Cereblon is a key E3 ubiquitin ligase that plays a central role in protein ubiquitination through its cooperation with other components of the ubiquitin-proteasome system. With the emergence of PROteolysis TArgeting Chimera (PROTAC) technology as a powerful strategy for inducing selective protein degradation, cereblon has become a highly attractive target in drug discovery. Although PROTAC-mediated degradation involves the assembly of a multi-protein complex, the Thalidomide-Binding Domain (TBD) of cereblon plays important roles in recruiting PROTAC molecules. In this study, we purified the human cereblon TBD for NMR studies and herein report its backbone resonance assignments. The purified TBD was shown to interact with the cereblon ligand lenalidomide, confirming its functional integrity. These resonance assignments provide a valuable foundation for characterizing ligand binding and for evaluating and optimizing small molecules targeting the cereblon TBD in targeted protein degradation strategies.

The A2 domain of von Willebrand factor (vWF A2) acts as a mechanosensor, unfolding under shear stress to enable cleavage by ADAMTS13. Dysfunction of this process causes von Willebrand disease (VWD) and thrombotic thrombocytopenic purpura (TTP). Although we previously reported the NMR assignments for mouse vWF A2, the human ortholog shares only 79% sequence identity (38 amino acid differences). Given that VWD and TTP are human pathologies, structural characterization of the human protein is essential. Here, we present the backbone 1H, 13C, and 15N resonance assignments of human vWF A2. Secondary structure propensity (SSP) analysis confirms that the solution structure retains the canonical Rossmann fold. Comparison with the mouse ortholog reveals distinct local differences in secondary structure propensities, particularly at the boundaries of α-helices and β-strands. These local variations, arising from sequence divergence, may influence the stability and unfolding dynamics relevant to human disease mechanisms.

Ubiquitin acts as a building block for a wide variety of poly-ubiquitin chains. Decoding the role of poly-ubiquitin chains in different cellular processes remains an active area of research. Here, we report amide 1H and 15N signal assignments of each ubiquitin unit in di-ubiquitins of all seven lysine linkages and in M1-linked di-ubiquitin determined by our lab over the last decade. These assignments can aid in NMR studies of the structure, dynamics, and function of various di-ubiquitins. Comparison of the NMR resonance assignments among all the di-ubiquitins revealed linkage-specific chemical shifts and isopeptide signals that can be used as "fingerprints" to directly identify using NMR spectroscopy the linkage type in a di-ubiquitin and potentially longer poly-ubiquitin chains. Our data highlight both the similarities and dissimilarities of NMR signals of ubiquitin units in di-ubiquitins of different linkages, as well as the importance of selective isotopic labeling of specific ubiquitin units in a poly-ubiquitin chain for NMR studies.

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.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12104-026-10260-x.

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 (NPTXRPTX) is expected to mediate interactions with synaptic partners but has not been structurally characterized. Here, we report near-complete backbone NMR resonance assignments of NPTXRPTX 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 1HN, 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.