Biomolecular NMR Assignments最新文献

The detection of nucleic acids that are present in atypical conformations is a crucial trigger of the innate immune response. Human Z-DNA binding protein 1 (ZBP1) is a pattern recognition receptor that harbors two Zα domains that recognize Z-DNA and Z-RNA. ZBP1 detects this alternate nucleic acid conformation as foreign, and upon stabilization of these substrates, it triggers activation of an immune response. Here, we present the backbone chemical shift assignment of a construct encompassing the Zα1 and Zα2 domains as well as the interconnecting linker of ZBP1. These assignments can be directly transferred to the isolated Zα1 and Zα2 domains, thereby demonstrating that these domains maintain virtually identical structures in the tandem context.

ProXp-ala is a key component of the translational machinery in all three Domains of life. This enzyme helps to maintain the fidelity of proline codon translation through aminoacyl-tRNA^Pro proofreading. In the first step of tRNA aminoacylation, the cognate aminoacyl-tRNA synthetase (aaRS) binds and activates an amino acid in the enzyme’s synthetic active site. If a non-cognate amino acid passes this first selection step and is charged onto the tRNA, a distinct aaRS editing active site may recognize the mischarged tRNA and deacylate it. Alternatively, this editing reaction may be carried out by a separate enzyme that deacylates the mischarged tRNA in trans. ProXp-ala is responsible for editing Ala mischarged onto tRNA^Pro. Since trans-editing domains such as ProXp-ala bind their substrates after release from the synthetase, they must recognize not only the mischarged amino acid, but also the specific tRNA. Previous studies showed that Caulobacter crescentus (Cc) ProXp-ala distinguishes tRNA^Pro from tRNA^Ala, in part, based on the unique tRNA^Pro acceptor stem base pair C1:G72. Previous crystallographic and NMR data also revealed a role for conformational selection by the ProXp-ala α2 helix in Ala- versus Pro-tRNA^Pro substrate discrimination. The α2 helix makes lattice contacts in the crystal, which left some uncertainty as to its position in solution. We report resonance assignments for the substrate-free Cc ProXp-ala and the NMR-derived three-dimensional structure of the protein. These data reveal the position of the α2 helix in solution, with implications for substrate binding and recognition.

Sorcin is a penta-EF hand calcium-binding protein that confers multidrug resistance in cancer cells. It regulates cellular Ca²⁺ homeostasis by interacting with calcium channels such as Ryanodine receptor 2 and Sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase in a calcium-dependent manner. The crystal structure of the Sorcin has been determined in both calcium-free and calcium-bound states to understand calcium-binding induced conformational change. However, due to its flexibility, most of the N-terminal domain is invisible in these crystal structures. Here we report the ¹H, ¹³C, and ¹⁵N backbone resonance assignments of full-length Sorcin in the calcium-free state using solution NMR. The protein secondary structure was predicted based on the assigned backbone chemical shifts using TALOS+ and CSI 3.0. Our backbone resonance assignment of the full-length Sorcin provides a foundation for future NMR spectroscopic studies to uncover the mechanism of Ca²⁺ sensing by Sorcin.

N-methyl-D-aspartate receptors (NMDARs) consist of glycine-binding GluN1 and glutamate-binding GluN2 subunits that form tetrameric ion channels. NMDARs in the brain are important for controlling neuronal excitability to promote synaptic plasticity. The cytoskeletal protein, α-actinin-1 (100 kDa, called ACTN1) binds to the cytosolic C0 domain of GluN1 (residues 841–865) that may play a role in the Ca²⁺-dependent desensitization of NMDAR channels. Mutations that disrupt NMDAR channel function are linked to Alzheimer’s disease, depression, stroke, epilepsy, and schizophrenia. NMR chemical shift assignments are reported here for the C-terminal EF-hand domain of ACTN1 (residues 824–892, called ACTN_EF34) and ACTN_EF34 bound to the GluN1 C0 domain (BMRB numbers 52385 and 52386, respectively).

Spider silk is a high-performance biomaterial known for its outstanding combination of strength and flexibility. Among the six distinct types of spider silk, eggcase silk stands out as it is exclusively produced from the tubuliform gland, playing a specialized role in offspring protection. In the spider species Latrodectus hesperus, eggcase silk is spun from a large spidroin complex, including the major silk component tubuliform spidroin 1 (TuSp1) and at least six different minor silk components. One of these minor components is eggcase protein 3 (ECP3), a small silk protein of 11.8 kDa that lacks the typical spidroin architecture. ECP3 shows very limited homology to all known spidroins. In this study, we report nearly complete backbone and side-chain resonance assignments of ECP3 as a basis for studying the structural mechanisms involved in eggcase silk formation.

The nucleocapsid (N) protein of SARS-CoV-2 is a multifunctional protein involved in nucleocapsid assembly and various regulatory functions. It is the most abundant protein during viral infection. Its functionality is closely related to its structure, which comprises two globular domains, the N-terminal domain (NTD) and the C-terminal domain (CTD), flanked by intrinsically disordered regions. The linker between the NTD and CTD includes a Serine-Arginine rich (SR) region, which is crucial for the regulation of the N protein’s function. Here, we report the near-complete assignment of the construct containing the NTD followed by the SR region (NTD-SR). Additionally, we describe the dynamic nature of the SR region and compare it with all other available chemical shift assignments reported for the SR region.

Single domain antibody (sdAb) is only composed of a variable domain of the heavy-chain-only antibody, which is devoid of light chain and naturally occurring in camelids and cartilaginous fishes. Variable New Antigen Receptor (VNAR), a type of single domain antibody present in cartilaginous fishes such as sharks, is the smallest functional antigen-binding fragment found in nature. The unique features, including flexible paratope, high solubility and outstanding stability make VNAR a promising prospect in antibody drug development and structural biology research. However, VNAR’s research has lagged behind camelid-derived sdAb, especially in the field of structural research. Here we report the ¹H,¹⁵N,¹³C resonance assignments of a VNAR derived from the immune library of Chiloscyllium plagiosum, termed B2-3, which recognizes the hyaluronan synthase. Analysis of the backbone chemical shifts demonstrates that the secondary structure of VNAR is predominately composed of β-sheets corresponding to around 40% of the B2-3 backbone. The Cβ chemical shift values of cysteine residues, combined with mass spectrometry data, clearly shows that B2-3 contains two pairs of disulfide bonds, which is import for protein stability. The assignments will be essential for determining the high resolution solution structure of B2-3 by NMR spectroscopy.

Amyloid fibrils from Alzheimer’s amyloid-beta peptides (Aβ) are found to be polymorphic. So far, 14 Aβ40 fibril structures have been determined. The mechanism of why one particular protein sequence adopts so many different three-dimensional structures is yet not understood. In this work, we describe the assignment of the NMR chemical shifts of two Alzheimer’s disease fibril polymorphs, P1 and P2, which are formed by the amyloid-beta peptide Aβ40. The assignment is based on ¹³C-detected 3D NCACX and NCOCX experiments MAS solid-state NMR experiments. The fibril samples are prepared using an extensive seeding protocol in the absence and presence of the small heat shock protein αB-crystallin. In addition to manual assignments, we obtain chemical shift assignments using the automation software ARTINA. We present an analysis of the secondary chemical shifts and a discussion on the differences between the manual and automated assignment strategies.

Klebsiella pneumoniae (Kp) poses an escalating threat to public health, particularly given its association with nosocomial infections and its emergence as a leading cause of neonatal sepsis, particularly in low- and middle-income countries (LMICs). Host cell adherence and biofilm formation of Kp is mediated by type 1 and type 3 fimbriae whose major fimbrial subunits are encoded by the fimA and mrkA genes, respectively. In this study, we focus on MrkA subunit, which is a 20 KDa protein whose 3D molecular structure remains elusive. We applied solution NMR to characterize a recombinant version of MrkA in which the donor strand segment situated at the protein’s N-terminus is relocated to the C-terminus, preceded by a hexaglycine linker. This construct yields a self-complemented variant of MrkA. Remarkably, the self-complemented MrkA monomer loses its capacity to interact with other monomers and to extend into fimbriae structures. Here, we report the nearly complete assignment of the ¹³C,¹⁵N labelled self-complemented MrkA monomer. Furthermore, an examination of its internal mobility unveiled that relaxation parameters are predominantly uniform across the polypeptide sequence, except for the glycine-rich region within loop 176–181. These data pave the way to a comprehensive structural elucidation of the MrkA monomer and to structurally map the molecular interaction regions between MrkA and antigen-induced antibodies.

Synucleinopathies are neurodegenerative diseases characterized by the accumulation of α-synuclein protein aggregates in the neurons and glial cells. Both ex vivo and in vitro α-synuclein fibrils tend to show polymorphism. Polymorphism results in structure variations among fibrils originating from a single polypeptide/protein. The polymorphs usually have different biophysical, biochemical and pathogenic properties. The various pathologies of a single disease might be associated with distinct polymorphs. Similarly, in the case of different synucleinopathies, each condition might be associated with a different polymorph. Fibril formation is a nucleation-dependent process involving the formation of transient and heterogeneous intermediates from monomers. Polymorphs are believed to arise from heterogeneous oligomer populations because of distinct selection mechanisms in different conditions. To test this hypothesis, we isolated and incubated different intermediates during in vitro fibrillization of α-synuclein to form different polymorphs. Here, we report ¹³C and ¹⁵N chemical shifts and the secondary structure of fibrils prepared from the helical intermediate using solid-state nuclear magnetic spectroscopy.