here a unified, easily adaptable, open-source NMR exchange format (NEF) for NMR restraints and associated data.