Biomolecular NMR Assignments最新文献

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.

The symbiotic nitrogen-fixing bacterium Bradyrhizobium japonicum (B.japonicum) enables high soybean yields with little or no nitrogen fertiliser. A two component regulatory system comprising FixL, a histidine kinase with O₂-sensing activity, and FixJ, a response regulator, controls the expression of genes involved in nitrogen fixation, such as fixK and nifA. Only under anaerobic conditions, the monophosphate group is transferred from FixL to the N-terminal receiver domain of FixJ (FixJ_N), which eventually promote the association of the C-terminal effector domain (FixJ_C) to the promoter regions of the nitrogen-fixation-related genes. Structural biological analyses carried out so far for rhizobial FixJ molecules have proposed a solution structure for FixJ that differs from the crystal structures, in which the two domains are extended. To understand the FixJ activation caused by phosphorylation of the N-terminal domain, which presumably regulates through the interactions between FixJ_N and FixJ_C, here we have performed backbone and sidechain resonance assignments of the unphosphorylated state of B. japonicum FixJ.

A comprehensive understanding of RNA-based gene regulation is a fundamental aspect for the development of innovative therapeutic options in medicine and for a more targeted response to environmental problems. Within the different mechanisms of RNA-based gene regulation, riboswitches are particularly interesting as they change their structure in response to the interaction with a low molecular weight ligand, often a well-known metabolite. Four distinct classes of riboswitches recognize the very small guanidinium cation. We are focused on the Guanidine-II riboswitch with the mini-ykkC motif. We report here the assignment of the ¹H, ¹³C, ¹⁵N and ³¹P chemical shifts of the 23 nucleotide-long sequence of the first stem-loop of the Guanidine-II riboswitch aptamer from Escherichia coli. Despite its small size, the assignment of the NMR signals of this RNA proved to be challenging as it has symmetrical base pairs and palindromic character.

The Alkbh7 protein, a member of the Alkylation B (AlkB) family of dioxygenases, plays a crucial role in epigenetic regulation of cellular metabolism. This paper focuses on the NMR backbone resonance assignment of Alkbh7, a fundamental step in understanding its three-dimensional structure and dynamic behavior at the atomic level. Herein, we report the backbone ¹H, ¹⁵N, ¹³C chemical shift assignment of the full-length human Alkbh7. Experiments were acquired at 25 °C by heteronuclear multidimensional NMR spectroscopy. Collectively, 70% of the backbone NH resonances were assigned, with 144 out of a possible 205 residues assigned in the ¹H-¹⁵N TROSY spectrum. Interestingly, peaks from the active site and the C-terminal end of Alkbh7 are not NMR visible, suggesting that these regions are dynamic on the intermediate exchange regime. Using the program TALOS+, a secondary structure prediction was generated from the assigned backbone resonance that is consistent with the previously reported X-ray structure of the enzyme. The reported assignment will permit investigations of the protein structural dynamics anticipated to provide crucial insight regarding fundamental aspects in the recognition and enzyme regulation processes.

In Arabidopsis thaliana, micro-RNA regulation is primarily controlled by DCL1, an RNase III enzyme, and its associated proteins. DCL1, together with DRB2, governs a specific group of miRNAs that induce the inhibition of target mRNA translation. DRB2 is a multi-domain protein containing two N-terminal dsRNA binding domains (dsRBD) separated by a linker, followed by an unstructured C-terminal tail. The two dsRBDs in DRB2 are involved in recognizing the miRNA precursor and aiding DCL1 in generating 21-nucleotide-long miRNA. Our study presents a nearly complete backbone chemical shift assignment of both dsRBDs and the side-chain assignment of the first dsRBD in DRB2. The data presented here lays the groundwork for future investigations into the structural, dynamic, and functional aspects of DRB2.

PhoCl is a photocleavable protein engineered from a green-to-red photoconvertible fluorescent protein by circular permutation, and has been used in various optogenetic applications including precise control of protein localization and activity in cells. Upon violet light illumination, PhoCl undergoes a β-elimination reaction to be cleaved at the chromophore, resulting in spontaneous dissociation into a large empty barrel and a small C-terminal peptide. However, the structural determinants and the mechanism of the PhoCl photocleavage remain elusive, hindering the further development of more robust photocleavable optogenetic tools. Here, we report the backbone resonance assignments of PhoCl as a basis for studying the violet-light-induced self-cleavage mechanism of PhoCl.

The nutrient germinant receptors (GRs) in spores of Bacillus species consist of a cluster of three proteins– designated A, B, and C subunits– that play a critical role in initiating the germination of dormant spores in response to specific nutrient molecules. The Bacillus cereus GerI GR is essential for inosine-induced germination; however, the roles of the individual subunits and the mechanism by which germinant binding activates GR function remain unclear. In this study, we report the backbone chemical shift assignments of the N-terminal domain (NTD) of the A subunit of GerI (GerIA^NTD). Furthermore, we derive the secondary structure of GerIA^NTD in solution and compare it with the crystal structure of the NTD of the A subunit of a Bacillus megaterium GR. These findings lay the foundation for further NMR studies aimed at investigating the structure-function relationship of the GerI subunits, with a broader goal of understanding the molecular mechanism underlying germinant recognition and signal transduction in GRs across Bacillus species.

Cyclic GMP-AMP synthase (cGAS) is a DNA-sensing enzyme that is a member of the nucleotidyltransferase (NTase) family and functions as a DNA sensor. The protein is comprised of a catalytic NTase core domain and an unstructured hypervariable N-terminal domain (NTD) that was reported to increase protein activity by providing an additional DNA-binding surface. We report nearly complete ¹H, ¹⁵N, and ¹³C backbone chemical-shift assignments of mouse cGAS NTD (residues 5-146), obtained with a set of 3D and 4D solution NMR experiments. Analysis of the chemical-shift values confirms that the NTD is intrinsically disordered. These resonance assignments can provide the basis for further studies such as activation by DNA and protein-protein interactions.

J-domain proteins (JDPs) are essential cochaperones of heat shock protein 70 (Hsp70), as they bind and deliver misfolded polypeptides while also stimulating ATPase activity, thereby mediating the refolding process and assisting Hsp70 in maintaining cellular proteostasis. Despite their importance, detailed structural information about JDP‒Hsp70 complexes is still being explored due to various technical challenges. One major challenge is the lack of more detailed structural data on full-length JDPs. Class A and B JDPs, the most extensively studied, are typically dimers of 300–400 residue polypeptides with central intrinsically disordered regions. These features complicate structural analysis via NMR and X-ray crystallography techniques. This work presents the ¹H, ¹⁵N, and ¹³C backbone resonance assignments of the full-length (352 residues long) Sis1, a dimeric class B JDP from S. cerevisiae. Our study achieved 70.5% residue assignment distributed across the entire protein, providing probes in all Sis1 domains for the first time. To overcome this challenging task, strategies such as deuteration and 3D BEST-TROSY correlation experiments were used. The methods and results are detailed within the text. We are confident that this achievement will significantly benefit both the structural biology and the proteostasis scientific communities.