Biomolecular NMR Assignments最新文献

Receptor-interacting protein kinase 1 (RIPK1) is a key regulator of necroptotic signalling that forms functional amyloid fibrils through its RIP Homotypic Interaction Motif (RHIM). Here, we report the solid-state NMR chemical shift assignments for the rigid amyloid core of human RIPK1 fibrils, encompassing residues 529–552 within the RHIM. Assignments were obtained from uniformly ¹³C,¹⁵N-labeled protein diluted with unlabeled protein and measured using cross-polarization magic angle spinning (CPMAS) experiments on a cryogenic probe. The dataset includes backbone and side-chain resonances for the ordered region and provides a basis for high-resolution structural and dynamics studies of RIPK1 and related RHIM-containing assemblies.

The human chaperonin system, Hsp60/Hsp10, is essential for maintaining protein homeostasis and is found mainly in mitochondria. Hsp60 forms a bowl-shaped structure that provides an enclosed environment for protein folding, while its co-chaperone, Hsp10, acts as a cap to seal the barrel. This coordinated process is crucial for the proper folding of many unfolded or misfolded proteins, making the Hsp60/Hsp10 complex an indispensable chaperone system. Changes in their expression levels have been linked to diseases such as neurodegenerative disorders and cancer. Although Hsp60 has gained increasing attention, its co-chaperone Hsp10 remains relatively underexplored and has often been assumed to play a passive role. However, emerging studies challenge this view, suggesting that Hsp10 alone may exert regulatory functions within the chaperonin cycle. Here, we present the near-complete NMR backbone assignment of the 102-residue human Hsp10, laying the groundwork for future investigations into its structure, interactions, and roles in facilitating protein folding and preventing aggregation.

The N-terminal tandem RNA Recognition Motifs (RRM1−2) of the splicing factor RNA Binding Motif 39 (RBM39) from Homo sapiens was expressed in E. coli with ¹³C and ¹⁵N labeling. A nearly complete assignment of backbone ¹H, ¹⁵N, and ¹³C resonances was obtained, along with a secondary structure prediction based on the assigned chemical shifts. The predicted secondary structures of the RBM39 tandem RRM1−2 closely matched the published solution structures of the individual RBM39 RRM1 and RRM2, and also aligned well with the AlphaFold2 model of RBM39.

Platelet Factor 4 (PF4), also known as CXCL4, is a CXC chemokine crucial for hemostasis, inflammation, and immune responses. Under physiological conditions PF4 assembles into asymmetric tetramers (31.2 kDa) that are dimers of dimers with highly flexible N-terminal regions. PF4 tetramers play a central role in prothrombotic autoimmune conditions, such as heparin-induced thrombocytopenia (HIT), as well as vaccine-induced immune thrombocytopenia and thrombosis (VITT). Here, we report the resonance assignments of ¹H, ¹⁵N, and ¹³C nuclei for wild-type asymmetric PF4 tetramers using TROSY-based triple resonance NMR experiments. We also used N_z-exchange spectroscopy to identify peaks split by slow-exchange between two distinct conformational states caused by the asymmetry of PF4 tetramers. Our NMR assignments establish a foundation for future investigations into the structural dynamics and functional mechanisms of PF4 as well as its pathological role in anti-PF4 disorders.

Flap endonuclease (FEN) enzymes are a group of metallonucleases that have essential roles in DNA repair and the maintenance of genomic resilience. They catalyse hydrolytic cleavage of a phosphodiester bond to remove 5′-flaps present on double-stranded DNA molecules formed during DNA replication. FEN locates a target scissile bond through the structural recognition of a pronounced bend in the double-stranded DNA substrate along with the presence of both a 5′-flap and a 1-nucleotide 3′-flap. FEN enzymes share a common structural architecture and are functionally conserved across all living organisms. In this work, we report the ¹H, ¹⁵N and ¹³C backbone resonance assignments of residues 2–349 of FEN from Plasmodium falciparum (PfFEN349) in its substrate-free state. Using heteronuclear multidimensional NMR spectroscopy, 90% of all backbone resonances of PfFEN349 were assigned, with 298 backbone amide resonances out of 337 theoretically-detectible resonances (which exclude 10 prolines and the N-terminal glycine) identified in the ¹H–¹⁵ N TROSY spectrum. Prediction of solution secondary structure content from a chemical shift analysis using the TALOS-N webserver is mostly in good agreement with an AlphaFold model of PfFEN349. The reported assignments provide a pathway for drug discovery as PfFEN349 is a potential target for the development of new inhibitors that could be utilised to control the incidence of malaria across the globe.

Due to the emergence of the SARS-CoV-2 virus, research on coronaviruses has been massively accelerated. In addition to SARS-CoV-2, there are other human coronaviruses, including HCoV-229E. In all coronaviruses, secondary structure predictions indicate the presence of conserved structural elements in the 5’-untranslated region (5’-UTR). These conserved elements play crucial roles in RNA translation and replication. Stem-loop 5 (SL5), consisting of three substructures (5a, 5b, 5c), is highly conserved and harbours the start codon for translation. SL5 has repetitive structural motifs (RSMs), 5’-UUYYGU-3’, which are conserved in many alpha- and betacoronaviruses. In the following, we present the ¹H, ¹³C and ¹⁵N NMR resonance assignment of the SL5a RNA element from HCoV-229E and variations in the RSMs to show the effect of loop mutations on the structure of the hexaloop, revealing the different impact of each loop nucleotide on RNA dynamics.

Over 7 million people worldwide are affected with Chagas disease, a lifelong debilitating and potentially fatal Neglected Tropical Disease caused by the single cell protozoan parasite Trypanosoma cruzi. To maintain viability and to reproduce under the harsh conditions within a host organism, pathogens express a variety of protecting enzymes and virulence factors that can serve as potential drug targets. To protect itself from redox stress, T. cruzi takes advantage of a unique thiol metabolism. For instance, a cytosolic peroxide clearance cascade is centered on the conserved oxidoreductase Tryparedoxin (Tpx). Tpx efficiently distributes reducing equivalents across the parasitic cell through the promiscuous yet selective binding of numerous up- and downstream clients. However, the exact structure and binding interfaces of this central protein binding hub remain unknown. To study the redox-dependent structural dynamics of T. cruzi Tpx, and its interactions with binding partners, we determined the ¹H, ¹³C, ¹⁵N-backbone NMR assignments of the enzyme in the reduced and oxidized state. In agreement with earlier NMR studies on Tpx from related protozoans, we report redox-dependent changes in the enzyme’s dithiol active site that could play a crucial role in the recognition of physiological substrates and should be considered in the rational design of small molecule inhibitors.

The INK4 family of proteins restricts uncontrolled cell cycle progression by inhibiting cyclin-dependent kinases 4 and 6. The family consists of small, monomeric and mainly alpha-helical proteins that are conserved across all vertebrate species. We recently discovered that the human INK4 protein p16 converts into amyloid structures upon oxidation of the single cysteine residue present. Here we investigate the Danio rerio (zebrafish) orthologue P18 protein. The 170-residue protein contains two cysteines which may similarly mediate transition into amyloids upon oxidation. We present the near complete backbone assignment of the reduced P18 protein in solution. These chemical shift data provide the foundation for studying oxidation-induced structural changes and protein interactions.

¹³C and ¹⁵N backbone chemical shift assignments are reported for the 28.5 kDa protein Toho-1 β-lactamase, a Class A extended spectrum β-lactamase. A very high level of assignment completeness (97% of the backbone) is enabled by the combined sensitivity and resolution gains of ultrahigh-field NMR spectroscopy (1.1 GHz), improved probe technology, and optimized pulse sequences. The assigned chemical shifts agree well with our previous solution-state NMR assignments, indicating that the secondary structure is conserved in the solid state. These assignments provide a foundation for future investigations of side-chain chemical shifts and catalytic mechanism.

UEV domains are catalytically dead variants of the E2 enzymes which play an intermediate role in ubiquitin signaling. UEV domain containing proteins, like the ESCRT-I factor Tsg101 often play critical roles in trafficking of ubiquitylated cargos or in modulating ubiquitin processivity, or in determining the type of signal that is transferred to a target protein. Ubiquitin-conjugating enzyme E2 variant (UEV) and lactate/malate dehydrogenase (UEVLD), also known as UEV3, is a human paralogue of Tsg101 with apparent associations to cancer, innate immunity, NF-κB signaling, and autophagy. It contains an N-terminal UEV domain with 56% identity to that of Tsg101 and a C-terminal lactate dehydrogenase domain. Here, we show the backbone assignments of the UEV domain from UEVLD and find that its Cα shifts are consistent with a UEV domain composition. Further experiments suggest that it may have regions corresponding to the known binding pockets of Tsg101, but further structural and functional work will be required to uncover critical determinants of UEV domain function, and the role of these domains in Ubiquitin signaling as a whole.