The tumor suppressor protein breast cancer type 1 susceptibility protein (BRCA1) plays a central role in maintaining genome stability through its involvement in DNA damage repair, transcriptional regulation, and cell-cycle control. BRCA1 functions as an obligate heterodimer with its binding partner, the BRCA1-associated RING domain protein 1 (BARD1), to coordinate accurate DNA repair. While the structured N- and C-terminal domains of BRCA1 have been well-characterized, the large central region encoded largely by exon 11 that comprises 80
The BRCA1-associated RING domain protein 1 (BARD1) is the obligate binding partner of the tumor suppressor breast cancer type 1 susceptibility protein (BRCA1) and plays a critical role in maintaining genome integrity. BARD1 contains structured N- and C-terminal domains that mediate heterodimerization with BRCA1, recognition of chromatin marks, and DNA repair functions. Approximately 40
Protein and nucleic acid alkylation are important genetic and epigenetic modifications. The dynamic balance between alkylation and dealkylation is regulated by distinct sets of enzymes and is essential for maintaining genomic stability. Escherichia coli AlkB is a member of the Alkylation B (AlkB) family of dioxygenases that dealkylates a wide range of nucleic acid substrates in E. coli, thereby playing a crucial role in cellular repair processes and epigenetic regulation. Here, we report the backbone 1H, 15N, 13C chemical shift assignment of E. coli AlkB in complex with Zn2+ and α-ketoglutarate. Experiments were acquired at 20 °C by heteronuclear multidimensional NMR spectroscopy. Collectively, 91
Disintegrins are a group of cysteine-rich proteins found in a wide variety of snake venoms. These proteins selectively bind to integrins, which play fundamental roles in the regulation of numerous physiological and pathological processes. Here, we report the NMR chemical shift assignments for 1H, 15N, and 13C nuclei in the backbone and side chains of the recombinant disintegrin jararacin (rJarc). The assignments were further validated by secondary structure prediction using the TALOS-N server. Despite sharing 86.4% sequence identity with jarastatin and 83.6% with trimestatin, rJarc displays a distinct dynamic behavior. Owing to this behavior, the assignment process was challenging due to the lack of observable correlations in triple-resonance experiments for several resonances. Taken together, these data provide an essential basis for NMR-based investigations, including structure determination and characterization of its unique dynamic properties.
Disintegrins are a group of cysteine-rich proteins found in a wide variety of snake venoms. These proteins selectively bind to integrins, which play fundamental roles in the regulation of numerous physiological and pathological processes. Here, we report the NMR chemical shift assignments for 1H, 15N, and 13C nuclei in the backbone and side chains of the recombinant disintegrin jararacin (rJarc). The assignments were further validated by secondary structure prediction using the TALOS-N server. Despite sharing 86.4
ADP-ribosylation is a reversible post-translational modification that regulates diverse cellular processes, including DNA damage repair, transcription, cell proliferation and innate immune responses, and is primarily catalyzed by members of the PARP family. While all 17 human PARPs contain a conserved C-terminal ADP-ribosyltransferase (ART) domain, only catalytically active members transfer ADP-ribose (ADPr) from nicotinamide adenine dinucleotide (NAD⁺) onto proteins or nucleic acids, and their N-terminal accessory domains, such as macro domains (MDs), WWE domains or RNA-binding motifs, mediate interactions that diversify PARP functions. Human PARP9 (hPARP9), known also as BAL1, is catalytically inactive due to sequence variations in catalytically important residues in the ART domain, but plays crucial roles in antiviral and antibacterial defense, stress responses and tumor progression through its heterodimeric interaction with the E3 ubiquitin ligase DTX3L. hPARP9 contains two tandem MDs (MD1 and MD2), with MD1 acting as a MacroD-type hydrolase “eraser” of mono-ADP-ribosylation (MARylation), while MD2 functions as an ADPr “reader”. Their different role in the ADP-ribosylation pathway highlights the importance of structural and functional characterization for understanding ADPr-mediated cellular signaling. In this study, we report the NMR backbone and side-chain resonance assignments of hPARP9 MD1 in both apo and ADPr bound states. In addition, the secondary structure predictions using TALOS+ server and the Chemical Shift Perturbation (CSP) analysis upon ADPr binding are presented. The latter illustrates the MD substrate’s accommodation mode and identifies the residues involved in ADPr binding, thus related to MDs’ hydrolytic activity.
Cytoplasmic polyadenylation element-binding protein 3 (CPEB3) is an RNA-binding protein that is essential for long-term memory formation. Its N-terminal intrinsically disordered region (residues 1-459) exhibits high aggregation propensity and regulates the translation of specific mRNAs, including those encoding AMPA receptor subunits, through processes such as liquid-liquid phase separation and the formation of fibrillar structures. However, the molecular basis of these regulatory mechanisms remains poorly understood. In this study, we present the backbone resonance assignments of three segments within the intrinsically disordered region of CPEB3 (residues 1-120, 186-315, and 400-459). In agreement with sequence-based secondary structure predictions, the three segments were predominantly disordered overall. However, short regions with partial helical propensity were identified at residues 3-7 in the 1-120 segment and residues 226-239 in the 186-315 segment.
Staphylococcal nuclease and Tudor domain‑containing protein 1 (SND1) is a multifunctional RNA‑binding protein implicated in transcriptional regulation, post‑transcriptional RNA control, oncogenesis, and viral infection. Initially identified as a transcriptional coactivator, SND1 was later established as a component of the RNA‑induced silencing complex, where it contributes to RNA turnover and microRNA regulation. SND1’s diverse activities stem from its modular architecture, comprising four staphylococcal nuclease‑like domains, capable of direct RNA binding, and an extended Tudor domain that together form an integrated RNA‑binding and catalytic platform. This versatility also underlies its role in viral infection: SND1 acts as an m⁶A reader and is exploited by RNA viruses, such as SARS‑CoV‑2. Recent work showed that SND1 depletion, particularly loss of its third structured domain (SN3), reduces recruitment of the viral protein Nsp9 to the 3′ untranslated region of the SARS‑CoV‑2 genome, impairing viral RNA synthesis through a direct SN3–Nsp9 interaction. Here, we report expression, purification, and near‑complete backbone NMR assignments of the SN3 domain of SND1. Secondary structure elements calculated by TALOS-N based on these assignments are in good agreement with the existing crystal structure of SN3. Our data provide an excellent foundation for future structural studies of SND1–RNA complexes and their roles in viral RNA priming in SARS-CoV-2.
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.
Knotted Methyltransferases (MTase) from the SpoU-TrmD (SPOUT) family offer a unique opportunity to study a protein knot topology and enzymatic function. The knotted methyltransferase from Staphylococcus aureus, MTTSA (PDB: 1vh0, 4fak), is an ɑ/β-knotted 23s rRNA MTase containing only the minimal scaffold among the SPOUT family members. This dimeric enzyme is a minimalist model to study the deep + 31 knot. Here, we report the non-proline backbone assignments with 98.8
Poly(ADP-ribose) polymerase 2 (PARP2) is a key sensor of DNA single-strand breaks that catalyzes ADP-ribosylation of itself and other substrates to initiate DNA repair. Human PARP2 contains an intrinsically disordered N-terminal domain (NTD) that mediates chromatin association and nuclear localization, a central WGR domain that recognizes DNA breaks, and a catalytic domain responsible for poly(ADP-ribose) synthesis. Despite its importance in genome maintenance and as a target of clinical PARP inhibitors, detailed information on the structural and dynamic properties of the NTD and WGR domains has remained limited. Here, we report the 1H, 13C, and 15N resonance assignments of the N-terminal intrinsically disordered region (residues 1–89) and the WGR domain (residues 90–212) of human PARP2. Resonance assignments were first obtained separately for each domain and subsequently transferred to a combined NTD–WGR construct. These assignments provide a foundation for future studies investigating the conformational dynamics, DNA recognition mechanisms, and allosteric regulation of PARP2 in chromatin and repair signaling.
Mucoricin is a key virulence factor in mucormycosis, as it targets host endothelial cells and inhibits protein translation through its N-glycosylase activity, leading to vascular leak and increased vascular permeability, thereby contributing to further disease progression. Its structure is quite unique, being structurally homologous to the Ricin B chain as confirmed by cross-reactivity with monoclonal anti-Ricin B chain antibodies, while exhibiting enzymatic activity similar to the Ricin A chain. Here, we report near-complete backbone (Soliman in Nat Microbiol 6(3):313–326, 2021) 1H, 15N, and 13C chemical shift assignments of Mucoricin from Rhizopus delemar at pH 7.5 using NMR spectroscopy. This represents a critical step toward identifying unique structural elements that contribute to its compact structure and toxin activity, which have remained evolutionarily conserved. The secondary structure probabilities derived from the chemical shift data are well aligned with the predicted AlphaFold model, reinforcing the accuracy of the assigned resonances and the structural integrity of recombinant Mucoricin.
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
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.
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.
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.
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.
