Structure of histone deacetylase complex Rpd3S bound to nucleosome.

Crosstalk between histone modifications represents a fundamental epigenetic mechanism in gene regulation. During the transcription elongation process, the histone deacetylase complex Rpd3S is recruited to H3K36-methylated nucleosomes to suppress cryptic transcription initiation. However, how subunits of Rpd3S are assembled and coordinated to recognize nucleosomal substrates and exert their deacetylation function remains unclear. Here we report the structure of Saccharomyces cerevisiae Rpd3S deacetylase bound to H3K36me3-modified nucleosome at 3.1 Å resolution. It shows that Sin3 and Rco1 subunits orchestrate the assembly of the complex and mediate its contact with nucleosome at multiple sites, with the Sin3-DNA interface as a pivotal anchor. The PHD1 domain of Rco1 recognizes the unmodified H3K4 and places the following H3 tail toward the active site of Rpd3, while the chromodomain of Eaf3 subunit recognizes the H3K36me3 mark and contacts both nucleosomal and linker DNA. The second copy of Eaf3-Rco1 is involved in neighboring nucleosome binding. Our work unravels the structural basis of chromatin targeting and deacetylation by the Rpd3S complex.

ClpC2 protects mycobacteria against a natural antibiotic targeting ClpC1-dependent protein degradation.

Mycobacterium tuberculosis Clp proteases are targeted by several antitubercular compounds, including cyclomarin A (CymA). CymA exerts its toxicity by binding to AAA + chaperone ClpC1. Here, we show that CymA can also bind a partial homologue of ClpC1, known as ClpC2, and we reveal the molecular basis of these interactions by determining the structure of the M. tuberculosis ClpC2:CymA complex. Furthermore, we show deletion of clpC2 in Mycobacterium smegmatis increases sensitivity to CymA. We find CymA exposure leads to a considerable upregulation of ClpC2 via a mechanism in which binding of CymA to ClpC2 prevents binding of ClpC2 to its own promoter, resulting in upregulation of its own transcription in response to CymA. Our study reveals that ClpC2 not only senses CymA, but that through this interaction it can act as a molecular sponge to counteract the toxic effects of CymA and possibly other toxins targeting essential protease component ClpC1 in mycobacteria.

Structures of prokaryotic ubiquitin-like protein Pup in complex with depupylase Dop reveal the mechanism of catalytic phosphate formation.

Pupylation is the post-translational modification of lysine side chains with prokaryotic ubiquitin-like protein (Pup) that targets proteins for proteasomal degradation in mycobacteria and other members of Actinobacteria. Pup ligase PafA and depupylase Dop are the two enzymes acting in this pathway. Although they share close structural and sequence homology indicative of a common evolutionary origin, they catalyze opposing reactions. Here, we report a series of high-resolution crystal structures of Dop in different functional states along the reaction pathway, including Pup-bound states in distinct conformations. In combination with biochemical analysis, the structures explain the role of the C-terminal residue of Pup in ATP hydrolysis, the process that generates the catalytic phosphate in the active site, and suggest a role for the Dop-loop as an allosteric sensor for Pup-binding and ATP cleavage.

Pupylated proteins are subject to broad proteasomal degradation specificity and differential depupylation.

In actinobacteria, post-translational modification of proteins with prokaryotic ubiquitin-like protein Pup targets them for degradation by a bacterial proteasome assembly consisting of the 20S core particle (CP) and the mycobacterial proteasomal ATPase (Mpa). Modification of hundreds of cellular proteins with Pup at specific surface lysines is carried out by a single Pup-ligase (PafA, proteasome accessory factor A). Pupylated substrates are recruited to the degradative pathway by binding of Pup to the N-terminal coiled-coil domains of Mpa. Alternatively, pupylation can be reversed by the enzyme Dop (deamidase of Pup). Although pupylated substrates outcompete free Pup in proteasomal degradation, potential discrimination of the degradation complex between the various pupylated substrates has not been investigated. Here we show that Mpa binds stably to an open-gate variant of the proteasome (oCP) and associates with bona fide substrates with highly similar affinities. The proteasomal degradation of substrates differing in size, structure and assembly state was recorded in real-time, showing that the pupylated substrates are processed by the Mpa-oCP complex with comparable kinetic parameters. Furthermore, the members of a complex, pupylated proteome (pupylome) purified from Mycobacterium smegmatis are degraded evenly as followed by western blotting. In contrast, analysis of the depupylation behavior of several pupylome members suggests substrate-specific differences in enzymatic turnover, leading to the conclusion that largely indiscriminate degradation competes with differentiated depupylation to control the ultimate fate of pupylated substrates.

Ionic interactions of Ba2+ blockades in the MthK K+ channel.

The movement and interaction of multiple ions passing through in single file underlie various fundamental K(+) channel properties, from the effective conduction of K(+) ions to channel blockade by Ba(2+) ions. In this study, we used single-channel electrophysiology and x-ray crystallography to probe the interactions of Ba(2+) with permeant ions within the ion conduction pathway of the MthK K(+) channel. We found that, as typical of K(+) channels, the MthK channel was blocked by Ba(2+) at the internal side, and the Ba(2+)-blocking effect was enhanced by external K(+). We also obtained crystal structures of the MthK K(+) channel pore in both Ba(2+)-Na(+) and Ba(2+)-K(+) environments. In the Ba(2+)-Na(+) environment, we found that a single Ba(2+) ion remained bound in the selectivity filter, preferably at site 2, whereas in the Ba(2+)-K(+) environment, Ba(2+) ions were predominantly distributed between sites 3 and 4. These ionic configurations are remarkably consistent with the functional studies and identify a molecular basis for Ba(2+) blockade of K(+) channels.

Nanoliter-scale protein crystallization and screening with a microfluidic droplet robot.

Large-scale screening of hundreds or even thousands of crystallization conditions while with low sample consumption is in urgent need, in current structural biology research. Here we describe a fully-automated droplet robot for nanoliter-scale crystallization screening that combines the advantages of both automated robotics technique for protein crystallization screening and the droplet-based microfluidic technique. A semi-contact dispensing method was developed to achieve flexible, programmable and reliable liquid-handling operations for nanoliter-scale protein crystallization experiments. We applied the droplet robot in large-scale screening of crystallization conditions of five soluble proteins and one membrane protein with 35-96 different crystallization conditions, study of volume effects on protein crystallization, and determination of phase diagrams of two proteins. The volume for each droplet reactor is only ca. 4-8 nL. The protein consumption significantly reduces 50-500 fold compared with current crystallization stations.