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 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. 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 from tRNA, in part, based on the unique tRNA 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 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.

The catalytic domain of the calcium-dependent endoribonuclease EndoU from Homo sapiens was expressed in E. coli with C and N labeling. A nearly complete assignment of backbone H, N, and C resonances was obtained, as well as a secondary structure prediction based on the assigned chemical shifts. The predicted secondary structures were almost identical to the published crystal structure of calcium-activated EndoU. This is the first NMR study of an eukaryotic member of the EndoU-like superfamily of ribonucleases.

Sorcin is a penta-EF hand calcium-binding protein that confers multidrug resistance in cancer cells. It regulates cellular Ca homeostasis by interacting with calcium channels such as Ryanodine receptor 2 and Sarcoplasmic/endoplasmic reticulum Ca-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 H, C, and N 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 Ca sensing by Sorcin.

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 C-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.

The Rho GTPase (Ras homolog GTPases) system is a crucial signal transducer that regulates various cellular processes, including cell cycle and migration, genetic transcription, and apoptosis. In this study, we investigated the unfolded state of the first FF domain (FF1) of P190A RhoGAP, which features four tandem FF domains. For signal transduction, FF1 is phosphorylated at tyrosine 308 (Y308), which is buried in the hydrophobic core and is inaccessible to kinases in the folded domain. It was proposed, therefore, that the phosphorylation occurs in a transiently populated unfolded state of FF1. To probe the folding pathway of the RhoGAP FF1 domain, here we have performed a nearly complete backbone resonance assignments of a putative partially unfolded state of FF1 in 5 M urea and its fully unfolded state in 8 M urea.

The CcdAB system expressed in the E.coli cells is a prototypical example of the bacterial toxin-antitoxin (TA) systems that ensure the survival of the bacterial population under adverse environmental conditions. The solution and crystal structures of CcdA, CcdB and of CcdB in complex with the toxin-binding C-terminal domain of CcdA have been reported. Our interest lies in the dynamics of CcdB-CcdA complex formation. Solution NMR studies have shown that CcdB_G100T, in presence of saturating concentrations of CcdA-c, a truncated C-terminal fragment of CcdA exists in equilibrium between two major populations. Sequence specific backbone resonance assignments of both equilibrium forms of the ~ 27 kDa complex, have been obtained from a suite of triple resonance NMR experiments acquired on H, C, N enriched samples of CcdB_G100T. Analysis of H, C, C secondary chemical shifts, shows that both equilibrium forms of CcdB_G100T have five beta-strands and one alpha-helix as the major secondary structural elements in the tertiary structure. The results of these studies are presented below.

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.

Diverse extracellular sensor domains enable cells to regulate their behavior, physiological processes, and interspecies interactions in response to environmental stimuli. These sensing mechanisms facilitate the ultimate adaptation of organisms to their surrounding conditions. Pseudomonas aeruginosa (PAO1) is a clinically significant opportunistic pathogen in hospital infection. The CHASE4 domain, a putative extracellular sensing module, is found in the N-terminus of GGDEF-EAL-containing PA2072, a transmembrane receptor from P. aeruginosa. However, the signal identification and sensing mechanism of monomeric PA2072 CHASE4 remains largely unknown. Here, we report backbone and side chain resonance assignments of PA2072 CHASE4 as a basis for studying the structural mechanism of CHASE4-mediated signal recognition.

Protein-water interactions profoundly influence protein structure and dynamics. Consequently, the function of many biomacromolecules is directly related to the presence and exchange of water molecules. While structural water molecules can be readily identified through X-ray crystallography, the dynamics within functional protein-water networks remain largely elusive. Therefore, to understand the role of biological water in protein dynamics and function, we have introduced S2A and H64A mutations in human Carbonic Anhydrase II (hCAII), a model system to study protein-water interactions. The mutations of serine to alanine at position 2 and histidine to alanine at position 64 cause an increase in hydrophobicity in the N-terminus and active site loop thereby restricting water entry and disrupting the water network in the Zn-binding pocket. To pave the way for a detailed investigation into the structural, functional, and mechanistic aspects of the Ser2Ala/His64Ala double mutant of hCAII, we present here almost complete sequence-specific resonance assignments for H, N, and C. These assignments serve as the basis for comprehensive studies on the dynamics of the protein-water network within the Zn-binding pocket and its role in catalysis.

Propionyl CoA carboxylase (PCC) is a multimeric enzyme composed of two types of subunits, α and β arranged in αβ stoichiometry. The α-subunit consists of an N-terminal carboxylase domain, a carboxyl transferase domains, and a C-terminal biotin carboxyl carrier protein domain (BCCP). The β-subunit is made up of an N- and a C- carboxyl transferase domain. During PCC catalysis, the BCCP domain plays a central role by transporting a carboxyl group from the α-subunit to the β-subunit, and finally to propionyl CoA carboxylase, resulting in the formation of methyl malonyl CoA. A point mutation in any of the subunits interferes with multimer assembly and function. Due to the association of this enzyme with propionic acidemia, a genetic metabolic disorder found in humans, PCC has become an enzyme of wide spread interest. Interestingly, unicellular eukaryotes like Leishmania also possess a PCC in their mitochondria that displays high sequence conservation with the human enzyme. Thus, to understand the function of this enzyme at the molecular level, we have initiated studies on Leishmania major PCC (LmPCC). Here we report chemical shift assignments of LmPCC BCCP domain using NMR. Conformational changes in LmPCC BCCP domain upon biotinylation, as well as upon interaction with its cognate biotinylating enzyme (Biotin protein ligase from L. major) have also been reported. Our studies disclose residues important for LmPCC BCCP interaction and function.

Transfer RNAs (tRNAs) are an essential component of the protein synthesis machinery. In order to accomplish their cellular functions, tRNAs go through a highly controlled biogenesis process leading to the production of correctly folded tRNAs. tRNAs in solution adopt the characteristic L-shape form, a stable tertiary conformation imperative for the cellular stability of tRNAs, their thermotolerance, their interaction with protein and RNA complexes and their activity in the translation process. The introduction of post-transcriptional modifications by modification enzymes, the global conformation of tRNAs, and their cellular stability are highly interconnected. We aim to further investigate this existing link by monitoring the maturation of bacterial tRNAs in E. coli extracts using NMR. Here, we report on the H, N chemical shift assignment of the imino groups and some amino groups of unmodified and modified E. coli tRNA, tRNA and tRNA, which are essential for characterizing their maturation process using NMR spectroscopy.

Chromosomal replication is a ubiquitous and essential cellular process. In bacteria, the master replication initiator DnaA plays a key role in promoting an open complex at the origin (oriC) and recruiting helicase in a tightly regulated process. The C-terminal domain IV specifically recognises consensus sequences of double-stranded DNA in oriC, termed DnaA-boxes, thereby facilitating the initial engagement of DnaA to oriC. Here, we report the Cβ and backbone H, N, and C chemical shift assignments of soluble DnaA domain IV from Bacillus subtilis at pH 7.6 and 298 K.

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 Ca-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).

Research on camelid-derived single-domain antibodies (sdAbs) has demonstrated their significant utility in diverse biotechnological applications, including therapy and diagnostic. This is largely due to their relative simplicity as monomeric proteins, ranging from 12 to 15 kDa, in contrast to immunoglobulin G (IgG) antibodies, which are glycosylated heterotetramers of 150-160 kDa. Single-domain antibodies exhibit high conformational stability and adopt the typical immunoglobulin domain fold, consisting of a two-layer sandwich of 7-9 antiparallel beta-strands. They contain three loops, known as complementary-determining regions (CDRs), which are assembled on the sdAb surface and are responsible for antigen recognition. The single-domain antibody examined in this study, sdAb-mrh-IgG, was engineered to recognize IgG from rats, mice, but it also weakly recognizes IgG from humans (Pleiner et al. 2018). A search of the Protein Data Bank revealed only one NMR structure of a single-domain antibody, which is unrelated to sdAb-mrh-IgG. The NMR chemical shift assignments of sdAb-mrh-IgG will be utilized to study its molecular dynamics and interactions with antigens in solution, which is fundamental for the rational design of novel single-domain antibodies.

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 81 kDa E. coli β clamp is a ring-shaped head-to-tail homodimer that encircles DNA and plays a central role in bacterial DNA replication by serving as a processivity factor for DNA polymerases and a binding platform for other DNA replication and repair proteins. Here we report the backbone H, N, and C NMR resonance assignments of the stabilized T45R/S107R β clamp variant obtained using standard TROSY-based triple-resonance experiments (BMRB 52548). The backbone assignments were aided by C and N edited NOESY experiments, allowing us to utilize our previously reported assignments of the β clamp ILV side-chain methyl groups (BMRB 51430, 51431). The backbone assignments of the T45R/S107R β clamp variant were transferred to the wild-type β clamp using a minimal set of TROSY-based N edited NOESY, NHCO and NHCA experiments (BMRB 52549). The reported backbone and previous ILV side-chain resonance assignments will enable NMR studies of the β clamp interactions and dynamics using amide and methyl groups as probes.

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 H,N,C 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.

In tumors, mutation in Ras proteins stimulates a signaling cascade through phosphorylation. Downstream of the cascade, many transcription and translation factors are up- or down-regulated by phosphorylation, leading to cancer progression. This phosphorylation cascade is sustained by 14-3-3ζ protein. 14-3-3ζ binds to its client proteins that are Ser/Thr-phosphorylated and prevents their dephosphorylation. One of those transcription factors is FOXO3a, whose transcriptional activity is suppressed in the phosphorylation cascade. FOXO3a binds to specific DNA sequences and activates the transcription of apoptosis-related proteins. In cancer cells, however, FOXO3a is phosphorylated, bound to 14-3-3ζ, and dissociated from the DNA, resulting in FOXO3a inactivation. To elucidate the mechanism of FOXO3a inactivation by the 14-3-3ζ binding, we aim to perform NMR analysis of the interaction between 14-3-3ζ and di-phosphorylated FOXO3a residues 1-284 (dpFOXO3a). Here, we report the backbone resonance assignments of dpFOXO3a, which are transferred from those of the N-terminal domain (NTD) and the DNA-binding domain (DBD) of dpFOXO3a.

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 C,N 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.

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.