SUMMARY Mammalian SWI/SNF (mSWI/SNF) ATP-dependent chromatin remodeling complexes are multi-subunit molecular machines that play vital roles in regulating genomic architecture and are frequently disrupted in human cancer and developmental disorders. To date, the modular organization and pathways of assembly of these chromatin regulators remain unknown, presenting a major barrier to structural and functional determination. Here, we elucidate the architecture and assembly pathway across three classes of mSWI/SNF complexes—canonical BRGI/BRM-associated factor (BAF), polybromo-associated BAF (PBAF), and newly defined ncBAF complexes—and define the requirement of each subunit for complex formation and stability. Using affinity purification of endogenous complexes from mammalian and Drosophila cells coupled with cross-linking mass spectrometry (CX-MS) and mutagenesis, we uncover three distinct and evolutionarily conserved modules, their organization, and the temporal incorporation of these modules into each complete mSWI/SNF complex class. Finally, we map human disease-associated mutations within subunits and modules, defining specific topological regions that are affected upon subunit perturbation.

l-Digitoxose is an unusual dideoxysugar found attached to various pharmacologically active natural products, including the antitumor antibiotic tetrocarcin A and the antibiotics kijanimicin and jadomycin B. Six enzymes are required for its production starting from glucose 1-phosphate. Here we describe a combined structural and functional investigation of KijD10, an NADPH-dependent C-3''-ketoreductase that catalyzes the third step of l-digitoxose biosynthesis in the African soil-dwelling bacterium Actinomadura kijaniata. KijD10 belongs to the glucose-fructose oxidoreductase superfamily. For this investigation, both binary and ternary complexes of KijD10 were crystallized, and their structures were determined to 2.0 Å resolution or better. On the basis of these high-resolution structures, two potential active site acids were identified, Lys 102 and Tyr 186. These residues were individually mutated and the resultant proteins investigated both kinetically and structurally. The Y186F mutant protein demonstrated significant catalytic activity, and its structure was virtually identical to that of the wild-type enzyme except for the positioning of the nicotinamide ring. All lysine mutations, on the other hand, resulted in proteins with either abolished or drastically reduced catalytic activities. Structures for the K102A and K102E mutant proteins were determined and showed that the abrogation of catalytic activity was not a result of large conformational changes. Taken together, these data suggest that Lys 102 donates a proton to the C-3'' keto group during the reaction and that Tyr 186 serves only an auxiliary role. This is in contrast to that proposed for glucose-fructose oxidoreductase and other family members in which the tyrosines, or in some cases similarly positioned histidines, are thought to play major catalytic roles.

Unusual deoxy sugars are often attached to natural products such as antibiotics, antifungals, and chemotherapeutic agents. One such sugar is mycinose, which has been found on the antibiotics chalcomycin and tylosin. An intermediate in the biosynthesis of mycinose is dTDP-6-deoxy-D-allose. Four enzymes are required for the production of dTDP-6-deoxy-D-allose in Streptomyces bikiniensis, a soil-dwelling microbe first isolated from the Bikini and Rongelap atolls. Here we describe a combined structural and functional study of the enzyme ChmJ, which reportedly catalyzes the third step in the pathway leading to dTDP-6-deoxy-D-allose formation. Specifically, it has been proposed that ChmJ is a 3'-epimerase that converts dTDP-4-keto-6-deoxyglucose to dTDP-4-keto-6-deoxyallose. This activity, however, has never been verified in vitro. As reported here, we demonstrate using (1)H nuclear magnetic resonance that ChmJ, indeed, functions as a 3'-epimerase. In addition, we determined the structure of ChmJ complexed with dTDP-quinovose to 2.0 Å resolution. The structure of ChmJ shows that it belongs to the well-characterized "cupin" superfamily. Two active site residues, His 60 and Tyr 130, were subsequently targeted for study via site-directed mutagenesis and kinetic analyses, and the three-dimensional architecture of the H60N/Y130F mutant protein was determined to 1.6 Å resolution. Finally, the structure of the apoenzyme was determined to 2.2 Å resolution. It has been previously suggested that the position of a conserved tyrosine, Tyr 130 in the case of ChmJ, determines whether an enzyme in this superfamily functions as a mono- or diepimerase. Our results indicate that the orientation of the tyrosine residue in ChmJ is a function of the ligand occupying the active site cleft.

The unusual dideoxy sugar d-anthrose has been identified as an important component in the endospores of infectious agents such as Bacillus anthracis and Bacillus cereus. Specifically, it is the terminal sugar on the bacterium's exosporium, and it provides a point of interaction between the spore and the host. The biosynthesis of d-anthrose involves numerous steps starting from α-d-glucose 1-phosphate. Here we present a combined structural and functional investigation of AntD from B. cereus. This enzyme plays a key role in d-anthrose biosynthesis by catalyzing the acylation of the C-4″ amino group of dTDP-4-amino-4,6-dideoxyglucose using 3-hydroxy-3-methylbutyryl-CoA as the acyl donor. For this investigation, two ternary complexes of AntD were determined to 1.8 Å resolution: one in which the protein contained bound β-hydroxybutyryl-CoA and dTDP and the second with CoA and dTDP-4-amino-4,6-dideoxyglucose. On the basis of these high-resolution structures, it was shown that the side chain of Asp 94 lies within hydrogen bonding distance of the sugar C-4″ amino group, and the side chain of Ser 84 resides near the carbonyl oxygen of β-hydroxybutyryl-CoA. To test the roles of these residues in the catalytic mechanism of AntD, various site-directed mutant proteins were prepared and subjected to kinetic and structural analyses. The D94A and D94N mutant proteins demonstrated enzymatic activity, albeit with significantly reduced catalytic efficiencies. The S84A mutant protein showed an approximate 10-fold decrease in activity. Interestingly, the S84C and S84T mutant proteins were both active but demonstrated substrate inhibition. The three-dimensional structures of all of the mutant proteins were nearly identical to that of the wild-type enzyme, indicating that the changes in their kinetic parameters were not due to major conformational changes. Taken together, these data suggest that Asp 94 is important for substrate binding, but probably does not function as an enzymatic base, and that Ser 84 most likely plays a role in the formation of an oxyanion hole.