NMR Chemical Shift Perturbation Research Articles

Correction to The Specialized Hsp70 (HscA) Interdomain Linker Binds to Its Nucleotide-Binding Domain and Stimulates ATP Hydrolysis in Both cis and trans Configurations

Proteins from the isc operon of Escherichia coli constitute the machinery used to synthesize iron-sulfur (Fe-S) clusters for delivery to recipient apoproteins. Efficient and rapid [2Fe-2S] cluster transfer from the holo-scaffold protein IscU depends on ATP hydrolysis in the nucleotide-binding domain (NBD) of HscA, a specialized Hsp70-type molecular chaperone with low intrinsic ATPase activity (0.02 min(-1) at 25 °C, henceforth reported in units of min(-1)). HscB, an Hsp40-type cochaperone, binds to HscA and stimulates ATP hydrolysis to promote cluster transfer, yet while the interactions between HscA and HscB have been investigated, the role of HscA's interdomain linker in modulating ATPase activity has not been explored. To address this issue, we created three variants of the 40 kDa NBD of HscA: NBD alone (HscA386), NBD with a partial linker (HscA389), and NBD with the full linker (HscA395). We found that the rate of ATP hydrolysis of HscA395 (0.45 min(-1)) is nearly 15-fold higher than that of HscA386 (0.035 min(-1)), although their apparent affinities for ATP are equivalent. HscA395, which contains the full covalently linked linker peptide, exhibited intrinsic tryptophan fluorescence emission and basal thermostability that were higher than those of HscA386. Furthermore, HscA395 displayed narrower (1)H(N) line widths in its two-dimensional (1)H-(15)N TROSY-HSQC spectrum in comparison to HscA386, indicating that the peptide in the cis configuration binds to and stabilizes the structure of the NBD. The addition to HscA386 of a synthetic peptide with a sequence identical to that of the interdomain linker (L(387)LLDVIPLS(395)) stimulated its ATPase activity and induced widespread NMR chemical shift perturbations indicative of a binding interaction in the trans configuration.

Solution structure of the SGTA dimerisation domain and investigation of its interactions with the ubiquitin-like domains of BAG6 and UBL4A.

Background The BAG6 complex resides in the cytosol and acts as a sorting point to target diverse hydrophobic protein substrates along their appropriate paths, including proteasomal degradation and ER membrane insertion. Composed of a trimeric complex of BAG6, TRC35 and UBL4A, the BAG6 complex is closely associated with SGTA, a co-chaperone from which it can obtain hydrophobic substrates. Methodology and Principal Findings SGTA consists of an N-terminal dimerisation domain (SGTA_NT), a central tetratricopeptide repeat (TPR) domain, and a glutamine rich region towards the C-terminus. Here we solve a solution structure of the SGTA dimerisation domain and use biophysical techniques to investigate its interaction with two different UBL domains from the BAG6 complex. The SGTA_NT structure is a dimer with a tight hydrophobic interface connecting two sets of four alpha helices. Using a combination of NMR chemical shift perturbation, isothermal titration calorimetry (ITC) and microscale thermophoresis (MST) experiments we have biochemically characterised the interactions of SGTA with components of the BAG6 complex, the ubiquitin-like domain (UBL) containing proteins UBL4A and BAG6. We demonstrate that the UBL domains from UBL4A and BAG6 directly compete for binding to SGTA at the same site. Using a combination of structural and interaction data we have implemented the HADDOCK protein-protein interaction docking tool to generate models of the SGTA-UBL complexes. Significance This atomic level information contributes to our understanding of the way in which hydrophobic proteins have their fate decided by the collaboration between SGTA and the BAG6 complex.

The specialized Hsp70 (HscA) interdomain linker binds to its nucleotide-binding domain and stimulates ATP hydrolysis in both cis and trans configurations.

Proteins from the isc operon of Escherichia coli constitute the machinery used to synthesize iron–sulfur (Fe–S) clusters for delivery to recipient apoproteins. Efficient and rapid [2Fe-2S] cluster transfer from the holo-scaffold protein IscU depends on ATP hydrolysis in the nucleotide-binding domain (NBD) of HscA, a specialized Hsp70-type molecular chaperone with low intrinsic ATPase activity (0.02 min−1 at 25 °C, henceforth reported in units of min–1). HscB, an Hsp40-type cochaperone, binds to HscA and stimulates ATP hydrolysis to promote cluster transfer, yet while the interactions between HscA and HscB have been investigated, the role of HscA’s interdomain linker in modulating ATPase activity has not been explored. To address this issue, we created three variants of the 40 kDa NBD of HscA: NBD alone (HscA386), NBD with a partial linker (HscA389), and NBD with the full linker (HscA395). We found that the rate of ATP hydrolysis of HscA395 (0.45 min–1) is nearly 15-fold higher than that of HscA386 (0.035 min–1), although their apparent affinities for ATP are equivalent. HscA395, which contains the full covalently linked linker peptide, exhibited intrinsic tryptophan fluorescence emission and basal thermostability that were higher than those of HscA386. Furthermore, HscA395 displayed narrower 1HN line widths in its two-dimensional 1H–15N TROSY-HSQC spectrum in comparison to HscA386, indicating that the peptide in the cis configuration binds to and stabilizes the structure of the NBD. The addition to HscA386 of a synthetic peptide with a sequence identical to that of the interdomain linker (L387LLDVIPLS395) stimulated its ATPase activity and induced widespread NMR chemical shift perturbations indicative of a binding interaction in the trans configuration.

The E6AP binding pocket of the HPV16 E6 oncoprotein provides a docking site for a small inhibitory peptide unrelated to E6AP, indicating druggability of E6.

The HPV E6 oncoprotein maintains the malignant phenotype of HPV-positive cancer cells and represents an attractive therapeutic target. E6 forms a complex with the cellular E6AP ubiquitin ligase, ultimately leading to p53 degradation. The recently elucidated x-ray structure of a HPV16 E6/E6AP complex showed that HPV16 E6 forms a distinct binding pocket for E6AP. This discovery raises the question whether the E6AP binding pocket is druggable, i. e. whether it provides a docking site for functional E6 inhibitors. To address these issues, we performed a detailed analysis of the HPV16 E6 interactions with two small peptides: (i) E6APpep, corresponding to the E6 binding domain of E6AP, and (ii) pep11**, a peptide that binds to HPV16 E6 and, in contrast to E6APpep, induces apoptosis, specifically in HPV16-positive cancer cells. Surface plasmon resonance, NMR chemical shift perturbation, and mammalian two-hybrid analyses coupled to mutagenesis indicate that E6APpep contacts HPV16 E6 amino acid residues within the E6AP pocket, both in vitro and intracellularly. Many of these amino acids were also important for binding to pep11**, suggesting that the binding sites for the two peptides on HPV16 E6 overlap. Yet, few E6 amino acids were differentially involved which may contribute to the higher binding affinity of pep11**. Data from the HPV16 E6/pep11** interaction allowed the rational design of single amino acid exchanges in HPV18 and HPV31 E6 that enabled their binding to pep11**. Taken together, these results suggest that E6 molecular surfaces mediating E6APpep binding can also accommodate pro-apoptotic peptides that belong to different sequence families. As proof of concept, this study provides the first experimental evidence that the E6AP binding pocket is druggable, opening new possibilities for rational, structure-based drug design.

Improved protocol to purify untagged amelogenin – Application to murine amelogenin containing the equivalent P70 → T point mutation observed in human amelogenesis imperfecta

Amelogenin is the predominant extracellular protein responsible for converting carbonated hydroxyapatite into dental enamel, the hardest and most heavily mineralized tissue in vertebrates. Despite much effort, the precise mechanism by which amelogenin regulates enamel formation is not fully understood. To assist efforts aimed at understanding the biochemical mechanism of enamel formation, more facile protocols to purify recombinantly expressed amelogenin, ideally without any tag to assist affinity purification, are advantageous. Here we describe an improved method to purify milligram quantities of amelogenin that exploits its high solubility in 2% glacial acetic acid under conditions of low ionic strength. The method involves heating the frozen cell pellet for two 15min periods at ∼70°C with 2min of sonication in between, dialysis twice in 2% acetic acid (1:250 v/v), and reverse phase chromatography. A further improvement in yield is obtained by resuspending the frozen cell pellet in 6M guanidine hydrochloride in the first step. The acetic acid heating method is illustrated with a murine amelogenin containing the corresponding P70→T point mutation observed in an human amelogenin associated with amelogenesis imperfecta (P71T), while the guanidine hydrochloride heating method is illustrated with wild type murine amelogenin (M180). The self-assembly properties of P71T were probed by NMR chemical shift perturbation studies as a function of protein (0.1–1.8mM) and NaCl (0–367mM) concentration. Relative to similar studies with wild type murine amelogenin, P71T self-associates at lower protein or salt concentrations with the interactions initiated near the N-terminus.

Characterization of two distinct modes of drug binding to human intestinal fatty acid binding protein.

The aqueous cytoplasm of cells poses a potentially significant barrier for many lipophilic drugs to reach their sites of action. Fatty acid binding proteins (FABPs) bind to poorly water-soluble fatty acids (FAs) and lipophilic compounds and facilitate their intracellular transport. Several structures of FA in complex with FABPs have been described, but data describing the binding sites of other lipophilic ligands including drugs are limited. Here the environmentally sensitive fluorophores, 1-anilinonapthalene 8-sulfonic acid (ANS), and 11-dansylamino undecanoic acid (DAUDA) were used to investigate drug binding to human intestinal FABP (hIFABP). Most drugs that bound hIFABP were able to displace both ANS and DAUDA. A notable exception was ketorolac, a non-steroidal anti-inflammatory drug that bound to hIFABP and displaced DAUDA but failed to displace ANS. Isothermal titration calorimetry revealed that for the majority of ligands including FA, ANS, and DAUDA, binding to hIFABP was exothermic. In contrast, ketorolac binding to hIFABP was endothermic and entropy-driven. The X-ray crystal structure of DAUDA-hIFABP revealed a FA-like binding mode where the carboxylate of DAUDA formed a network of hydrogen bonds with residues at the bottom of the binding cavity and the dansyl group interacted with residues in the portal region. In contrast, NMR chemical shift perturbation (CSP) data suggested that ANS bound only toward the bottom of the hIFABP cavity, whereas ketorolac occupied only the portal region. The CSP data further suggested that ANS and ketorolac were able to bind simultaneously to hIFABP, consistent with the lack of displacement of ANS observed by fluorescence and supported by a model of the ternary complex. The NMR solution structure of the ketorolac-hIFABP complex therefore describes a newly characterized, hydrophobic ligand binding site in the portal region of hIFABP.

The periplasmic domain of Escherichia coli outer membrane protein A can undergo a localized temperature dependent structural transition

Gram-negative bacteria such as Escherichia coli are surrounded by two membranes with a thin peptidoglycan (PG)-layer located in between them in the periplasmic space. The outer membrane protein A (OmpA) is a 325-residue protein and it is the major protein component of the outer membrane of E. coli. Previous structure determinations have focused on the N-terminal fragment (residues 1–171) of OmpA, which forms an eight stranded transmembrane β-barrel in the outer membrane. Consequently it was suggested that OmpA is composed of two independently folded domains in which the N-terminal β-barrel traverses the outer membrane and the C-terminal domain (residues 180–325) adopts a folded structure in the periplasmic space. However, some reports have proposed that full-length OmpA can instead refold in a temperature dependent manner into a single domain forming a larger transmembrane pore. Here, we have determined the NMR solution structure of the C-terminal periplasmic domain of E. coli OmpA (OmpA180–325). Our structure reveals that the C-terminal domain folds independently into a stable globular structure that is homologous to the previously reported PG-associated domain of Neisseria meningitides RmpM. Our results lend credence to the two domain structure model and a PG-binding function for OmpA, and we could indeed localize the PG-binding site on the protein through NMR chemical shift perturbation experiments. On the other hand, we found no evidence for binding of OmpA180–325 with the TonB protein. In addition, we have also expressed and purified full-length OmpA (OmpA1–325) to study the structure of the full-length protein in micelles and nanodiscs by NMR spectroscopy. In both membrane mimetic environments, the recombinant OmpA maintains its two domain structure that is connected through a flexible linker. A series of temperature-dependent HSQC experiments and relaxation dispersion NMR experiments detected structural destabilization in the bulge region of the periplasmic domain of OmpA above physiological temperatures, which may induce dimerization and play a role in triggering the previously reported larger pore formation.

Constitutively Oxidized CXXC Motifs within the CD3 Heterodimeric Ectodomains of the T Cell Receptor Complex Enforce the Conformation of Juxtaposed Segments

The CD3ϵγ and CD3ϵδ heterodimers along with the CD3ζζ homodimer are the signaling components of the T cell receptor (TCR). These invariant dimers are non-covalently associated on the T cell plasma membrane with a clone-specific (i.e. clonotypic) αβ heterodimer that binds its cognate ligand, a complex between a particular antigenic peptide, and an MHC molecule (pMHC). These four TCR dimers exist in a 1:1:1:1 stoichiometry. At the junction between the extracellular and transmembrane domains of each mammalian CD3ϵ, CD3γ, and CD3δ subunit is a highly conserved CXXC motif previously found to be important for thymocyte and T cell activation. The redox state of each CXXC motif is presently unknown. Here we show using LC-MS and a biotin switch assay that these CXXC segments are constitutively oxidized on resting and activated T cells, consistent with their measured reduction potential. NMR chemical shift perturbation experiments comparing a native oxidized CD3δ CXXC-containing segment with that of a mutant SXXS-containing CD3δ segment in LPPG (1-palmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt)) micelles show extensive chemical shift differences in residues within the membrane-proximal motif as well as throughout the transmembrane and cytoplasmic domains as a result of the elimination of the native disulfide. Likewise, direct comparison of the native CD3δ segment in oxidizing and reducing conditions reveals numerous spectral differences. The oxidized CXXC maintains the structure within the membrane-proximal stalk region as well as that of its contiguous transmembrane and cytoplasmic domain, inclusive of the ITAM (immunoreceptor tyrosine-based activation motif) involved in signaling. These results suggest that preservation of the CD3 CXXC oxidized state may be essential for TCR mechanotransduction.

Novel Interaction of the Z-DNA Binding Domain of Human ADAR1 with the Oncogenic c-Myc Promoter G-Quadruplex

Both G-quadruplex and Z-DNA can be formed in G-rich and repetitive sequences on genome, and their formation and biological functions are controlled by specific proteins. Z-DNA binding proteins, such as human ADAR1, have a highly conserved Z-DNA binding domain having selective affinity to Z-DNA. Here, our study identifies the Z-DNA binding domain of human ADAR1 (hZαADAR1) as a novel G-quadruplex binding protein that recognizes c-myc promoter G-quadruplex formed in NHEIII1 region and represses the gene expression. An electrophoretic migration shift assay shows the binding of hZαADAR1 to the intramolecular c-myc promoter G-quadruplex-forming DNA oligomer. To corroborate the binding of hZαADAR1 to the G-quadruplex, we conducted CD and NMR chemical shift perturbation analyses. CD results indicate that hZαADAR1 stabilizes the parallel-stranded conformation of the c-myc G-quadruplex. The NMR chemical shift perturbation data reveal that the G-quadruplex binding region in hZαADAR1 was almost identical with the Z-DNA binding region. Finally, promoter assay and Western blot analysis show that hZαADAR1 suppresses the c-myc expression promoted by NHEIII1 region containing the G-quadruplex-forming sequence. This finding suggests a novel function of Z-DNA binding protein as a regulator of G-quadruplex-mediated gene expression.

Mapping the binding interactions between a specialized Hsp70 chaperone and its Hsp40 co‐chaperone: a direct role for the Hsp40 HPD motif (567.5)

Proteins from the ISC operon of E. coli constitute the machinery used for the synthesis of iron‐sulfur clusters and their delivery to recipient apo‐proteins. [2Fe‐2S] cluster transfer from the holo‐scaffold protein IscU depends on ATP hydrolysis in the nucleotide‐binding domain (NBD) of HscA, a specialized Hsp70 chaperone. HscB, an Hsp40‐type co‐chaperone, binds to HscA and stimulates ATP hydrolysis to promote cluster transfer. However, the interactions between HscA and HscB have remained uncharacterized, and, moreover, the role of HscA’s interdomain linker in modulating ATPase activity has not been explored. We have created three variants of the isolated HscA NBD truncated at different residues within the linker, and have shown that the linker binds to the NBD and autoactivates ATP hydrolysis. Using solution‐state NMR spectroscopy and chemical shift perturbations, we mapped the apo‐HscA, HscA‐ADP and HscA‐ATP binding sites onto HscB. Our NMR data provide the first evidence of a structural role for the ubiquitously conserved J‐domain residues HPD of HscB, which we found selectively bind to the ATP‐bound form of HscA. Additionally, we have identified and characterized a previously unreported interaction between HscA and IscA, an iron‐binding protein putatively involved in [4Fe‐4S] cluster transfer, which competes with HscB for binding to HscA.Grant Funding Source: Supported by NIH grants U01 GM094622 and P41 GM103399

Structure-Guided Activity Enhancement and Catalytic Mechanism of Yeast Grx8

Glutaredoxins (Grxs) are wide-spread oxidoreductases that are found in all kingdoms of life. The yeast Saccharomyces cerevisiae encodes eight Grxs, among which, Grx8 shares a sequence identity of 30 and 23% with typical dithiol Grx1 and Grx2, respectively, but it exhibits a much lower GSH-dependent oxidoreductase activity. To elucidate its catalytic mechanism, we solved the solution structure of Grx8, which displays a typical Grx fold. Structural analysis indicated that Grx8 possesses a negatively charged CXXC motif (Cys(33)-Pro(34)-Asp(35)-Cys(36)) and a GSH-recognition site, which are distinct from Grx1 and Grx2. Subsequent structure-guided site mutations revealed that the D35Y single mutant and N80T/L81V double mutant possess increased activity of 10- and 11-fold, respectively; moreover, the D35Y/N80T/L81V triple mutant has increased activity of up to 44-fold, which is comparable to that of canonical Grx. Biochemical analyses suggested that the increase in catalytic efficiency resulted from a decreased pKa value of catalytic cysteine Cys33 and/or enhancement of the putative GSH-recognition site. Moreover, NMR chemical shift perturbation analyses combined with GSH analogue inhibition assays enabled us to elucidate that wild-type Grx8 and all mutants adopt a ping-pong mechanism of catalysis. All together, these findings provide structural insights into the catalytic mechanism of dithiol Grxs.

A conserved mechanism for binding of p53 DNA-binding domain and anti-apoptotic Bcl-2 family proteins.

The molecular interaction between tumor suppressor p53 and the anti-apoptotic Bcl-2 family proteins plays an essential role in the transcription-independent apoptotic pathway of p53. In this study, we investigated the binding of p53 DNA-binding domain (p53DBD) with the anti-apoptotic Bcl-2 family proteins, Bcl-w, Mcl-1, and Bcl-2, using GST pull-down assay and NMR spectroscopy. The GST pull-down assays and NMR experiments demonstrated the direct binding of the p53DBD with Bcl-w, Mcl-1, and Bcl-2. Further, NMR chemical shift perturbation data showed that Bcl-w and Mcl-1 bind to the positively charged DNA-binding surface of p53DBD. Noticeably, the refined structural models of the complexes between p53DBD and Bcl-w, Mcl-1, and Bcl-2 showed that the binding mode of p53DBD is highly conserved among the anti-apoptotic Bcl-2 family proteins. Furthermore, the chemical shift perturbations on Bcl-w, Mcl-1, and Bcl-2 induced by p53DBD binding occurred not only at the p53DBD-binding acidic region but also at the BH3 peptide-binding pocket, which suggests an allosteric conformational change similar to that observed in Bcl-XL. Taken altogether, our results revealed a structural basis for a conserved binding mechanism between p53DBD and the anti-apoptotic Bcl-2 family proteins, which shed light on to the molecular understanding of the transcription-independent apoptosis pathway of p53.

Post-Translational Modifications Modulate Ligand Recognition by the Third PDZ Domain of the MAGUK Protein PSD-95

The relative promiscuity of hub proteins such as postsynaptic density protein-95 (PSD-95) can be achieved by alternative splicing, allosteric regulation, and post-translational modifications, the latter of which is the most efficient method of accelerating cellular responses to environmental changes in vivo. Here, a mutational approach was used to determine the impact of phosphorylation and succinimidation post-translational modifications on the binding affinity of the postsynaptic density protein-95/discs large/zonula occludens-1 (PDZ3) domain of PSD-95. Molecular dynamics simulations revealed that the binding affinity of this domain is influenced by an interplay between salt-bridges linking the α3 helix, the β2–β3 loop and the positively charged Lys residues in its high-affinity hexapeptide ligand KKETAV. The α3 helix is an extra structural element that is not present in other PDZ domains, which links PDZ3 with the following SH3 domain in the PSD-95 protein. This regulatory mechanism was confirmed experimentally via thermodynamic and NMR chemical shift perturbation analyses, discarding intra-domain long-range effects. Taken together, the results presented here reveal the molecular basis of the regulatory role of the α3 extra-element and the effects of post-translational modifications of PDZ3 on its binding affinity, both energetically and dynamically.

Structural basis for the conserved binding mechanism of MDM2-inhibiting peptides and anti-apoptotic Bcl-2 family proteins

The interaction between tumor suppressor p53 and the anti-apoptotic Bcl-2 family proteins serves a critical role in the transcription-independent apoptosis mechanism of p53. Our previous studies showed that an MDM2-inhibiting motif (residues 15–29) in the p53 transactivation domain (p53TAD) mediates the interaction with anti-apoptotic Bcl-2 family proteins. In this study, we provided structural models of the complexes between the MDM2-inhibiting p53TAD peptide and Mcl-1, Bcl-w, and Kaposi sarcoma-associated herpes virus (KSHV) Bcl-2 using NMR chemical shift perturbation data. The binding mode of the MDM2-inhibiting p53TAD peptide is highly conserved among the anti-apoptotic Bcl-2 family proteins despite their distinct specificities for pro-apoptotic Bcl-2 family proteins. We also identified the binding of a phage-display-derived MDM2-inhibiting peptide 12-1 to anti-apoptotic Bcl-XL protein by using NMR spectroscopy. The structural model of the Bcl-XL/12-1 peptide complex revealed that the conserved residues Phe4, Trp8, and Leu11 in the MDM2-inhibiting peptide fit into a hydrophobic cleft of Bcl-XL in a manner similar to that of pro-apoptotic Bcl-2 homology 3 (BH3) peptides. Our results shed light on the mechanism underlying dual-targeting of the FxxxWxxL-based α-helical motif to MDM2 and anti-apoptotic Bcl-2 family proteins for anticancer therapy.

C60@Lysozyme: Direct Observation by Nuclear Magnetic Resonance of a 1:1 Fullerene Protein Adduct

Integrating carbon nanoparticles (CNPs) with proteins to form hybrid functional assemblies is an innovative research area with great promise for medical, nanotechnology, and materials science. The comprehension of CNP-protein interactions requires the still-missing identification and characterization of the 'binding pocket' for the CNPs. Here, using Lysozyme and C60 as model systems and NMR chemical shift perturbation analysis, a protein-CNP binding pocket is identified unambiguously in solution and the effect of the binding, at the level of the single amino acid, is characterized by a variety of experimental and computational approaches. Lysozyme forms a stoichiometric 1:1 adduct with C60 that is dispersed monomolecularly in water. Lysozyme maintains its tridimensional structure upon interaction with C60 and only a few identified residues are perturbed. The C60 recognition is highly specific and localized in a well-defined pocket.

Calorimetric and NMR Characterization of a Rem2 G-Domain Interaction with the Sh3-GK Core of the Ca2+ Channel Beta4 Subunit

The RGK (Rad, Rem1, Rem2 and Gem/Kir) subfamily of small GTPases has potent inhibitory effects on high voltage-activated Ca2+ channels. The inhibition has been shown to occur through a mechanism that is dependent on the Ca2+ channel beta subunit. In this study, we characterized an interaction between the G domain of Rem2 and the beta4 guanylate kinase (GK) domain using NMR and isothermal titration calorimetry (ITC) techniques. We determined that the interaction is endothermic, has a stoichiometry of 1, and a Kd 156 uM, which is three orders of magnitude weaker than the beta4 interaction with the pore-forming alpha1 subunit. NMR chemical shift perturbation analysis reveals that Rem2 interacts exclusively with the GK domain of the SH3-GK core by way of residues located in the former GMP binding region of the inactive kinase. The site is composed of residues just proximal to 310 helix sequences 2 and 3, and residues in alpha helix 6. Thus, the RGK binding site is clearly distinct from the binding site for the alpha1 subunit AID peptide. Site-directed mutagenesis of surface residues combined with ITC indicate that multiple GK sequences contribute to Rem2 binding collectively. These results support the idea that Ca2+ channel inhibition by Rem2 is unlikely to be the result of direct competition with alpha1 binding to the beta subunit. Instead, it argues in favor of a mechanism in which beta subunit-anchored Rem2 executes its inhibitory effect through other contacts with alpha1 subunits. This study demonstrates that NMR combined with ITC can provide detailed information on important weak protein-protein interactions involved in Ca2+ channel regulation.

Targeting of p53 peptide analogues to anti-apoptotic Bcl-2 family proteins as revealed by NMR spectroscopy

Inhibition of the interaction between the p53 tumor suppressor and its negative regulator MDM2 is of great importance to cancer therapy. The anti-apoptotic Bcl-2 family proteins are also attractive anti-cancer molecular targets, as they are key regulators of apoptotic cell death. Previously, we reported the interactions between the p53 transactivation domain (p53TAD) and diverse members of the anti-apoptotic Bcl-2 family proteins. In this study, we investigated the binding of MDM2-inhibiting p53TAD peptide analogues, p53-MDM2/MDMX inhibitor (PMI) and pDI, with anti-apoptotic Bcl-2 family proteins, Bcl-XL and Bcl-2, by using NMR spectroscopy. The NMR chemical shift perturbation data demonstrated the direct binding of the p53 peptide analogues to Bcl-XL and Bcl-2 and showed that the PMI and pDI peptides bind to a conserved hydrophobic groove of the anti-apoptotic Bcl-2 family proteins. Furthermore, the structural model of the Bcl-XL/PMI peptide complex showed that the binding mode of the PMI peptide is highly similar to that of pro-apoptotic Bcl-2 homology 3 (BH3) peptides. Finally, our structural comparison provided a molecular basis for how the same PMI peptide can bind to two distinct anti-cancer target proteins Bcl-XL and MDM2, which may have potential applications for multi-targeting cancer therapy.

Steric Mechanism of Auto-Inhibitory Regulation of Specific and Non-Specific DNA Binding by the ETS Transcriptional Repressor ETV6

DNA binding by the ETS transcriptional repressor ETV6 (or TEL) is auto-inhibited ~50-fold due to an α-helix that sterically blocks its ETS domain binding interface. Using NMR spectroscopy, we demonstrate that this marginally stable helix is unfolded, and not displaced to a non-inhibitory position, when ETV6 is bound to DNA containing a consensus 5′GGAA3′ recognition site. Although significantly lower in affinity, binding to non-specific DNA is auto-inhibited ~5-fold and is also accompanied by helix unfolding. Based on NMR chemical shift perturbations, both specific and non-specific DNA are bound via the same canonical ETS domain interface. However, spectral perturbations are smaller for the non-specific complex, suggesting weaker and less well-defined interactions than in the specific complex. In parallel, the crystal structure of ETV6 bound to a specific DNA duplex was determined. The structure of this complex reveals that a non-conserved histidine residue in the ETS domain recognition helix helps establish the specificity of ETV6 for DNA-binding sites containing 5′GGAA3′versus5′GGAT3′. These studies provide a unified steric mechanism for attenuating ETV6 binding to both specific and non-specific DNA and expand the repertoire of characterized auto-inhibitory strategies utilized to regulate ETS factors.

Decoding The BH3-Mimetic Pro-Apoptotic Activity Of Quercetin In Jurkat Cells

Deregulation of apoptosis contributes largely to the pathogenesis of lymphoid neoplasms allowing the expansion of cell clones that develop resistance to drug-induced cell death. Therefore, subverting inherent resistance to apoptosis is a very challenging therapeutic issue and developing small molecules that can mimic the BH3 domain of the pro-apoptotic Bcl-2 family proteins appears to offer such potentials. Natural products, a rich source of compounds with BH3 mimetic activity, make a sound basis for such an approach. Quercetin, a natural polyphenol, has drawn much attention because it exerts anticancer cytotoxic effects, while sparing normal cells. Pro-apoptotic effects of quercetin have been shown in human lymphoma and leukemic cells and have been associated with Bcl-2 and Mcl-1 downregulation and Bax conformational activation. We undertook a multidisciplinary approach to elucidate the mode of action of quercetin, including cytotoxicity cell assays, biochemical approaches (pull down assays), NMR spectroscopy and docking calculations. We demonstrate that quercetin binds directly to the BH3 domain of the anti-apoptotic Bcl-2 and Bcl-xL proteins, exhibiting BH3-mimetic properties. Specifically, functional cytotoxicity assays of quercetin in Jurkat T-cell leukemic lines, Jurkat Bcl-2 and Jurkat Puro, indicated that quercetin binds to Bcl-2 protein. The direct binding of quercetin to Bcl-2 and to Bcl-xL was biochemically validated using pull down assays. NMR chemical shift perturbation experiments and docking calculations finally revealed that quercetin was bound to the pro-apoptotic BH3-binding site of the anti-apoptotic Bcl-2 family proteins (Figure 1). This property classifies quercetin as a natural flavonoid with BH3 mimetic activity, capable of driving leukemic cells to apoptosis. These results explain the cytotoxic effects of quercetin on Jurkat cells. These data can serve as a basis to consider studying BH3 mimetic natural products as adjuncts in the therapeutic approaches of lymphoid neoplasms in combination with chemotherapy, and can also provide a starting structure for developing novel potent analogues for the treatment of blood cancers. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Protein Dielectric Constants Determined from NMR Chemical Shift Perturbations

Understanding the connection between protein structure and function requires a quantitative understanding of electrostatic effects. Structure-based electrostatic calculations are essential for this purpose, but their use has been limited by a long-standing discussion on which value to use for the dielectric constants (ε(eff) and ε(p)) required in Coulombic and Poisson-Boltzmann models. The currently used values for ε(eff) and ε(p) are essentially empirical parameters calibrated against thermodynamic properties that are indirect measurements of protein electric fields. We determine optimal values for ε(eff) and ε(p) by measuring protein electric fields in solution using direct detection of NMR chemical shift perturbations (CSPs). We measured CSPs in 14 proteins to get a broad and general characterization of electric fields. Coulomb's law reproduces the measured CSPs optimally with a protein dielectric constant (ε(eff)) from 3 to 13, with an optimal value across all proteins of 6.5. However, when the water-protein interface is treated with finite difference Poisson-Boltzmann calculations, the optimal protein dielectric constant (ε(p)) ranged from 2 to 5 with an optimum of 3. It is striking how similar this value is to the dielectric constant of 2-4 measured for protein powders and how different it is from the ε(p) of 6-20 used in models based on the Poisson-Boltzmann equation when calculating thermodynamic parameters. Because the value of ε(p) = 3 is obtained by analysis of NMR chemical shift perturbations instead of thermodynamic parameters such as pK(a) values, it is likely to describe only the electric field and thus represent a more general, intrinsic, and transferable ε(p) common to most folded proteins.