NMR <Superscript>1</Superscript>H,<Superscript>13</Superscript>C, <Superscript>15</Superscript>N backbone and <Superscript>13</Superscript>C side chain resonance assignment of the G12C mutant of human K-Ras bound to GDP

K-Ras is a key driver of oncogenesis, accounting for approximately 80% of Ras-driven human cancers. The small GTPase cycles between an inactive, GDP-bound and an active, GTP-bound state, regulated by guanine nucleotide exchange factors and GTPase activating proteins, respectively. Activated K-Ras regulates cell proliferation, differentiation and survival by signaling through several effector pathways, including Raf-MAPK. Oncogenic mutations that impair the GTPase activity of K-Ras result in a hyperactivated state, leading to uncontrolled cellular proliferation and tumorogenesis. A cysteine mutation at glycine 12 is commonly found in K-Ras associated cancers, and has become a recent focus for therapeutic intervention. We report here ¹H^{N, 15}N, and ¹³C resonance assignments for the 19.3 kDa (aa 1–169) human K-Ras protein harboring an oncogenic G12C mutation in the GDP-bound form (K-RAS^G12C-GDP), using heteronuclear, multidimensional NMR spectroscopy. Backbone ¹H–¹⁵N correlations have been assigned for all non-proline residues, except for the first methionine residue.     

Different domain organization of two main conformational states of Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase

The Na⁺/K⁺-ATPase generates an electrochemical gradient of Na⁺ and K⁺, which is necessary for the functioning of animal cells. During the catalytic act, the enzyme passes through two principal conformational states, E₁ and E₂. To assess the domain organization of the protein in these conformations, thermal denaturation of Na⁺/K⁺-ATPases from duck salt gland and from rabbit kidney has been studied in the absence and in the presence of Na⁺ or K⁺, which induce the transition to E₁ or E₂. The melting curves for the ion-free forms of the two ATPases have different shapes: the rabbit protein shows one transition at 56.1°C, whereas the duck protein shows two transitions, at 49.8 and 56.9°C. Addition of Na⁺ or K⁺ ions abolishes the difference in thermal behavior between these enzymes, but through opposite effects. The melting curves for the E₂ conformation (K⁺ bound) in both cases exhibit a single peak of heat absorption at ∼63°C. For the E₁ conformation (Na⁺ bound), each melting curve has three peaks, indicating denaturation of three domains. The difference in the domain organization of Na⁺/K⁺-ATPase in the E1 and E2 states may account for the different sensitivity to temperature, proteolysis, and oxidative stress observed for the two enzyme conformations.     

Towards extracellular Ca<Superscript>2+</Superscript> sensing by MRI: synthesis and calcium-dependent <Superscript>1</Superscript>H and <Superscript>17</Superscript>O relaxation studies of two novel bismacrocyclic Gd<Superscript>3+</Superscript> complexes

Two new bismacrocyclic Gd³⁺ chelates containing a specific Ca²⁺ binding site were synthesized as potential MRI contrast agents for the detection of Ca²⁺ concentration changes at the millimolar level in the extracellular space. In the ligands, the Ca²⁺-sensitive BAPTA-bisamide central part is separated from the DO3A macrocycles either by an ethylene (L¹) or by a propylene (L²) unit [H₄BAPTA is 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; H₃DO₃A is 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid]. The sensitivity of the Gd³⁺ complexes towards Ca²⁺ and Mg²⁺ was studied by ¹H relaxometric titrations. A maximum relaxivity increase of 15 and 10% was observed upon Ca²⁺ binding to Gd₂L¹ and Gd₂L², respectively, with a distinct selectivity of Gd₂L¹ towards Ca²⁺ compared with Mg²⁺. For Ca²⁺ binding, association constants of log K = 1.9 (Gd₂L¹) and log K = 2.7 (Gd₂L²) were determined by relaxometry. Luminescence lifetime measurements and UV–vis spectrophotometry on the corresponding Eu³⁺ analogues proved that the complexes exist in the form of monohydrated and nonhydrated species; Ca²⁺ binding in the central part of the ligand induces the formation of the monohydrated state. The increasing hydration number accounts for the relaxivity increase observed on Ca²⁺ addition. A ¹H nuclear magnetic relaxation dispersion and ¹⁷O NMR study on Gd₂L¹ in the absence and in the presence of Ca²⁺ was performed to assess the microscopic parameters influencing relaxivity. On Ca²⁺ binding, the water exchange is slightly accelerated, which is likely related to the increased steric demand of the central part leading to a destabilization of the Ln–water binding interaction. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Simultaneous <Superscript>1</Superscript>H- or <Superscript>2</Superscript>H-, <Superscript>15</Superscript>N- and multiple-band-selective <Superscript>13</Superscript>C-decoupling during acquisition in <Superscript>13</Superscript>C-detected experiments with proteins and oligonucleotides

Significant resolution improvement in ¹³C,¹³C-TOCSY spectra of uniformly deuterated and ¹³C, ¹⁵N-labeled protein and ¹³C,¹⁵N-labeled RNA samples is achieved by introduction of multiple-band-selective ¹³C-homodecoupling applied simultaneously with ¹H- or ²H- and ¹⁵N-decoupling at all stages of multidimensional experiments including signal acquisition period. The application of single, double or triple band-selective ¹³C-decoupling in 2D-[¹³C,¹³C]-TOCSY experiments during acquisition strongly simplifies the homonuclear splitting pattern. The technical aspects of complex multiple-band homonuclear decoupling and hardware requirements are discussed. The use of this technique (i) facilitates the resonance assignment process as it reduces signal overlap in homonuclear ¹³C-spectra and (ii) possibly improves the signal-to-noise ratio through multiplet collapse. It can be applied in any ¹³C-detected experiment.     

Backbone <Superscript>1</Superscript>H, <Superscript>15</Superscript>N,and <Superscript>13</Superscript>C resonance assignments of the Tom1 VHS domain

Efficient trafficking of ubiquitinated receptors (cargo) to endosomes requires the recruitment of adaptor proteins that exhibit ubiquitin-binding domains for recognition and transport. Tom1 is an adaptor protein that not only associates with ubiquitinated cargo but also represents a phosphoinositide effector during specific bacterial infections. This phosphoinositide-binding property is associated with its N-terminal Vps27, Hrs, STAM (VHS) domain. Despite its biological relevance, there are no resonance assignments of Tom1 VHS available that can fully characterize its molecular interactions. Here, we report the nearly complete ¹H, ¹⁵N, and ¹³C backbone resonance assignments of the VHS domain of human Tom1.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N chemical shift assignments of Saccharomyces cerevisiae type 1 thioredoxin in the oxidized state by solution NMR spectroscopy

Thioredoxins (Trx) are ubiquitous proteins that regulate several biochemical processes inside the cell. Trx is an important player, displaying oxidoreductase activity and helping to keep and regulate the oxidative state of the cellular environment. Trx also participates in the regulation of many cellular functions, such as DNA synthesis, protection against oxidative stress, cell cycle and signal transduction. The oxidized Trx is the target for another set of proteins, such as thioredoxin reductase (TrR), which used the reductive potential of NADPH. The oxidized state of Trx also plays important role in regulation of redox state in the cells. In this regard, the oxidized form of Trx is a putative conformer that contributes to the cellular redox environment. Here we report the chemical shift assignments (¹H, ¹³C and ¹⁵N) in solution at 15 °C. We also showed the secondary structure analysis of the oxidized form of yeast thioredoxin (yTrx1) as basis for future NMR studies of protein–target interactions and dynamics. The assignment was done at low concentration (200 µM) because it is important to keep intact the water cavity.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N resonance assignments of a C-terminal domain of human CHD1

Chromatin remodelling proteins are an essential family of eukaryotic proteins. They harness the energy from ATP hydrolysis and apply it to alter chromatin structure in order to regulate all aspects of genome biology. Chromodomain helicase DNA-binding protein 1 (CHD1) is one such remodelling protein that has specialised nucleosome organising abilities and is conserved across eukaryotes. CHD1 possesses a pair of tandem chromodomains that directly precede the core catalytic Snf2 helicase-like domain, and a C-terminal SANT-SLIDE DNA-binding domain. We have identified an additional conserved domain in the C-terminal region of CHD1. Here, we report the backbone and side chain resonance assignments for this domain from human CHD1 at pH 6.5 and 25 °C (BMRB No. 25638).     

Regulation of durum wheat Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchanger TdSOS1 by phosphorylation

We have identified a plasma membrane Na⁺/H⁺ exchanger from durum wheat, designated TdSOS1. Heterologous expression of TdSOS1 in a yeast strain lacking endogenous Na⁺ efflux proteins showed complementation of the Na⁺- and Li⁺-sensitive phenotype by a mechanism involving cation efflux. Salt tolerance conferred by TdSOS1 was maximal when co-expressed with the Arabidopsis protein kinase complex SOS2/SOS3. In vitro phosphorylation of TdSOS1 with a hyperactive form of the Arabidopsis SOS2 kinase (T/DSOS2∆308) showed the importance of two essential serine residues at the C-terminal hydrophilic tail (S1126, S1128). Mutation of these two serine residues to alanine decreased the phosphorylation of TdSOS1 by T/DSOS2∆308 and prevented the activation of TdSOS1. In addition, deletion of the C-terminal domain of TdSOS1 encompassing serine residues at position 1126 and 1128 generated a hyperactive form that had maximal sodium exclusion activity independent from the regulatory SOS2/SOS3 complex. These results are consistent with the presence of an auto-inhibitory domain at the C-terminus of TdSOS1 that mediates the activation of TdSOS1 by the protein kinase SOS2. Expression of TdSOS1 mRNA in young seedlings of the durum wheat variety Om Rabia3, using different abiotic stresses (ionic and oxidative stress) at different times of exposure, was monitored by RT–PCR.     

Automated amino acid side-chain NMR assignment of proteins using <Superscript>13</Superscript>C- and <Superscript>15</Superscript>N-resolved 3D [<Superscript>1</Superscript>H,<Superscript>1</Superscript>H]-NOESY

ASCAN is a new algorithm for automatic sequence-specific NMR assignment of amino acid side-chains in proteins, which uses as input the primary structure of the protein, chemical shift lists of (1)H(N), (15)N, (13)C(alpha), (13)C(beta) and possibly (1)H(alpha) from the previous polypeptide backbone assignment, and one or several 3D (13)C- or (15)N-resolved [(1)H,(1)H]-NOESY spectra. ASCAN has also been laid out for the use of TOCSY-type data sets as supplementary input. The program assigns new resonances based on comparison of the NMR signals expected from the chemical structure with the experimentally observed NOESY peak patterns. The core parts of the algorithm are a procedure for generating expected peak positions, which is based on variable combinations of assigned and unassigned resonances that arise for the different amino acid types during the assignment procedure, and a corresponding set of acceptance criteria for assignments based on the NMR experiments used. Expected patterns of NOESY cross peaks involving unassigned resonances are generated using the list of previously assigned resonances, and tentative chemical shift values for the unassigned signals taken from the BMRB statistics for globular proteins. Use of this approach with the 101-amino acid residue protein FimD(25-125) resulted in 84% of the hydrogen atoms and their covalently bound heavy atoms being assigned with a correctness rate of 90%. Use of these side-chain assignments as input for automated NOE assignment and structure calculation with the ATNOS/CANDID/DYANA program suite yielded structure bundles of comparable quality, in terms of precision and accuracy of the atomic coordinates, as those of a reference structure determined with interactive assignment procedures. A rationale for the high quality of the ASCAN-based structure determination results from an analysis of the distribution of the assigned side chains, which revealed near-complete assignments in the core of the protein, with most of the incompletely assigned residues located at or near the protein surface.     

Sequence-specific <Superscript>1</Superscript>H, <Superscript>15</Superscript>N,and <Superscript>13</Superscript>C resonance assignments of the autophagy-related protein LC3C

Autophagy is a versatile catabolic pathway for lysosomal degradation of cytoplasmic material. While the phenomenological and molecular characteristics of autophagic non-selective (bulk) decomposition have been investigated for decades, the focus of interest is increasingly shifting towards the selective mechanisms of autophagy. Both, selective as well as bulk autophagy critically depend on ubiquitin-like modifiers belonging to the Atg8 (autophagy-related 8) protein family. During evolution, Atg8 has diversified into eight different human genes. While all human homologues participate in the formation of autophagosomal membrane compartments, microtubule-associated protein light chain 3C (LC3C) additionally plays a unique role in selective autophagic clearance of intracellular pathogens (xenophagy), which relies on specific protein–protein recognition events mediated by conserved motifs. The sequence-specific ¹H, ¹⁵N, and ¹³C resonance assignments presented here form the stepping stone to investigate the high-resolution structure and dynamics of LC3C and to delineate LC3C’s complex network of molecular interactions with the autophagic machinery by NMR spectroscopy.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N and <Superscript>13</Superscript>C sequence specific backbone assignment of the vanadate inhibited hematopoietic tyrosine phosphatase

The sequence-specific backbone assignment of hematopoietic protein tyrosine phosphatase (HePTP; PTPN7) in presence of vanadate has been determined, based on triple-resonance experiments using uniformly [¹³C,¹⁵N]-labeled protein. These assignments facilitate further studies of HePTP in the presence of inhibitors to target leukemia and provide further insights into the function of protein tyrosine phosphatases.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N and <Superscript>13</Superscript>C assignment of the amyloidogenic protein medin using fast-pulsing NMR techniques

Thirty-one proteins are known to form extracellular fibrillar amyloid in humans. Molecular information about many of these proteins in their monomeric, intermediate or fibrillar form and how they aggregate and interact to form the insoluble fibrils is sparse. This is because amyloid proteins are notoriously difficult to study in their soluble forms, due to their inherent propensity to aggregate. Using recent developments in fast NMR techniques, band-selective excitation short transient and band-selective optimized flip-angle short-transient heteronuclear multiple quantum coherence we have been able to assign a 5 kDa full-length amyloidogenic protein called medin. Medin is the key protein component of the most common form of localised amyloid with a proposed role in aortic aneurysm and dissection. This assignment will now enable the study of the early interactions that could influence initiation and progression of medin aggregation. The chemical shifts have been deposited in the BioMagRes-Bank accession Nos. 25399 and 26576.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N,and <Superscript>13</Superscript>C chemical shift assignments of the regulatory domain of human calcineurin

Calcineurin (CaN) plays an important role in T-cell activation, cardiac system development and nervous system function. Previous studies have demonstrated that the regulatory domain (RD) of CaN binds calmodulin (CaM) towards the N-terminal end. Calcium-loaded CaM activates the serine/threonine phosphatase activity of CaN by binding to the RD, although the mechanistic details of this interaction remain unclear. It is thought that CaM binding at the RD displaces the auto-inhibitory domain (AID) from the active site of CaN, activating phosphatase activity. In the absence of calcium-loaded CaM, the RD is disordered, and binding of CaM induces folding in the RD. In order to provide mechanistic detail about the CaM–CaN interaction, we have undertaken an NMR study of the RD of CaN. Complete ¹³C, ¹⁵N and ¹H assignments of the RD of CaN were obtained using solution NMR spectroscopy. The backbone of RD has been assigned using a combination of ¹³C-detected CON-IPAP experiments as well as traditional HNCO, HNCA, HNCOCA and HNCACB-based 3D NMR spectroscopy. A ¹⁵N-resolved TOCSY experiment has been used to assign Hα and Hβ chemical shifts.     

Breast cancer cells expressing stem cell markers CD44<Superscript>+</Superscript> CD24<Superscript>lo</Superscript> are eliminated by Numb-1 peptide-activated T cells

Cancer stem cells (CSC) are resistant to chemo- and radiotherapy. To eliminate cells with phenotypic markers of CSC-like we characterized: (1) expression of CD44, CD24, CD133 and MIC-A/B (NKG2 receptors) in breast (MCF7) and ovarian (SK-OV-3) cells resistant to gemcitabine (GEM), paclitaxel (PTX) and 5-fluorouracil (5-FU) and (2) their elimination by Numb- and Notch-peptide activated CTL. The number of cells in all populations with the luminal CSC phenotype [epithelial specific antigen⁺ (ESA) CD44^hi CD24^lo, CD44^hi CD133⁺, and CD133⁺ CD24^lo] increased in drug-resistant MCF7 and SK-OV-3 cells. Similarly, the number of cells with expressed MIC-A/B increased 4 times in drug-resistant tumor cells compared with drug-sensitive cells. GEM^Res MCF7 cells had lower levels of the Notch-1-extracellular domain (NECD) and Notch trans-membrane intracellular domain (TMIC) than GEM^Sens MCF7. The levels of Numb, and Numb-L-[P]-Ser²⁶⁵ were similar in GEM^Res and GEM^Sens MCF7 cells. Only the levels of Numb-L (long)-Ser²⁹⁵ decreased slightly. This finding suggests that Notch-1 cleavage to TMIC is inhibited in GEM^Res MCF7 cells. PBMC activated by natural immunogenic peptides Notch-1 (2112–2120) and Numb-1 (87–95) eliminated NICD^positive, CD24^hi CD24^lo MCF7 cells. It is likely that the immunogenic Numb-1 peptide in MCF7 cells originated from Numb, [P]-lated by an unknown kinase, because staurosporine but not wortmannin and MAPK-inhibitors decreased peptide presentation. Numb and Notch are antagonistic proteins which degrade each other to stop and activate cell proliferation, respectively. Their peptides are presented alternatively. Targeting both antagonistic proteins should be useful to prevent metastases in patients whose tumors are resistant to conventional treatments. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.