Biomolecular NMR Assignments最新文献

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.

Ephrin receptors regulate intercellular communication and are thus involved in tumor development. Ephrin receptor A2 (EphA2), in particular, is overexpressed in a variety of cancers and is a proven target for anti-cancer drugs. The N-terminal ligand-binding domain of ephrin receptors is responsible for the recognition of their ligands, ephrins, and is directly involved in receptor activation. Here, we report on the complete ¹H, ¹⁵N and ¹³C NMR chemical shift assignment of EphA2 ligand binding domain that provides the basis for NMR-assisted drug design.

Natural macrocyclic peptides produced by microorganisms serve as valuable resources for therapeutic compounds, including antibiotics, anticancer agents, and immune suppressive agents. Nonribosomal peptide synthetases (NRPSs) are responsible for the biosynthesis of macrocyclic peptides. NRPSs are large multimodular enzymes, and each module recognizes and incorporates one specific amino acid into the polypeptide product. In the final biosynthetic step, the mature linear peptide precursor is subject to head-to-tail cyclization by the thioesterase (TE) domain in the C-terminal module. Since the TE domains can autonomously catalyze the cyclization of diverse linear peptide substrates, isolated TE domains can be used to produce natural product derivatives. To understand the mechanism of TE domains in NRPSs as a base for therapeutic applications, we investigated the TE domain (residues 6236-6486) of tyrocidine synthetase TycC by NMR. Tyrocidine is a cyclic decapeptide with antibiotic activity, and TycC-TE catalyzes the cyclization of the linear decapeptide precursor. Here, we report the backbone resonance assignments of TycC-TE. The assignments of TycC-TE provide the basis for NMR investigations of the structure and substrate-recognition mode of the TE domain in NRPS.

It has been shown that human seminal fluid is a major factor in enhancing HIV activity. The SEM2(49-107) peptide is a product of cleavage after ejaculation by internal prostheses of the semenogelin 2 protein, expressed in seminal vesicles. It is established that the peptide SEM2(49-107) forms amyloid fibrils, which increase probability of contracting HIV infection. In this nuclear magnetic resonance (NMR) study, we present almost complete (86%) resonance assignments for the ¹H ¹⁵N and ¹³C atoms of the backbone and side-chain of the SEM2(49-107) peptide (BioMagResBank accession number 52356). The secondary structure of SEM2(49-107) peptide was estimated by using two approaches, secondary chemical shifts analysis (CSI) and TALOS-N prediction. Analysis of the secondary structure of the SEM2(49-107) peptide using both methods revealed that the peptide contains helical segments at the C-terminus. Also in this work, we used phase-sensitive 2D HSQC ¹H- ¹⁵N experiments measuring longitudinal T₁ and transverse T₂ NMR relaxation times to report predicted secondary structure and backbone dynamics of the SEM2(49-107) peptide. This resonance assignment will form the basis of future NMR research, contributing to a better understanding of the peptide structure and internal dynamics of the molecule.

Zika virus has raised global concerns due to its link to microcephaly and Guillain-Barré syndrome in adults. One of viral nonstructural proteins-NS4B, an integral membrane protein, plays crucial roles in viral replication by interacting with both viral and host proteins, rendering it an attractive drug target for antiviral development. We purified the N-terminal region of ZIKV NS4B (NS4B NTD) and reconstituted it into detergent micelles. Here, we report the assignments of the backbone resonances of NS4B NTD in detergent micelles. The available assignment is useful for understanding its structure and ligand binding to provide useful information for developing NS4B inhibitors.