Characterization of the ATP-binding domain of the sarco(endo)plasmic reticulum Ca2+-ATPase: probing nucleotide binding by multidimensional NMR

ABSTRACT: The skeletal muscle sarco(endo)plasmic reticulum Ca 2+ -ATPase (SERCA1a) mediates muscle relaxation by pumping Ca 2+ from the cytosol to the ER/SR lumen. In efforts aimed at understanding the structural basis for the conformational changes accompanying the reaction cycle catalyzed by SERCA1a, we have studied the ATP-binding domain of SERCA1a in both nucleotide-bound and -free forms by NMR. Limited proteolysis analyses guided us to express a 28 kDa stably folded fragment containing the nucleotide-binding domain of SERCA1a spanning residues Thr357-Leu600. ATP binding activity was demonstrated for this fragment by a FITC competition assay. A nearly complete backbone resonance assignment of this 28 kDa ATP-binding fragment, in both the AMP-PNP-bound and -free forms, was obtained by means of heteronuclear multidimensional NMR techniques. NMR titration experiments with AMP-PNP revealed a confined nucleotide-binding site which coincides with a cytoplasmic pocket region identified in the crystal structure of apo-SERCA1a. These results are consistent with previous site-directed mutagenesis studies of SERCA1a

Force-dependent allostery of the α-catenin actinbinding domain controls adherens junction dynamics and functions

α-catenin is a key mechanosensor that forms force-dependent interactions with F-actin, thereby coupling the cadherin-catenin complex to the actin cytoskeleton at adherens junctions (AJs). However, the molecular mechanisms by which α-catenin engages F-actin under tension remained elusive. Here we show that the α1-helix of the α-catenin actin-binding domain (αcat-ABD) is a mechanosensing motif that regulates tension-dependent F-actin binding and bundling. αcat-ABD containing an α1-helix-unfolding mutation (H1) shows enhanced binding to F-actin in vitro. Although full-length α-catenin-H1 can generate epithelial monolayers that resist mechanical disruption, it fails to support normal AJ regulation in vivo. Structural and simulation analyses suggest that α1-helix allosterically controls the actin-binding residue V796 dynamics. Crystal structures of αcat-ABD-H1 homodimer suggest that α-catenin can facilitate actin bundling while it remains bound to E-cadherin. We propose that force-dependent allosteric regulation of αcat-ABD promotes dynamic interactions with F-actin involved in actin bundling, cadherin clustering, and AJ remodeling during tissue morphogenesis

Rational design and validation of a Tip60 histone acetyltransferase inhibitor

Histone acetylation is required for many aspects of gene regulation, genome maintenance and metabolism and dysfunctional acetylation is implicated in numerous diseases, including cancer. Acetylation is regulated by histone acetyltransferases (HATs) and histone deacetylases and currently, few general HAT inhibitors have been described. We identified the HAT Tip60 as an excellent candidate for targeted drug development, as Tip60 is a key mediator of the DNA damage response and transcriptional co-activator. Our modeling of Tip60 indicated that the active binding pocket possesses opposite charges at each end, with the positive charges attributed to two specific side chains. We used structure based drug design to develop a novel Tip60 inhibitor, TH1834, to fit this specific pocket. We demonstrate that TH1834 significantly inhibits Tip60 activity in vitro and treating cells with TH1834 results in apoptosis and increased unrepaired DNA damage (following ionizing radiation treatment) in breast cancer but not control cell lines. Furthermore, TH1834 did not affect the activity of related HAT MOF, as indicated by H4K16Ac, demonstrating specificity. The modeling and validation of the small molecule inhibitor TH1834 represents a first step towards developing additional specific, targeted inhibitors of Tip60 that may lead to further improvements in the treatment of breast cancer

Mapping interactions with the chaperone network reveals factors that protect against tau aggregation.

A network of molecular chaperones is known to bind proteins ('clients') and balance their folding, function and turnover. However, it is often unclear which chaperones are critical for selective recognition of individual clients. It is also not clear why these key chaperones might fail in protein-aggregation diseases. Here, we utilized human microtubule-associated protein tau (MAPT or tau) as a model client to survey interactions between ~30 purified chaperones and ~20 disease-associated tau variants (~600 combinations). From this large-scale analysis, we identified human DnaJA2 as an unexpected, but potent, inhibitor of tau aggregation. DnaJA2 levels were correlated with tau pathology in human brains, supporting the idea that it is an important regulator of tau homeostasis. Of note, we found that some disease-associated tau variants were relatively immune to interactions with chaperones, suggesting a model in which avoiding physical recognition by chaperone networks may contribute to disease

Imaging of Oxidation-Specific Epitopes in Atherosclerosis and Macrophage-Rich Vulnerable Plaques

Oxidative stress, and in particular oxidation of lipoproteins, is a hallmark of atherosclerosis. Upon entry of lipoproteins into the vessel wall, a cascade of pro-atherogenic pathways is initiated whereby the reaction of reactive oxygen species with substrates amenable to oxidation, such as polyunsaturated fatty acids, generates a variety of oxidation-specific epitopes on lipoproteins, proteins in the vessel wall, and apoptotic macrophages. Several of these oxidation-specific epitopes have been well characterized and specific murine and fully human antibodies have been generated in our laboratory to detect them in the vessel wall. We have developed radionuclide, gadolinium and iron oxide based MRI techniques to noninvasively image oxidation-specific epitopes in atherosclerotic lesions. These approaches quantitate plaque burden and also allow detection of atherosclerosis regression and plaque stabilization. In particular, gadolinium micelles or lipid-coated ultrasmall superparamagnetic iron oxide particles containing oxidation-specific antibodies accumulate within macrophages in the artery wall, suggesting they may image the most unstable plaques. Translation of these approaches to humans may allow a sensitive technique to image and monitor high-risk atherosclerotic lesions and may guide optimal therapeutic interventions

Structural mechanism of JH delivery in hemolymph by JHBP of silkworm, Bombyx mori

Juvenile hormone (JH) plays crucial roles in many aspects of the insect life. All the JH actions are initiated by transport of JH in the hemolymph as a complex with JH-binding protein (JHBP) to target tissues. Here, we report structural mechanism of JH delivery by JHBP based upon the crystal and solution structures of apo and JH-bound JHBP. In solution, apo-JHBP exists in equilibrium of multiple conformations with different orientations of the gate helix for the hormone-binding pocket ranging from closed to open forms. JH-binding to the gate-open form results in the fully closed JHBP-JH complex structure where the bound JH is completely buried inside the protein. JH-bound JHBP opens the gate helix to release the bound hormone likely by sensing the less polar environment at the membrane surface of target cells. This is the first report that provides structural insight into JH signaling

Function of the Active Site Lysine Autoacetylation in Tip60 Catalysis

The 60-kDa HIV-Tat interactive protein (Tip60) is a key member of the MYST family of histone acetyltransferases (HATs) that plays critical roles in multiple cellular processes. We report here that Tip60 undergoes autoacetylation at several lysine residues, including a key lysine residue (i.e. Lys-327) in the active site of the MYST domain. The mutation of K327 to arginine led to loss of both the autoacetylation activity and the cognate HAT activity. Interestingly, deacetylated Tip60 still kept a substantial degree of HAT activity. We also investigated the effect of cysteine 369 and glutamate 403 in Tip60 autoacetylation in order to understand the molecular pathway of the autoacetylation at K327. Together, we conclude that the acetylation of K327 which is located in the active site of Tip60 regulates but is not obligatory for the catalytic activity of Tip60. Since acetylation at this key residue appears to be evolutionarily conserved amongst all MYST proteins, our findings provide an interesting insight into the regulatory mechanism of MYST activities

ATM Modulates the Loading of Recombination Proteins onto a Chromosomal Translocation Breakpoint Hotspot

Chromosome translocations induced by DNA damaging agents, such as ionizing radiation and certain chemotherapies, alter genetic information resulting in malignant transformation. Abrogation or loss of the ataxia-telangiectasia mutated (ATM) protein, a DNA damage signaling regulator, increases the incidence of chromosome translocations. However, how ATM protects cells from chromosome translocations is still unclear. Chromosome translocations involving the MLL gene on 11q23 are the most frequent chromosome abnormalities in secondary leukemias associated with chemotherapy employing etoposide, a topoisomerase II poison. Here we show that ATM deficiency results in the excessive binding of the DNA recombination protein RAD51 at the translocation breakpoint hotspot of 11q23 chromosome translocation after etoposide exposure. Binding of Replication protein A (RPA) and the chromatin remodeler INO80, which facilitate RAD51 loading on damaged DNA, to the hotspot were also increased by ATM deficiency. Thus, in addition to activating DNA damage signaling, ATM may avert chromosome translocations by preventing excessive loading of recombinational repair proteins onto translocation breakpoint hotspots

The p400 ATPase regulates nucleosome stability and chromatin ubiquitination during DNA repair

p400 unwinds chromatin from nucleosomes flanking double-strand breaks to facilitate recruitment of the DNA repair components brca1 and 53BP1

Mapping Site-Specific Changes that Affect Stability of the NTerminal Domain of Calmodulin

Biophysical tools have been invaluable in formulating therapeutic proteins. These tools characterize protein stability rapidly in a variety of solution conditions, but in general, the techniques lack the ability to discern site-specific information to probe how solution environment acts to stabilize or destabilize the protein. NMR spectroscopy can provide site-specific information about subtle structural changes of a protein under different conditions, enabling one to assess the mechanism of protein stabilization. In this study, NMR was employed to detect structural perturbations at individual residues as a result of altering pH and ionic strength. The N-terminal domain of calmodulin (N-CaM) was used as a model system, and the 1H-15N heteronuclear single quantum coherence (HSQC) experiment was used to investigate effects of pH and ionic strength on individual residues. NMR analysis revealed that different solution conditions affect individual residues differently, even when the amino acid sequence and structure are highly similar. This study shows that addition of NMR to the formulation toolbox has the ability to extend understanding of the relationship between site-specific changes and overall protein stability