Backbone NMR Research Articles

Backbone NMR resonance assignment of the apo human Tsg101-UEV domain.

The Endosomal Sorting Complex Required for Transport (ESCRT) pathway, through inverse topology membrane remodeling, is involved in many biological functions, such as ubiquitinated membrane receptor trafficking and degradation, multivesicular bodies (MVB) formation and cytokinesis. Dysfunctions in ESCRT pathway have been associated to several human pathologies, such as cancers and neurodegenerative diseases. The ESCRT machinery is also hijacked by many enveloped viruses to bud away from the plasma membrane of infected cells. Human tumor susceptibility gene 101 (Tsg101) protein is an important ESCRT-I complex component. The structure of the N-terminal ubiquitin E2 variant (UEV) domain of Tsg101 (Tsg101-UEV) comprises an ubiquitin binding pocket next to a late domain [P(S/T)AP] binding groove. These two binding sites have been shown to be involved both in the physiological roles of ESCRT-I and in the release of the viral particles, and thus are attractive targets for antivirals. The structure of the Tsg101-UEV domain has been characterized, using X-ray crystallography or NMR spectroscopy, either in its apo-state or bound to ubiquitin or late domains. In this study, we report the backbone NMR resonance assignments, including the proline signals, of the apo human Tsg101-UEV domain, that so far was not publicly available. These data, that are in good agreement with the crystallographic structure of Tsg101-UEV domain, can therefore be used for further NMR studies, including protein-protein interaction studies and drug discovery.

Predicting protein flexibility with AlphaFold.

AlphaFold2 has revolutionized protein structure prediction from amino-acid sequence. In addition to protein structures, high-resolution dynamics information about various protein regions is important for understanding protein function. Although AlphaFold2 has neither been designed nor trained to predict protein dynamics, it is shown here how the information returned by AlphaFold2 can be used to predict dynamic protein regions at the individual residue level. The approach, which is termed cdsAF2, uses the 3D protein structure returned by AlphaFold2 to predict backbone NMR NH S2 order parameters using a local contact model that takes into account the contacts made by each peptide plane along the backbone with its environment. By combining for each residue AlphaFold2's pLDDT confidence score for the structure prediction accuracy with the predicted S2 value using the local contact model, an estimator is obtained that semi-quantitatively captures many of the dynamics features observed in experimental backbone NMR NH S2 order parameter profiles. The method is demonstrated for a set nine proteins of different sizes and variable amounts of dynamics and disorder.

NMR study of human macroPARPs domains: 1H, 15N and 13C resonance assignment of hPARP14 macro domain 2 in the free and the ADPr bound state

hPARP14 is a human ADP-ribosyl-transferase (ART) that belongs to the macroPARPs family, together with hPARP9 and hPARP15. It contains a tandem of three macro domains (MD) while each of them has different properties. The first one, namely MD1, has not been reported to exhibit a high binding affinity for ADP-ribose (ADPr) in contrast to the following two (MD2 and MD3). All three MDs exhibit an α/β/α sandwich-like fold as reported by the deposited crystallographic structures. MD2 and MD3 recognize mono-ADP-ribosylated (MARylated) but not poly-ADP-ribosylated (PARylated) substrates and thus they allow hPARP14 to bind its targets, which can be potentially MARylated by its catalytic domain (CD). hPARP14 participates in DNA damage repair process and immune response against viruses like SARS-CoV-2, which also harbors an MD fold. Furthermore, hPARP14 like the other two macroPARPs (hPARP9 and hPARP15), is implicated in numerous types of cancer, such as B-aggressive lymphoma and sarcoma, rendering its MDs as potential important drug targets. Herein, we report the complete NMR backbone and side chain assignment (1H, 13C, 15N) of hPARP14 MD2 in the free and ADPr bound states and the NMR chemical shift-based prediction of its secondary structure elements. This is the first reported NMR study of a hPARP macro domain, paving the way to screen by NMR chemical compounds which may alter the ability of hPARP14 to interact with its substrates affecting its function.

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

The ribosomal maturation factor (RimP) is a 17.7kDa 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 β-strands and three α-helices with the topology α1-β1-β2-α2- β3- α3- β4- β5- β6- β7- β8. 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.

NMR Observation of Sulfhydryl Signals in SARS-CoV-2 Main Protease Aids Structural Studies.

The 68‐kDa homodimeric 3C‐like protease of SARS‐CoV‐2, Mpro (3CLpro/Nsp5), is a key antiviral drug target. NMR spectroscopy of this large system proved challenging and resonance assignments have remained incomplete. Here we present the near‐complete (>97 %) backbone assignments of a C145A variant of Mpro (Mpro C145A) both with, and without, the N‐terminal auto‐cleavage substrate sequence, in its native homodimeric state. We also present SILLY (Selective Inversion of thioL and Ligand for NOESY), a simple yet effective pseudo‐3D NMR experiment that utilizes NOEs to identify interactions between Cys‐thiol or aliphatic protons, and their spatially proximate backbone amides in a perdeuterated protein background. High protection against hydrogen exchange is observed for 10 of the 11 thiol groups in Mpro C145A, even those that are partially accessible to solvent. A combination of SILLY methods and high‐resolution triple‐resonance NMR experiments reveals site‐specific interactions between Mpro, its substrate peptides, and other ligands, which present opportunities for competitive binding studies in future drug design efforts.

Backbone and side chain NMR assignment of the heme-nitric oxide/oxygen binding (H-NOX) domain from Nostoc punctiforme

Soluble guanylate cyclase (sGC) is considered as the primary NO receptor across several known eukaryotes. The main interest regarding the biological role and its function, focuses on the H-NOX domain of the β1 subunit. This domain in its active form bears a ferrous b type heme as prosthetic group, which facilitates the binding of NO and other diatomic gases. The key point that still needs to be answered is how the protein selectively binds the NO and how the redox state of heme and coordination determines H-NOX active state upon binding of diatomic gases. H-NOX domain is present in the genomes of both prokaryotes and eukaryotes, either as a stand-alone protein domain or as a partner of a larger polypeptide. The biological functions of these signaling modules for a wide range of genomes, diverge considerably along with their ligand binding properties. In this direction, we examine the prokaryotic H-NOX protein domain from Nostoc punctiforme (Npun H-NOX). Herein, we first report the almost complete NMR backbone and side-chain resonance assignment (1H, 13C, 15 N) of Npun H-NOX domain together with the NMR chemical shift-based prediction of the domain’s secondary structure elements.

NMR resonance assignment of the N-terminal GTPase domain of human Miro2 Bound to GTP

Miro2 and Miro1 are mitochondrial-associated proteins critical for regulating mitochondrial movement within the cell. Both Miro1 and Miro2 have roles in promoting neuron function, but recently Miro2 has been shown to have additional roles in response to nutrient starvation in tumor cells. Miro1 and 2 consist of two small GTPase domains flanking a pair of EF-hands. The N-terminal GTPase (nGTPase) domain is responsible for initiating mitochondrial trafficking and interactions with GCN1 in prostate cancer. The crystal structure of Miro1 nGTPase bound to GTP has been solved. However, no structural data is available for the nGTPase domain of Miro2. To better understand the similarities and differences in the functions of Miro1 and Miro2, we have initiated structural studies of Miro2. Here we report the backbone NMR chemical shift assignments of a 22 KDa construct of the nGTPase domain of Miro2 bound to GTP that includes residues 1–180 of the full-length protein. We affirm that the overall secondary structure of this complex closely resembles that of Miro1 nGTPase bound to GTP. Minor variations in the overall structures can be attributed to crystal packing interactions in the structure of Miro1. These NMR studies will form the foundation for future work identifying the specific interaction sites between Miro2 and its cellular binding partners.

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 β-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 15N 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.

Sequence-specific 1H, 13C and 15N backbone NMR assignments for the N-terminal IgV-like domain (D1) and full extracellular region (D1D2) of PD-L1

The co-inhibitory immune checkpoint interaction between programmed cell death-protein 1 (PD-1) and programmed cell death-ligand 1 (PD-L1) serves to regulate T-cell activation, promoting self-tolerance. Over-expression of PD-L1 is a mechanism through which tumour cells can evade detection by the immune system. Several therapeutic antibodies targeting PD-L1 or PD-1 have been approved for the treatment of a variety of cancers, however, the discovery and development of small-molecule inhibitors of PD-L1 remains a challenge. Here we report comprehensive sequence-specific backbone resonance assignments (1H, 13C, and 15N) obtained for the N-terminal IgV-like domain of PD-L1 (D1) and the full two domain extracellular region (D1D2). These NMR assignments will serve as a useful tool in the discovery of small-molecule therapeutics targeting PD-L1 and in the characterisation of functional interactions with other protein partners, such as CD80.

Extent of intrinsic disorder and NMR chemical shift assignments of the distal N-termini from human TRPV1, TRPV2 and TRPV3 ion channels

The mammalian Transient Receptor Potential Vanilloid (TRPV) channels are a family of six tetrameric ion channels localized at the plasma membrane. The group I members of the family, TRPV1 through TRPV4, are heat-activated and exhibit remarkable polymodality. The distal N-termini of group I TRPV channels contain large intrinsically disordered regions (IDRs), ranging from ~ 75 amino acids (TRPV2) to ~ 150 amino acids (TRPV4), the vast majority of which is invisible in the structural models published so far. These IDRs provide important binding sites for cytosolic partners, and their deletion is detrimental to channel activity and regulation. Recently, we reported the NMR backbone assignments of the distal TRPV4 N-terminus and noticed some discrepancies between the extent of disorder predicted solely based on protein sequence and from experimentally determined chemical shifts. Thus, for an analysis of the extent of disorder in the distal N-termini of all group I TRPV channels, we now report the NMR assignments for the human TRPV1, TRPV2 and TRPV3 IDRs.

1H, 13C, 15 N backbone and side-chain NMR assignments for three MAX effectors from Magnaporthe oryzae

Effectors are small and very diverse proteins secreted by fungi and translocated in plant cells during infection. Among them, MAX effectors (for Magnaporthe Avrs and ToxB) were identified as a family of effectors that share an identical fold topology despite having highly divergent sequences. They are mostly secreted by ascomycetes from the Magnaporthe genus, a fungus that causes the rice blast, a plant disease leading to huge crop losses. As rice is the first source of calories in many countries, especially in Asia and Africa, this constitutes a threat for world food security. Hence, a better understanding of these effectors, including structural and functional characterization, constitutes a strategic milestone in the fight against phytopathogen fungi and may give clues for the development of resistant varieties of rice. We report here the near complete 1H, 15N and 13C NMR resonance assignment of three new putative MAX effectors (MAX47, MAX60 and MAX67). Secondary structure determination using TALOS-N and CSI.3 demonstrates a high content of β-strands in all the three proteins, in agreement with the canonic ß-sandwich structure of MAX effectors. This preliminary study provides foundations for further structural characterization, that will help in turn to improve sequence predictions of other MAX effectors through data mining.

Backbone and ILV side-chain NMR resonance assignments of the catalytic domain of human deubiquitinating enzyme USP7.

Ubiquitin specific protease 7 (USP7) is a deubiquitinating enzyme, which removes ubiquitin tag from numerous protein substrates involved in diverse cellular processes such as cell cycle regulation, apoptosis and DNA damage response. USP7 affects stability, interaction network and cellular localization of its cellular and viral substrates by controlling their ubiquitination status. The large 41kDa catalytic domain of USP7 harbors the active site of the enzyme. Here we present a nearly complete (93%) NMR resonance assignment of isoleucine, leucine and valine (ILV) side-chains of the USP7 catalytic domain along with a refined nearly complete (93%) assignment of its backbone resonances. The reported ILV methyl group assignment will facilitate further NMR investigations of structure, interactions and conformational dynamics of the USP7 enzyme.

1H, 15N and 13C chemical shift assignments of the N-terminal domain of the two isoforms of the human apolipoprotein E.

Apolipoprotein E (ApoE) is one of the major lipid transporters in humans. It is also implicated in pathological conditions like Alzheimer's and cardiovascular diseases. The N-terminal domain of ApoE binds low-density lipoprotein receptors (LDLR) while the C-terminal domain binds to the lipid. I report the backbone and aliphatic side-chain NMR chemical shifts of the N-terminal domain of two isoforms of ApoE, namely ApoE3 NTD (BMRB No. 51,122) and ApoE4 NTD (BMRB No. 51,123) at pH 3.5 (20°C).

Backbone NMR assignments of the extensive human and chicken TRPV4 N-terminal intrinsically disordered regions as important players in ion channel regulation

Transient receptor potential (TRP) channels are important pharmacological targets due to their ability to act as sensory transducers on the organismic and cellular level, as polymodal signal integrators and because of their role in numerous diseases. However, a detailed molecular understanding of the structural dynamics of TRP channels and their integration into larger cellular signalling networks remains challenging, in part due to the systematic absence of highly dynamic regions pivotal for channel regulation from available structures. In human TRP vanilloid 4 (TRPV4), a ubiquitously expressed homotetrameric cation channel involved in temperature, osmo- and mechano-sensation and in a multitude of (patho)physiological processes, the intrinsically disordered N-terminus encompasses 150 amino acids and thus represents > 17% of the entire channel sequence. Its deletion renders the channel significantly less excitable to agonists supporting a crucial role in TRPV4 activation and regulation. For a structural understanding and a comparison of its properties across species, we determined the NMR backbone assignments of the human and chicken TRPV4 N-terminal IDRs.

Structure and DNA binding analysis of AT-rich interaction domain present in human BAF-B specific subunit BAF250b.

BAF250b and its paralog BAF250a are the DNA-binding central hub proteins present in BAF-B and BAF-A classes of SWI/SNF chromatin-remodeling complexes. BAF250b contains an AT-rich interaction domain (ARID) and C-terminal BAF250_C domain, and it is found mutated in several cancers. ARID is a conserved helix-turn-helix motif-containing DNA-binding domain present in several eukaryotic proteins. The ARID of BAF250b has been proposed to play roles in recruiting SWI/SNF to the target gene promoters for their activation. BAF250b ARID structures had been deposited in the protein data bank by a structural genomics consortium. However, it is not well-studied for its DNA-binding and solution dynamic properties. Here, we report complete backbone NMR resonance assignments of human BAF250b ARID. NMR chemical shifts and the backbone dynamics showed that the solution structure of the protein matched the reported crystal structures. The structure and chemical shift indexing revealed the presence of a short β-sheet in the DNA-binding region of BAF250b ARID that was absent in the structure of its paralog BAF250a ARID. NMR chemical shift perturbations identified DNA-binding residues and revealed the DNA-binding interface on BAF250b ARID. NMR data-driven HADDOCK models of BAF250b ARID - DNA complexes revealed its plausible mode of DNA-binding. Isothermal titration calorimetry experiments showed that BAF250b ARID interacts with DNA sequences with moderate affinities like BAF250a ARID. However, distinct thermodynamic signatures were observed for binding of BAF250a ARID and BAF250b ARID to AT-rich DNA sequence, suggesting that subtle sequence and structural differences in these two proteins influence their DNA-binding.

Backbone NMR resonance assignment of the intrinsically disordered UBact protein from Nitrospira nitrosa.

Ubiquitin signaling in eukaryotes is responsible for a variety of cellular outcomes, most notably proteasomal degradation. A recent bioinformatic study has revealed the existence of a new proteasomal operon in certain gram-negative bacteria phyla. This operon contains genes similar to those included in the prokaryotic ubiquitin-like protein (Pup) proteasomal operon, but do not themselves contain Pup. Instead, they encode for a protein termed UBact with 30% sequence similarity to Pup. Here, we report the near-complete NMR assignment of the backbone and partial assignment of the side chain chemical shifts of the UBact protein from Nitrospira nitrosa. The 1H-15N HSQC spectrum shows a narrow spread of proton NMR signals, characteristic of an intrinsically disordered protein. This chemical shift assignment will facilitate further NMR studies to explore the role of UBact in this new putative proteasomal operon.

Solution NMR backbone assignments of disordered Olduvai protein domain CON1 employing Hα-detected experiments.

Olduvai protein domains, encoded by the NBPF gene family, are responsible for the largest increase in copy number of any protein-coding region in the human genome. This has spawned various genetics studies which have linked these domains to human brain development and divergence from our primate ancestors, as well as currently relevant cognitive diseases such as schizophrenia and autismspectrum disorder (ASD). There are six separate Olduvai domains which together form the majority of the various protein products of the NBPF genes. The six domains include three conserved domains (CON1-3), and three human-lineage-specific domains (HLS1-3) which occur in triplet. Here, we present the solution nuclear magnetic resonance backbone assignments for the CON1 domain, which has been linked to the severity of ASD. The data confirm that CON1 is an intrinsically disordered protein (IDP). Additionally, we use innovative Hα-detected experiments which allow us to not only assign the Hα atoms and N atoms of proline residues, but also to assign residues where HN-experiments suffered from peak overlap or broadening.

NMR Backbone Assignment of VIM-2 and Identification of the Active Enantiomer of a Potential Inhibitor

Carbapenem resistance caused by metallo-β-lactamases is a serious global challenge that, if not tackled efficiently, is expected to lead to millions of deaths in the coming decades. Verona-integron encoded metallo-β-lactamase 2 (VIM-2) is a bacterial enzyme that has been reported from multidrug-resistant nosocomial isolates of Pseudomonas aeruginosa and other Gram-negative pathogens. As it hydrolyzes most β-lactams efficiently, including carbapenems, it is a major threat to current antimicrobial chemotherapies. So far, there is no clinically applicable inhibitor for this enzyme. In this work, the backbone NMR resonance assignment of VIM-2 is disclosed, opening up NMR investigations of this clinically important enzyme and its potential inhibitors for solutions, enabling a rational improvement of inhibitor candidates. Making use of the assignment, we identified the active enantiomer of a VIM-2 inhibitor candidate as well as its possible binding site and Kd, utilizing NMR chemical shift titration experiments.

Metallo-β-Lactamase Inhibitor Phosphonamidate Monoesters.

Being the second leading cause of death and the leading cause of disability-adjusted life years worldwide, infectious diseases remain—contrary to earlier predictions—a major consideration for the public health of the 21st century. Resistance development of microbes to antimicrobial drugs constitutes a large part of this devastating problem. The most widely spread mechanism of bacterial resistance operates through the degradation of existing β-lactam antibiotics. Inhibition of metallo-β-lactamases is expected to allow the continued use of existing antibiotics, whose applicability is becoming ever more limited. Herein, we describe the synthesis, the metallo-β-lactamase inhibition activity, the cytotoxicity studies, and the NMR spectroscopic determination of the protein binding site of phosphonamidate monoesters. The expression of single- and double-labeled NDM-1 and its backbone NMR assignment are also disclosed, providing helpful information for future development of NDM-1 inhibitors. We show phosphonamidates to have the potential to become a new generation of antibiotic therapeutics to combat metallo-β-lactamase-resistant bacteria.