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Binding hotspots of BAZ2B bromodomain: Histone interaction revealed by solution NMR driven docking.

ABSTRACT: Bromodomains are epigenetic reader domains, which have come under increasing scrutiny both from academic and pharmaceutical research groups. Effective targeting of the BAZ2B bromodomain by small molecule inhibitors has been recently reported, but no structural information is yet available on the interaction with its natural binding partner, acetylated histone H3K14ac. We have assigned the BAZ2B bromodomain and studied its interaction with H3K14ac acetylated peptides by NMR spectroscopy using both chemical shift perturbation (CSP) data and clean chemical exchange (CLEANEX-PM) NMR experiments. The latter was used to characterize water molecules known to play an important role in mediating interactions. Besides the anticipated Kac binding site, we consistently found the bromodomain BC loop as hotspots for the interaction. This information was used to create a data-driven model for the complex using HADDOCK. Our findings provide both structure and dynamics characterization that will be useful in the quest for potent and selective inhibitors to probe the function of the BAZ2B bromodomain.

SUBMITTER: Ferguson FM

PROVIDER: S-EPMC4458377 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Binding hotspots of BAZ2B bromodomain: Histone interaction revealed by solution NMR driven docking.

Ferguson Fleur M FM Dias David M DM Rodrigues João P G L M JP Wienk Hans H Boelens Rolf R Bonvin Alexandre M J J AM Abell Chris C Ciulli Alessio A

Biochemistry 20141015 42

Bromodomains are epigenetic reader domains, which have come under increasing scrutiny both from academic and pharmaceutical research groups. Effective targeting of the BAZ2B bromodomain by small molecule inhibitors has been recently reported, but no structural information is yet available on the interaction with its natural binding partner, acetylated histone H3K14ac. We have assigned the BAZ2B bromodomain and studied its interaction with H3K14ac acetylated peptides by NMR spectroscopy using bot ...[more]

PMID: 25266743

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Project description:BackgroundBrd2 is a transcriptional regulator and belongs to BET family, a less characterized novel class of bromodomain-containing proteins. Brd2 contains two tandem bromodomains (BD1 and BD2, 46% sequence identity) in the N-terminus and a conserved motif named ET (extra C-terminal) domain at the C-terminus that is also present in some other bromodomain proteins. The two bromodomains have been shown to bind the acetylated histone H4 and to be responsible for mitotic retention on chromosomes, which is probably a distinctive feature of BET family proteins. Although the crystal structure of Brd2 BD1 is reported, no structure features have been characterized for Brd2 BD2 and its interaction with acetylated histones.ResultsHere we report the solution structure of human Brd2 BD2 determined by NMR. Although the overall fold resembles the bromodomains from other proteins, significant differences can be found in loop regions, especially in the ZA loop in which a two amino acids insertion is involved in an uncommon pi-helix, termed piD. The helix piD forms a portion of the acetyl-lysine binding site, which could be a structural characteristic of Brd2 BD2 and other BET bromodomains. Unlike Brd2 BD1, BD2 is monomeric in solution. With NMR perturbation studies, we have mapped the H4-AcK12 peptide binding interface on Brd2 BD2 and shown that the binding was with low affinity (2.9 mM) and in fast exchange. Using NMR and mutational analysis, we identified several residues important for the Brd2 BD2-H4-AcK12 peptide interaction and probed the potential mechanism for the specific recognition of acetylated histone codes by Brd2 BD2.ConclusionBrd2 BD2 is monomeric in solution and dynamically interacts with H4-AcK12. The additional secondary elements in the long ZA loop may be a common characteristic of BET bromodomains. Surrounding the ligand-binding cavity, five aspartate residues form a negatively charged collar that serves as a secondary binding site for H4-AcK12. We suggest that Brd2 BD1 and BD2 may possess distinctive roles and cooperate to regulate Brd2 functions. The structure basis of Brd2 BD2 will help to further characterize the functions of Brd2 and its BET members.

Project description:BackgroundBiological interaction affects diverse facets of microbial life by modulating the activity, diversity, abundance, and composition of microbial communities. Aerobic methane oxidation is a community function, with emergent community traits arising from the interaction of the methane-oxidizers (methanotrophs) and non-methanotrophs. Yet little is known of the spatial and temporal organization of these interaction networks in naturally-occurring complex communities. We hypothesized that the assembled bacterial community of the interaction network in methane hotspots would converge, driven by high substrate availability that favors specific methanotrophs, and in turn influences the recruitment of non-methanotrophs. These environments would also share more co-occurring than site-specific taxa.ResultsWe applied stable isotope probing (SIP) using 13C-CH4 coupled to a co-occurrence network analysis to probe trophic interactions in widespread methane-emitting environments, and over time. Network analysis revealed predominantly unique co-occurring taxa from different environments, indicating distinctly co-evolved communities more strongly influenced by other parameters than high methane availability. Also, results showed a narrower network topology range over time than between environments. Co-occurrence pattern points to Chthoniobacter as a relevant yet-unrecognized interacting partner particularly of the gammaproteobacterial methanotrophs, deserving future attention. In almost all instances, the networks derived from the 13C-CH4 incubation exhibited a less connected and complex topology than the networks derived from the unlabelledC-CH4 incubations, likely attributable to the exclusion of the inactive microbial population and spurious connections; DNA-based networks (without SIP) may thus overestimate the methane-dependent network complexity.ConclusionWe demonstrated that site-specific environmental parameters more strongly shaped the co-occurrence of bacterial taxa than substrate availability. Given that members of the interactome without the capacity to oxidize methane can exert interaction-induced effects on community function, understanding the co-occurrence pattern of the methane-driven interaction network is key to elucidating community function, which goes beyond relating activity to community composition, abundances, and diversity. More generally, we provide a methodological strategy that substantiates the ecological linkages between potentially interacting microorganisms with broad applications to elucidate the role of microbial interaction in community function.

Project description:'In dopaminergic neurons, alpha-synuclein (alphaS) partitions between a disordered cytosolic state and a lipid-bound state. Binding of alphaS to membrane phospholipids is implicated in its functional role in synaptic regulation, but also impacts fibril formation associated with Parkinson's disease. We describe here a solution NMR study in which alphaS is added to small unilamellar vesicles of a composition mimicking synaptic vesicles; the results provide evidence for multiple distinct phospholipid-binding modes of alphaS. Exchange between the free state and the lipid-bound alphaS state, and between different bound states is slow on the NMR timescale, being in the range of 1-10 s(-1). Partitioning of the binding modes is dependent on lipid/alphaS stoichiometry, and tight binding with slow-exchange kinetics is observed at stoichiometries as low as 2:1. In all lipid-bound states, a segment of residues starting at the N-terminus of alphaS adopts an alpha-helical conformation, while succeeding residues retain the characteristics of a random coil. The 40 C-terminal residues remain dynamically disordered, even at high-lipid concentrations, but can also bind to lipids to an extent that appears to be determined by the fraction of cis X-Pro peptide bonds in this region. While lipid-bound alphaS exhibits dynamic properties that preclude its direct observation by NMR, its exchange with the NMR-visible free form allows for its indirect characterization. Rapid amide-amide nuclear Overhauser enhancement buildup points to a large alpha-helical conformation, and a distinct increase in fluorescence anisotropy attributed to Tyr39 indicates an ordered environment for this "dark state." Titration of alphaS with increasing amounts of lipids suggests that the binding mode under high-lipid conditions remains qualitatively similar to that in the low-lipid case. The NMR data appear incompatible with the commonly assumed model where alphaS lies in an alpha-helical conformation on the membrane surface and instead suggest that considerable remodeling of the vesicles is induced by alphaS.

Dataset Information

Binding hotspots of BAZ2B bromodomain: Histone interaction revealed by solution NMR driven docking.

Publications

Binding hotspots of BAZ2B bromodomain: Histone interaction revealed by solution NMR driven docking.

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