Biomolecular NMR Assignments最新文献

Human K-Ras protein, which is a member of the GTPase Ras family, hydrolyzes GTP to GDP and concomitantly converts from its active to its inactive state. It is a key oncoprotein, because several mutations, particularly those at residue position 12, occur with a high frequency in a wide range of human cancers. The K-Ras protein is therefore an important target for developing therapeutic anti-cancer agents. In this work we report the almost complete sequence-specific resonance assignments of wild-type and the oncogenic G12C and G12D mutants in the GTP-complexed active forms, including the functionally important Switch I and Switch II regions. These assignments serve as the basis for a comprehensive functional dynamics study of wild-type K-Ras and its G12 mutants.

Fibrils of the protein α-synuclein (Asyn) are implicated in the pathogenesis of Parkinson Disease, Lewy Body Dementia, and Multiple System Atrophy. Numerous forms of Asyn fibrils have been studied by solid-state NMR and resonance assignments have been reported. Here, we report a new set of ¹³C, ¹⁵N assignments that are unique to fibrils obtained by amplification from postmortem brain tissue of a patient diagnosed with Lewy Body Dementia.

S. aureus resistance to antibiotics has increased rapidly. MRSA strains can simultaneously be resistant to many different classes of antibiotics, including the so-called “last-resort” drugs. Resistance complicates treatment, increases mortality and substantially increases the cost of treatment. The need for new drugs against (multi)resistant S. aureus is high. M23B family peptidoglycan hydrolases, enzymes that can kill S. aureus by cleaving glycine-glycine peptide bonds in S. aureus cell wall are attractive targets for drug development because of their binding specificity and lytic activity. M23B enzymes lysostaphin, LytU and LytM have closely similar catalytic domain structures. They however differ in their lytic activities, which can arise from non-conserved residues in the catalytic groove and surrounding loops or differences in dynamics. We report here the near complete ¹H/¹³C/¹⁵N resonance assignment of the catalytic domain of LytM, residues 185–316. The chemical shift data allow comparative structural and functional studies between the enzymes and is essential for understanding how these hydrolases degrade the cell wall.

Adhesin P1 (aka AgI/II) plays a pivotal role in mediating Streptococcus mutans attachment in the oral cavity, as well as in regulating biofilm development and maturation. P1’s naturally occurring truncation product, Antigen II (AgII), adopts both soluble, monomeric and insoluble, amyloidogenic forms within the bacterial life cycle. Monomers are involved in important quaternary interactions that promote cell adhesion and the functional amyloid form promotes detachment of mature biofilms. The heterologous, 51-kD C123 construct comprises most of AgII and was previously characterized by X-ray crystallography. C123 contains three structurally homologous domains, C1, C2, and C3. NMR samples made using the original C123 construct, or its C3 domain, yielded moderately resolved NMR spectra. Using Alphafold, we re-analyzed the P1 sequence to better identify domain boundaries for C123, and in particular the C3 domain. We then generated a more tractable construct for NMR studies of the monomeric form, including quaternary interactions with other proteins. The addition of seven amino acids at the C-terminus greatly improved the spectral dispersion for C3 relative to the prior construct. Here we report the backbone NMR resonance assignments for the new construct and characterize some of its quaternary interactions. These data are in good agreement with the structure predicted by Alphafold, which contains additional β-sheet secondary structure compared to the C3 domain in the C123 crystal structure for a construct lacking the seven C-terminal amino acids. Its quaternary interactions with known protein partners are in good agreement with prior competitive binding assays. This construct can be used for further NMR studies, including protein-protein interaction studies and assessing the impact of environmental conditions on C3 structure and dynamics within C123 as it transitions from monomer to amyloid form.

Kinesin is a motor protein, comprised of two heavy and two light chains that transports cargo along the cytoskeletal microtubule filament network. The heavy chain has a neck domain connecting the ATPase motor head responsible for walking along microtubules, with the stalk and subsequent tail domains that bind cargo. The neck domain consists of a coiled coli homodimer with about five heptad repeats, preceded by a linker region that joins to the ATPase head. Here we report ¹H, ¹⁵N, and ¹³C NMR assignments and a solution structure for the kinesin neck domain from rat isoform Kif5c. The calculation of the NMR structure of the homodimer was facilitated by unambiguously assigning sidechain NOEs between heptad a and d positions to interchain contacts, since these positions are too far apart to give sidechain contacts in the monomers. The dimeric coiled coil NMR structure is similar to the previously described X-ray structure, whereas the linker region is disordered in solution but contains a short segment with β-strand propensity— the β-linker. Only the coiled coil is protected from solvent exchange, with ∆G values for hydrogen exchange on the order of 4–6 kcal/mol. The high stability of the hydrogen-bonded α-helical structure makes it unlikely that unzippering of the coiled coil is involved in kinesin walking. Rather, the linker region serves as a flexible hinge between the kinesin head and neck.

Protein p53 is mostly known for playing a key role in tumour suppression, and mutations in the p53 gene are amongst the most frequent genomic events accompanying oncogenic transformation. Continuous research is conducted to target disordered proteins/protein regions for cancer therapy, for which atomic level information is also necessary. The disordered N-terminal part of p53 contains the transactivation and the proline-rich domains—which besides being abundant in proline residues—contains repetitive Pro-Ala motifs. NMR assignment of such repetitive, proline-rich regions is challenging due to the lack of amide protons in the ¹H^N-detected approaches, as well as due to the small chemical shift dispersion. In the present study we perform the full assignment of the p53^1–100 region by applying a combination of ¹H^N- and ¹H^α-detected NMR experiments. We also show the increased information content when using real-time homo- and heteronuclear decoupled acquisition schemes. On the other hand, we highlight the presence of minor proline species, and using Pro-selective experiments we determine the corresponding cis or trans conformation. Secondary chemical shifts for (C^α–C^β) atoms indicate the disordered nature of this region, with expected helical tendency for the TAD1 region. As the role of the proline-rich domain is yet not well understood our results can contribute to further successful investigations.

hHR23a (human homolog of Rad23 a) functions in nucleotide excision repair and proteasome-mediated protein degradation. It contains an N-terminal ubiquitin-like (UBL) domain, an xeroderma pigmentosum C (XPC)-binding domain, and a ubiquitin-associated (UBA) domain preceding and following the XPC-binding domain. Each of the four structural domains are connected by flexible linker regions. We report in this NMR study, the ¹H, ¹⁵N and ¹³C resonance assignments for the backbone and sidechain atoms of the hHR23a full-length protein with BioMagResBank accession number 52059. Assignments are 97% and 87% for the backbone (^NH, N, C′, Cα, and Hα) and sidechain atoms of the hHR23a structured regions. The secondary structural elements predicted from the NMR data fit well to the hHR23a NMR structure. The assignments described in this manuscript can be used to apply NMR for studies of hHR23a with its binding partners.

Leptin is an adipose tissue-expressed 16-kDa hormone encoded by the ob/ob gene. It serves a crucial role in regulating diverse physiological processes, including body weight control, energy homeostasis regulation, promotion of cell proliferation, and more. Emerging research has also revealed potential implications of leptin in various aging-related diseases, suggesting multifaceted physiological roles of leptin. Structural investigation of wild-type leptin in apo form is of particular importance to understand its conformational plasticity for receptor interaction and recognition. Here, we report backbone and side-chain resonance assignments of wild-type human leptin as a basis for structural and functional studies on leptin-mediated signaling.

FapA is an accessory protein within the biofilm forming functional bacterial amyloid related fap-operon in Pseudomonas, and maybe a chaperone for FapC controlling its fibrillization. To allow further structural analysis, here we present a complete sequential assignment of ¹H_amide, ¹³C_α, ¹³C_β, and ¹⁵N NMR resonances for the functional form of the monomeric soluble FapA protein, comprising amino acids between 29 and 152. From these observed chemical shifts, the secondary structure propensities (SSPs) were determined. FapA predominantly adopts a random coil conformation, however, we also identified small propensities for α-helical and β-strand conformations. Notably, these observed SSPs are smaller compared to the ones we recently observed for the monomeric soluble FapC protein. These NMR results provide valuable insights into the activity of FapA in functional amyloid formation and regulation, that will also aid developing strategies targeting amyloid formation within biofilms and addressing chronic infections.