Backbone assignments of the biotin carboxyl carrier protein domain of Propionyl CoA carboxylase of Leishmania major and its interaction with its cognate Biotin protein ligase.

Propionyl CoA carboxylase (PCC) is a multimeric enzyme composed of two types of subunits, α and ß arranged in α6ß6 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 ß-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 ß-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.

1H, 13C and 15N assignment of self-complemented MrkA protein antigen from Klebsiella pneumoniae.

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.

Backbone triple resonance assignments of the dimerization domain of NF-kappaB p52 subunit.

NF-kappaB is a family of inducible transcription factors playing an important role in immune response in vertebrates. All the five members of the family function as dimers in various combinations. Though all the family members recognize and bind to similar DNA elements to regulate the transcription of its target genes, the dimer composition can lead to differential transcriptional outcomes. Here we report the backbone resonance assignment of the 24.2 kDa homodimer of p52 subunit of the NF-kB family. The p52 subunit of NF-kB is a crucial player in the non-canonical NF-kB pathway and its dysregulation has shown detrimental effects in immune response leading to various inflammatory diseases and cancers. While the ß-strands predicted using the backbone chemical shifts in this study largely conform with the available crystal structure, the helical turns present in the crystal structure are not observed in our results.

Resonance assignments of cytochrome MtoD from the extracellular electron uptake pathway of sideroxydans lithotrophicus ES-1.

The contribution of Fe(II)-oxidizing bacteria to iron cycling in freshwater, groundwater, and marine environments has been widely recognized in recent years. These organisms perform extracellular electron transfer (EET), which constitutes the foundations of bioelectrochemical systems for the production of biofuels and bioenergy. It was proposed that the Gram-negative bacterium Sideroxydans lithotrophicus ES-1 oxidizes soluble ferrous Fe(II) at the surface of the cell and performs EET through the Mto redox pathway. This pathway is composed by the periplasmic monoheme cytochrome MtoD that is proposed to bridge electron transfer between the cell exterior and the cytoplasm. This makes its functional and structural characterization, as well as evaluating the interaction process with its physiological partners, essential for understanding the mechanisms underlying EET. Here, we report the complete assignment of the heme proton and carbon signals together with a near-complete assignment of 1H, 13C and 15N backbone and side chain resonances for the reduced, diamagnetic form of the protein. These data pave the way to identify and structurally map the molecular interaction regions between the cytochrome MtoD and its physiological redox partners, to explore the EET processes of S. lithotrophicus ES-1.

Backbone 1H, 13C and 15N resonance assignment of the ubiquitin specific protease 7 catalytic domain (residues 208-554) in complex with a small molecule ligand.

The backbone 1H, 13C and 15N resonance assignment of Ubiquitin Specific Protease 7 catalytic domain (residues 208-554) was performed in its complex with a small molecule ligand and in its apo form as a reference. The amide 1H-15N signal intensities were boosted by an amide hydrogen exchange protocol, where expressed 2H, 13C, 15N-labeled protein was unfolded and re-folded to ensure exchange of amide deuterons to protons. The resonance assignments were used to determine chemical shift perturbations on ligand binding, which are consistent with the binding site observed by crystallography.

5D solid-state NMR spectroscopy for facilitated resonance assignment.

1H-detected solid-state NMR spectroscopy has been becoming increasingly popular for the characterization of protein structure, dynamics, and function. Recently, we showed that higher-dimensionality solid-state NMR spectroscopy can aid resonance assignments in large micro-crystalline protein targets to combat ambiguity (Klein et al., Proc. Natl. Acad. Sci. U.S.A. 2022). However, assignments represent both, a time-limiting factor and one of the major practical disadvantages within solid-state NMR studies compared to other structural-biology techniques from a very general perspective. Here, we show that 5D solid-state NMR spectroscopy is not only justified for high-molecular-weight targets but will also be a realistic and practicable method to streamline resonance assignment in small to medium-sized protein targets, which such methodology might not have been expected to be of advantage for. Using a combination of non-uniform sampling and the signal separating algorithm for spectral reconstruction on a deuterated and proton back-exchanged micro-crystalline protein at fast magic-angle spinning, direct amide-to-amide correlations in five dimensions are obtained with competitive sensitivity compatible with common hardware and measurement time commitments. The self-sufficient backbone walks enable efficient assignment with very high confidence and can be combined with higher-dimensionality sidechain-to-backbone correlations from protonated preparations into minimal sets of experiments to be acquired for simultaneous backbone and sidechain assignment. The strategies present themselves as potent alternatives for efficient assignment compared to the traditional assignment approaches in 3D, avoiding user misassignments derived from ambiguity or loss of overview and facilitating automation. This will ease future access to NMR-based characterization for the typical solid-state NMR targets at fast MAS.

Backbone 1H, 15N and 13C resonance assignments for an E2 ubiquitin conjugating enzyme-UBE2T.

Ubiquitin-conjugating enzyme E2 T (UBE2T) plays important roles in ubiquitination of proteins through participation in transferring ubiquitin to its substrate. Due to its importance in protein modifications, UBE2T associates with diverse diseases and serves as an important target for drug discovery and development. The crystal structure of UBE2T has been determined and the structure reveals the lack of a druggable pocket for binding to small molecules for clinical applications. Despite the challenge, effort has been made to develop UBE2T inhibitors. We obtained UBE2T constructs with and without the C-terminal region which is flexible in solution. Herein, we report the backbone resonance assignments for human UBE2T without the C-terminal region. The backbone dynamics of UBE2T was also explored. The available assignments will be helpful for hit identification, determining ligand binding site and understanding the mechanism of action of UBE2T inhibitors.

Resonance assignments of the PUB domain of the RNF31 protein.

E3 ubiquitin protein ligase RNF31 is present in human proteins and is involved in linear ubiquitin chain assembly complex (LUBAC) activity and cell growth. RNF31 is involved in ubiquitination, which is the post-translational modification of proteins. Ubiquitin molecules connect with amino acid residues of target proteins under the action of ubiquitin-activating enzyme E1, ubiquitin binding enzyme E2 and ubiquitin ligase E3, so as to achieve certain physiological functions. The abnormal expression of ubiquitination promotes the formation of cancer. In studies of breast cancer, RNF31 mRNA levels were found to be higher in cancer cells than in other tissues. The PUB domain of RNF31 is the binding site of the ubiquitin thioesterase otulin. Here, we report the backbone and side-chain resonance assignments of the PUB domain of RNF31 and study the backbone relaxation of the domain. These studies will contribute to further understanding of the structural and functional relationship of RNF31 protein, which may also be a target for drug research.

1H, 13C, and 15N resonance assignments of SarA monomer from Staphylococcus aureus in complex with DNA.

SarA is a global transcription regulator in S. aureus which regulates the expression of over 120 genes related to quorum sensing, biofilm synthesis, drug resistance and many other important physiological processes during host infection. SarA can bind to the promoter region of agr and other target genes to activate or repress the transcription. The crystal structure of SarA uncovered a MarR protein-like conformation with two symmetrical winged helix domains, while its DNA binding mechanism is still unknown. We have constructed a monomeric DNA binding domain of SarA (SarAΔN19) for the study of the interaction between SarA and DNA with NMR spectroscopy. Here, we report the 1H, 13C and 15N NMR assignment of SarAΔN19/DNA complex which is the first step towards further structure and function analysis.

Complete non-proline backbone resonance assignments of the S. aureus neutrophil serine protease inhibitor, EapH1.

The S. aureus extracellular adherence protein (Eap) and its homologs, EapH1 and EapH2, serve roles in evasion of the human innate immune system. EapH1 binds with high-affinity and inhibits the neutrophil azurophilic granule proteases neutrophil elastase, cathepsin-G and proteinase-3. Previous structural studies using X-ray crystallography have shown that EapH1 binds to neutrophil elastase and cathepsin-G using a globally similar binding mode. However, whether the same holds true in solution is unknown and whether the inhibitor experiences dynamic changes following binding remains uncertain. To facilitate solution-phase structural and biochemical studies of EapH1 and its complexes with neutrophil granule proteases, we have characterized EapH1 by multidimensional NMR spectroscopy. Here we report a total of 100% of the non-proline backbone resonance assignments of EapH1 with BMRB accession number 50,304.

1H, 15N, and 13C chemical shift backbone resonance NMR assignment of the accumulation-associated protein (Aap) lectin domain from Staphylococcus epidermidis.

Staphylococcus epidermidis is the leading causative agent for hospital-acquired infections, especially device-related infections, due to its ability to form biofilms. The accumulation-associated protein (Aap) of S. epidermidis is primarily responsible for biofilm formation and consists of two domains, A and B. It was found that the A domain is responsible for the attachment to the abiotic/biotic surface, whereas the B domain is responsible for the accumulation of bacteria during biofilm formation. One of the parts of the A domain is the Aap lectin, which is a carbohydrate-binding domain having 222 amino acids in its structure. Here we report the near complete backbone chemical shift assignments for the lectin domain, as well as its predicted secondary structure. This data will provide a platform for future NMR studies to explore the role of lectin in biofilm formation.

Utilizing the 1H-15N NMR Methods for the Characterization of Isomeric Human Milk Oligosaccharides.

Human milk oligosaccharides (HMOs) are structurally complex unconjugated glycans that are the third largest solid fraction in human milk after lactose and lipids. HMOs are in the forefront of research since they have been proven to possess beneficial health effects, especially on breast-fed neonates. Although HMO research is a trending topic nowadays, readily available analytical methods suitable for the routine investigation of HMOs are still incomplete. NMR spectroscopy provides detailed structural information that can be used to indicate subtle structural differences, particularly for isomeric carbohydrates. Herein, we propose an NMR-based method to identify the major isomeric HMOs containing GlcNAc and/or Neu5Ac building blocks utilizing their amide functionality. Experimental conditions were optimized (H2O:D2O 9:1 v/v solvent at pH 3.0) to obtain 1H-15N HSQC and 1H-15N HSQC-TOCSY NMR spectra of the aforementioned building blocks in HMOs. Four isomeric HMO pairs, LNT/LNnT, 3'SL/6'SL, LNFP II/LNFP III, and LSTa/LSTb, were investigated, and complete NMR resonance assignments were provided. In addition, 1H and 15N NMR resonances were found to be indicative of various linkages, thereby facilitating the distinction of isomeric tri-, tetra-, and pentasaccharide HMOs. The rapid growth of HMO products (from infant formulas and dietary supplements to cosmetics) undoubtedly requires expanding the range of applicable analytical methods. Thus, our work provides a 15N NMR-based method to advance this challenging field of carbohydrate analysis.

1H, 15N and 13C resonance assignments of a minimal CPSF73-CPSF100 C-terminal heterodimer.

The initial pre-mRNA transcript in eukaryotes is processed by a large multi-protein complex in order to correctly cleave the 3' end, and to subsequently add the polyadenosine tail. This cleavage and polyadenylation specificity factor (CPSF) is composed of separate subunits, with structural information available for both isolated subunits and also larger assembled complexes. Nevertheless, certain key components of CPSF still lack high-resolution atomic data. One such region is the heterodimer formed between the first and second C-terminal domains of the endonuclease CPSF73, with those from the catalytically inactive CPSF100. Here we report the backbone and sidechain resonance assignments of a minimal C-terminal heterodimer of CPSF73-CPSF100 derived from the parasite Encephalitozoon cuniculi. The assignment process used several amino-acid specific labeling strategies, and the chemical shift values allow for secondary structure prediction.

1H, 13C, 15N Backbone and sidechain chemical shift assignments of the C-terminal domain of human UDP-glucuronosyltransferase 2B17 (UGT2B17-C).

UDP-glucuronosyltransferases are the principal enzymes involved in the glucuronidation of metabolites and xenobiotics for physiological clearance in humans. Though glucuronidation is an indispensable process in the phase II metabolic pathway, UGT-mediated glucuronidation of most prescribed drugs (> 55%) and clinical evidence of UGT-associated drug resistance are major concerns for therapeutic development. While UGTs are highly conserved enzymes, they manifest unique substrate and inhibitor specificity which is poorly understood given the dearth of experimentally determined full-length structures. Such information is important not only to conceptualize their specificity but is central to the design of inhibitors specific to a given UGT in order to avoid toxicity associated with pan-UGT inhibitors. Here, we provide the 1H, 13C and 15N backbone (~ 90%) and sidechain (~ 62%) assignments for the C-terminal domain of UGT2B17, which can be used to determine the molecular binding sites of inhibitor and substrate, and to understand the atomic basis for inhibitor selectivity between UGT2B17 and other members of the UGT2B subfamily. Given the physiological relevance of UGT2B17 in the elimination of hormone-based cancer drugs, these assignments will contribute towards dissecting the structural basis for substrate specificity, selective inhibitor recognition and other aspects of enzyme activity with the goal of selectively overcoming glucuronidation-based drug resistance.

The early days of ex vivo 1 H, 13 C, and 31 P nuclear magnetic resonance in the laboratory of Dr. Robert G. Shulman from 1975 to 1995.

This paper provides a brief description of the early use of ex vivo nuclear magnetic resonance (NMR) studies of tissue and tissue extracts performed in the laboratory of Dr. Robert G. Shulman from 1975 through 1995 at Bell Laboratories, then later at Yale University. During that period, ex vivo NMR provided critical information in support of resonance assignments and the quantitation of concentrations for magnetic resonance spectroscopy studies. The period covered saw rapid advances in magnet technology, starting with studies of microorganisms in vertical bore high-resolution NMR studies, then by 1981 studies of small mammals in a horizontal bore magnet, and then studies of humans in 1984. Ex vivo NMR played a critical role in all these studies. A general strategy developed in the lab for using ex vivo NMR to support in vivo studies is presented, as well as illustrative examples.

AssignSLP_GUI, a software tool exploiting AI for NMR resonance assignment of sparsely labeled proteins.

Not all proteins are amenable to uniform isotopic labeling with 13C and 15N, something needed for the widely used, and largely deductive, triple resonance assignment process. Among them are proteins expressed in mammalian cell culture where native glycosylation can be maintained, and proper formation of disulfide bonds facilitated. Uniform labeling in mammalian cells is prohibitively expensive, but sparse labeling with one or a few isotopically enriched amino acid types is an option for these proteins. However, assignment then relies on accessing the best match between a variety of measured NMR parameters and predictions based on 3D structure, often from X-ray crystallography. Finding this match is a challenging process that has benefitted from many computational tools, including trained neural nets for chemical shift prediction, genetic algorithms for searches through a myriad of assignment possibilities, and now AI-based prediction of high-quality structures for protein targets. AssignSLP_GUI, a new version of a software package for assignment of resonances from sparsely-labeled proteins, uses many of these tools. These tools and new additions to the package are highlighted in an application to a sparsely-labeled domain from a glycoprotein, CEACAM1.

Backbone and side chain NMR assignments for the ribosome maturation factor P (RimP) from Staphylococcus aureus.

The ribosomal maturation factor (RimP) is a 17.7 kDa protein and is the assembly factor of the 30S subunit. RimP is essential for efficient processing of 16S rRNA and maturation (assembly) of the 30S ribosome. It was suggested that RimP takes part in stabilization of the central pseudoknot at the early stages of the 30S subunit maturation, and this process may occur before the head domain assembly and later stages of the 30S assembly, but the mechanism of this interaction is still not fully understood. Here we report the assignment of the 1H, 13C and 15N chemical shift in the backbone and side chains of RimP from Staphylococcus aureus. Analysis of chemical shifts of the main chain using TALOS + suggests that the RimP contains eight ß-strands and three α-helices with the topology α1-ß1-ß2-α2- ß3- α3- ß4- ß5- ß6- ß7- ß8. Structural studies of RimP and its complex with the ribosome by integrated structural biology approaches (NMR spectroscopy, X-ray diffraction analysis and cryoelectron microscopy) will allow further screening of highly selective inhibitors of the translation of S. aureus.

Resonance assignments of the ORC2-WH domain of the human ORC protein.

ORC2 is a small subunit of the origin recognition complex (ORC), which is important for gene replication. The ORC2 WH domain recognizes dsDNA sequences with its flexible ß-sheet hairpins as anchors. Here, we report near-complete NMR backbone and side chain resonance assignments of the WH domain and study the backbone relaxation of the WH domain. These studies will contribute to further understanding of the structure-function relationship of the ORC protein.

1H, 15N and 13C backbone and side chain solution NMR assignments of the truncated small hepatitis delta antigen Δ60-S-HDAg.

Hepatitis D virus (HDV) is a defective virus that relies on hepatitis B virus envelope proteins to complete its replication cycle. The HDV genome contains two isoforms of hepatitis delta antigen: the small and the large hepatitis delta antigens (S- and L-HDAg). Here we report the 1H, 13C and 15 N backbone and side chain resonance assignments of an N-terminally truncated form of S-HDAg (SΔ60), which lacks the 1-60 oligomerization domain. We derived secondary structures based on NMR chemical shifts, which will be used in further structural and functional studies. We show that SΔ60 is partially disordered, and that the central structured part contains two well-defined α-helices of 22 and 17 residues, respectively. A temperature titration allowed to identify the residues involved in hydrogen bonds.

1H, 13C and 15N resonance assignments of TbBDF5-bromo1 domain from Trypanosoma brucei.

1H, 13C and 15N resonance assignments are presented for the first bromo domain of TbBDF5 (TbBDF5-bromo1) from Trypanosoma brucei. TbBDF5 is localized in the nucleus and plays a potential role in transcription regulation. Bromo domains can recognize acetylated histone through a conserved binding pocket. Here we report the NMR resonance assignments of TbBDF5-bromo1 domain for further studies of the relationship between its structure and function.