Biomolecular NMR Assignments最新文献

The Rib domain, a conserved structural element found in Gram-positive bacterial cell surface proteins, plays a role in bacterial virulence and is a potential target for vaccine development. Despite the availability of high-resolution crystallographic structures, the precise functional role of the Rib domain remains elusive. Here, we report the chemical shift assignments of the Rib domain from a cell surface protein of Limosilactobacillus reuteri, providing a foundational step toward understanding its potential involvement in host-bacteria interactions.

Prokaryotes, eukaryotes, and certain viruses with positive single-stranded RNA genomes are among the forms of life that have been found to possess macro domains (MDs). There are claims that viral MDs inhibit the immune response mediated by PARPs, such as PARP12 and PARP14, and are involved in the formation of the viral replication transcription complex (RTC). Rubella virus (RuV) is included in this group of viruses. Its MD acts as an “eraser” of the posttranslation modification (PTM) ADP-ribosylation by binding to and hydrolyzing ADP-ribose (ADPr) from ADP-ribosylated substrates including proteins and nucleic acids. Consequently, it represents an attractive pharmacological target. Currently, no inhibitors exist for RuV MD’s de-ADP-ribosylation activity, which may play a crucial role in viral replication and pathogenesis, as observed in severe acute respiratory syndrome coronavirus (SARS-CoV) and Chikungunya virus (CHIKV). RuV remains a serious threat, particularly to unvaccinated children, with approximately 10,000 of the 18,000 global cases in 2022 reported in Africa. Alarmingly, no FDA-approved drugs are available for RuV treatment. In this study, we present the almost complete NMR backbone and side-chain resonance assignment of RuV MD in both free and ADPr bound forms, along with the NMR chemical shift-based secondary structure element prediction. These findings will support the efficient screening of fragments or chemical libraries using NMR spectroscopy to identify compounds that are strong binders and potentially exhibit antiviral activity.

G protein-coupled receptors (GPCRs) are highly dynamic seven-transmembrane (7TM) proteins that respond to various extracellular stimuli and elicit diverse intracellular signaling cascades. The third intracellular loops (ICL3s) of the GPCRs are intrinsically disordered and play important roles in signaling. The muscarinic acetylcholine receptors (mAChRs) harbor extremely long ICL3s, which comprise over a hundred amino acid residues and contain multiple phosphorylation sites. Due to their intrinsic flexibility, ICL3s are commonly absent or unobservable in cryo-EM or X-ray structures, and there has been a lack of structural and dynamics study of these regions. Herein, we report the ¹H, ¹³C and ¹⁵N chemical shift assignments of the M1 muscarinic receptor ICL3, which provides a basis for further NMR studies of its conformational dynamics, post-translational modifications and interactions.

The increasing prevalence of antibiotic resistance represents a significant public health concern, underscoring the urgent need for the development of novel therapeutic strategies. The antibiotic effects of macrolides, the second most widely used class of antibiotics, are counteracted by Erm proteins through the methylation of adenosine 2058 of the 23S ribosomal RNA (rRNA) (~ 2900 nucleotides), yielding either monomethylated or dimethylated A2058. This methylation is the molecular basis for preventing macrolides from binding and leads to the development of resistance of bacteria including Staphylococcus, Streptococcus and Enterococcus. While the function of Erm proteins have been thoroughly investigated, the role of the ribosomal RNA in acquiring antibiotic resistance is frequently underestimated, given that the ribosomal RNA is the actual target for methylation. Here, we present the comprehensive ¹H, ¹³C and ¹⁵N NMR resonance assignment for the part of the 23S rRNA that serves as the Erm substrate in antimicrobial resistance. Furthermore, we compare the chemical shift signature of the unmethylated to the monomethylated and dimethylated RNA construct and show that changes in the RNA upon methylation are locally restricted. The resonance assignments provide a starting point for investigating and targeting the molecular mechanism of the resistance-conferring Erm proteins.

Cytoplasmic polyadenylation element-binding protein 3 (CPEB3) is an RNA-binding protein that plays a pivotal role in the formation of long-term memory. The N-terminal region (residues 1–459) of CPEB3 is a highly aggregative intrinsically disordered region (IDR) that regulates the translation of specific targets, such as AMPA subunits, through mechanisms including liquid-liquid phase separation (LLPS) and the formation of fibrous aggregates. Despite its significance, the underlying mechanisms remain poorly understood. In this study, we present the backbone resonance assignments of residues 101–200 and 294–410 segments of the CPEB3 IDR. In agreement with sequence-based predictions, CPEB3 [101–200] was found to be disordered, whereas two partial α-helices were identified within CPEB3 [294–410].

Serpine mRNA-Binding Protein 1 (SERBP1) is an RNA-binding protein implicated in diverse cellular functions, including translational regulation, tumor progression, and stress response. It interacts with ribosomal subunits, RNA, and proteins involved in stress granules, contributing to processes such as phase separation and epigenetic regulation. Recent studies have shown SERBP1’s role in glioblastoma progression and its involvement in ribosomal regulation. Structurally, SERBP1 contains N- and C-terminal hyaluronan-binding domains, two RG/RGG motifs, and is predicted to be predominantly disordered. Here, we report the backbone resonance assignment and secondary structure propensities of SERBP1’s N-terminal residues (1–149). Using NMR spectroscopy, we identified a stable α-helix (residues 28–40) and transient structural elements. These findings provide insight into the structural features of SERBP1 that may mediate its interactions with ribosomal subunits, RNA, and other binding partners, laying a foundation for future structural studies of its functional mechanisms.

Double-stranded RNA (dsRNA) binding proteins (dsRBPs) are among the key players that act along with other components involved in the RNA interference (RNAi) pathway for mediating gene silencing. Additionally, members of the dsRBP family of proteins play divergent roles in the broader array of biological processes. In Arabidopsis thaliana, dsRNA binding protein 5 (DRB5), along with DRB2 and DRB3, serves in recognition of viral RNA invasion and co-localizes with viral replication complexes. However, the functional role of DRB5 in such complexes is yet to be explored. DRB5 is a multidomain protein containing two tandem dsRNA binding domains (dsRBDs) at its N-terminus. Our current study presents the near-complete backbone and sidechain assignment of the dsRBD1 and dsRBD2 of DRB5 using solution NMR. The study will further contribute to determining the solution structure of dsRBDs and open new avenues to investigate the functional role of DRB5 in gene silencing pathways.

Dopamine N-acetyltransferase (Dat), belonging to the GCN5-related N-acetyltransferase (GNAT) superfamily, is an arylalkylamine N-acetyltransferase (AANAT) that is involved in insects neurotransmitter inactivation and the development of insect cuticle sclerotization. By using the cofactor acetyl coenzyme A (Ac-CoA) as an acetyl group donor, Dat produces acetyl-dopamine through the reaction with dopamine. Although AANATs share similar structural features with the GNAT family, they have low sequence identities among insect AANATs (~ 40%) and between insect AANATs and vertebrate AANATs (~ 12%). A common noticed feature in GNATs is the Ac-CoA-binding induced conformational change, and this is important for further selection and catalysis of its substrate. In AANATs, the conformational changes help the sequential binding mechanism. Here, we report the ¹H, ¹³C and ¹⁵N backbone resonance assignments of the 24 kDa Dat from Drosophila melanogaster in the free and Ac-CoA-bound states, and the chemical shift differences revealed a significant conformational change in the α1 region of Dat. These assignments provide a foundation for further investigations of the catalysis and structural regulation of Dat in solution.

CpxA is an extensively studied histidine kinase implicated in cellular stress responses. The highly conserved CA domain of CpxA (CpxA^CA) is an essential domain for the hydrolysis of ATP and the binding of inhibitors and considered to be a promising target for broad-spectrum antimicrobial drugs development. The ATP-binding pocket in the CA domain contains a flexible ATP lid motif. Although the crystal structure of CA domain has been defined, the structure of the ATP lid remains uncertain, posing a challenge to the study of its catalytic mechanism. In this study, we report the backbone ¹H, ¹³C and ¹⁵N chemical shift assignments of CpxA^CA by heteronuclear multidimensional spectroscopy and predict its secondary structure in solution using TALOS⁺. The residues of ATP lid motif are well-assigned. Therefore, this study provides a foundation for understanding the role of CpxA^CA in cellular signaling and the development of novel antimicrobial therapies.