Biomolecular NMR Assignments最新文献

Molten globules are compact, partially-folded proteins postulated to be general intermediates in protein folding. Human α-lactalbumin (α-LA) is a Ca²⁺-binding, four-disulphide protein whose native structure is divided into two lobes, one is largely helical, the α-domain, and the other has a significant β-sheet content, the β-domain. α-LA forms a “classical” molten globule at low pH which has been studied widely as a model system of a partially-folded protein. The α-LA molten globule is compact and has a native-like helical secondary structure content. All-Ala α-LA, which has all eight native cysteines mutated to alanine, also adopts a partially-folded molten globule conformation and gives a high-quality ¹H-¹⁵N HSQC spectrum at pH 2 and 40 °C. The lack of cysteine residues makes all-Ala α-LA a suitable template for spin-labelling studies. In this report we present ¹H, ¹³C and ¹⁵N assignments for human all-Ala α-LA in its molten globule and 8 M urea-denatured states. Analysis of the chemical shift data for the molten globule state shows they are consistent with high populations of conformations in the α region of φ,ψ space for residues in the α domain of the protein. In contrast, the data for the urea-denatured state are closely similar to those expected for a random coil.

Neuronal pentraxin receptor (NPTXR) is a synaptic organizing protein important for excitatory neurotransmission, yet its structural properties remain poorly defined. The conserved C-terminal pentraxin (PTX) domain of NPTXR (NPTXR^PTX) is expected to mediate interactions with synaptic partners but has not been structurally characterized. Here, we report near-complete backbone NMR resonance assignments of NPTXR^PTX using uniformly ¹⁵N, ¹³C-labeled protein. These assignments provide a foundation for further studies of NPTXR-ligand interactions that drive NPTXR-dependent synapse organization and will advance our understanding of the molecular mechanisms underlying synaptic assembly and maintenance.

Vitamin A is essential for vision and many other biological processes required for human health and survival. Extracellular retinol binding protein (RBP) delivers vitamin A into the cell upon binding to the vitamin A transporter, STRA6. However, when retinol free RBP binds to STRA6, it induces vitamin A transport out of the cell. The bi-directionality of vitamin A transport is thought to be regulated further by an intracellular protein-protein interaction (PPI) between STRA6 and the EF-hand Ca²⁺-binding protein, calmodulin (CaM). Insights regarding how CaM regulates vitamin A transport were originally provided at atomic resolution by a cryoEM structure of the zebrafish STRA6-CaM complex. This cryoEM structure, together with NMR studies, confirmed that three STRA6 helices (i.e., BP0, BP1, and BP2) comprised the CaM-STRA6 binding interface, with BP2 providing the major set of interactions. NMR and other biophysical methods demonstrated that zebrafish BP2 peptide (zfBP2) binding to CaM involved a Ca²⁺-dependent type 2 binding and functional folding mechanism of action, which could influence structural, dynamic, and allosteric functions of STRA6. To expand our understanding of vitamin A transport to a mammalian STRA6 transporter, the backbone and sidechain ¹H^N, ¹³C, and ¹⁵N resonances were assigned here for ^CaCaM (148 residues) when bound to a sheep BP2 peptide (32 residues) (shBP2). Interestingly, the NMR data showed ^CaCaM resonances were affected differently upon binding shBP2 versus zfBP2. Such differences may be useful for distinguishing important features regarding ^CaCaM complexes with mammalian versus zebrafish STRA6.

Adhesin P1 (aka AgI/II) is an extracellular protein regulating adherence and detachment of Streptococcus mutans in the oral cavity and thus plays a pivotal role in biofilm development and maturation. P1’s naturally occurring C-terminal truncation product, Antigen II (AgII), adopts both soluble, monomeric and insoluble, amyloidogenic forms during the bacterial life cycle. Monomeric AgII forms important quaternary interactions with P1’s A3VP1 segment that is projected from the bacterial cell surface to promote cell adhesion, while the functional amyloid form of AgII promotes detachment of mature biofilms. The heterologous recombinant 51-kD C123 construct, comprising most of AgII, has been characterized by X-ray crystallography and serves as a functional surrogate of AgII in studies of adhesion and biofilm regulation. C123 contains three structurally similar domains, C1, C2, and C3. Using Alphafold prediction and the C123 crystal structure, we identified domain boundaries within C123 to develop more tractable constructs for NMR studies, including quaternary interactions with other proteins. The C2 domain is of particular interest because it contains several unique helices in addition to the β-sheet fold it shares with the C1 and C3 domains. Here we report the backbone NMR resonance assignments for the C2 construct. Secondary structure predictions from NMR assignments are in good agreement with those anticipated by Alphafold and the observed crystal structure, except for some of the helices suggesting they are more dynamic. We then compare C2 chemical shift perturbations caused by quaternary interactions with recombinant A3VP1, as well as by a monoclonal antibody, MAb 6–8C, known to inhibit bacterial adherence and C123 binding to A3VP1. We note the C2 chemical shift perturbations are markedly different from previously observed interactions of C3 with A3VP1 and MAb 6–8C, providing further insight on how the individual domains of C123 may vary in their ability to mediate bacterial adhesion and formation of functional amyloid. The prior NMR assignment and characterization of C3 combined with the NMR assignment and characterization of C2 described here provide a foundation for further NMR studies, including assignment of C23 and C123 constructs, protein-protein interaction studies of C23 and C123, assessing the impact of environmental conditions on structure and dynamics within C123 as it transitions from monomer to amyloid form, and the functional relevance of having three successive domains with similar tertiary folds.

Transcription mediated by RNA polymerase II (RNAPII) involves multiple stages, including initiation, promoter-proximal pausing for capping, elongation, and termination. The C-terminal domain of RNAPII (CTD) contains repetitions of the heptad consensus sequence, such as Y₁S₂P₃T₄S₅P₆S₇ with some variety, and the phosphorylated positions in the heptad sequences are altered according to the transcriptional stages. The interaction between several regulatory protein factors and the phosphorylated heptad sequence plays an important role in the accurate progress of transcription. A subset of these regulatory proteins possesses a CTD-interacting domain (CID) that specifically recognizes the phosphorylated CTD and mediates stage-specific transcriptional control. Among them, SCAF8 (RBM16), which also contains a CID, plays a key role in accurate transcriptional termination in conjunction with its paralog SCAF4. Despite their importance, the precise molecular mechanisms through which SCAF8 and SCAF4 coordinate transcriptional termination via their CID domains remain poorly understood. In this study, we report the ¹H, ¹⁵N, and ¹³C NMR resonance assignments and solution structure of the human SCAF8 CID domain. The structure exhibits an α1–α2–α3–α4–α5–α6–α7–α8 helical topology, consistent with the previously determined crystal structure. These assignments provide a valuable foundation for understanding how SCAF8 interacts with the RNAPII CTD and contributes to transcriptional elongation and termination.

ATPase inhibitory factor 1 (IF1) is the only known endogenous, proteinaceous inhibitor of mitochondrial ATP synthase in mammals. The inhibitor forms an antiparallel coiled-coil, which binds ATP synthase through an N-terminal α-helix extension that is disordered in the free protein. Because the IF1 dimer affects mitochondrial bioenergetics through its modulation of ATP synthase, it is a therapeutic target for cancer and cardiac disease. Here, we report ¹H, ¹³C and ¹⁵N NMR assignments for the mature dimeric form of human IF1. Secondary structure analyses based on chemical shifts and short-range NOE patterns indicate the N-terminal half of the 81-residue IF1 is intrinsically disordered, while the C-terminal half adopts a continuous α-helix. The chemical shift assignments for human IF1 provide a foundation for future mechanistic structure-function studies and NMR-based drug screening.

Lytic polysaccharide monooxygenases (LPMOs) are mono-copper binding enzymes involved in the degradation of carbohydrates. The 25 kDa sized LPMO LsAA9A from the basidiomycete Lentinus similis is known to oxidate cellulose and cellooligomers at the C4 position and thus leading to a breakage of the glycosidic bond. LsAA9A has been recombinantly expressed in Escherichia coli with ¹³C and ¹⁵N labelling. Here, we present the ¹H, ¹³C and ¹⁵N backbone resonance assignment of the apo form. The secondary structure was predicted using the TALOS-N software and it was overall in agreement with the crystal structure of LsAA9A expressed in E. coli. A few shorter α-helices and β-sheets present in the crystal structure are missing in the NMR prediction and vice versa. LsAA9A resembles the typical structural elements of LPMOs with a core β-sandwich.

Chronic infections with hepatitis B virus (HBV) are a leading cause of liver cirrhosis and hepatocellular carcinoma worldwide. Among the four viral proteins encoded by HBV, the X protein (HBx) has remained resistant to atomic-level characterization. HBx is a small, non-structural protein that interacts with various human host proteins. It is essential for HBV replication and implicated in HBV-induced carcinogenesis. Here, we present the sequential backbone resonance assignments of a C-terminally truncated HBx isoform (residues 1–120), which is frequently found in chronically infected patients. Three-dimensional NMR experiments were recorded in the presence of residual urea (1 M), and the assignments of amide moieties were subsequently transferred to a low-urea condition (< 0.2 M) compatible with HBx interaction studies. We compare manual assignments with automated predictions using the ARTINA software. These results reveal secondary structure propensities in the truncated HBx isoform and lay the groundwork for future NMR-based studies of HBx interactions in solution.

LCC^ICCG, a bioengineered variant of a cutinase called LCC (Leaf-branch Compost Cutinase), is a high-performance, industrial-grade enzyme capable of efficiently degrading polyethylene terephthalate (PET). This engineered enzyme exhibits significantly enhanced thermal stability and PET hydrolysis activity compared to its predecessor and competing PETases. Here, we report the comprehensive resonance assignment of the polypeptide backbone and the side chain methyl groups of the active LCC^ICCG. Taking advantage of its exceptional thermostability all the experiments were conducted at 60 °C on a single, uniformly ¹⁵N-¹³C-labeled sample of this 27 kDa serine-hydrolase enzyme. LCC^ICCG represents a leap forward in enzymatic PET recycling, combining speed, efficiency, and scalability. The residue-specific information through both backbone and methyl side chain assignment represents a critical step toward detailed structural and dynamic NMR analyses.

p47/NSFL1C is a key adaptor protein of p97/VCP, a AAA+ ATPase that is involved in a diverse array of cellular functions. p47 directs p97 activity towards membrane remodelling and contributes to the regulation of cellular pathways such as NF-κB signalling and autophagy. p47 consists of three folded domains (UBA, SEP, UBX) connected by extended intrinsically disordered regions (IDRs). The dynamic and flexible nature of p47 has hindered high-resolution structural characterization of the full-length protein and its complex with p97 by X-ray crystallography or cryo-EM. Here, we report NMR backbone resonance assignments and secondary structure propensities of full-length p47, as well as of a truncated construct containing a SEP-interacting motif (SIM) and the SEP domain (residues 101–266). These findings provide the basis for future NMR studies aimed at elucidating the structural mechanisms by which p47 regulates p97 activity and for rational design of molecular binders to selectively target p47 and modulate specific p47-dependent pathways.