Structure of RadB recombinase from a hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1: an implication for the formation of a near-7-fold helical assembly

The X-ray crystal structure of RadB from Thermococcus kodakaraensis KOD1, an archaeal homologue of the RecA/Rad51 family proteins, have been determined in two crystal forms. The structure represents the core ATPase domain of the RecA/Rad51 proteins. Two independent molecules in the type 1 crystal were roughly related by 7-fold screw symmetry whereas non-crystallographic 2-fold symmetry was observed in the type 2 crystal. The dimer structure in the type 1 crystal is extended to construct a helical assembly, which resembles the filamentous structures reported for other RecA/Rad51 proteins. The molecular interface in the type 1 dimer is formed by facing a basic surface patch of one monomer to an acidic one of the other. The empty ATP binding pocket is located at the interface and barely concealed from the outside similarly to that in the active form of the RecA filament. The model assembly has a positively charged belt on one surface bordering the helical groove suitable for facile binding of DNA. Electron microscopy has revealed that, in the absence of ATP and DNA, RadB forms a filament with a similar diameter to that of the hypothetical assembly, although its helical properties were not confirmed

Structural basis for recognition of the matrix attachment region of DNA by transcription factor SATB1

Special AT-rich sequence binding protein 1 (SATB1) regulates gene expression essential in immune T-cell maturation and switching of fetal globin species, by binding to matrix attachment regions (MARs) of DNA and inducing a local chromatin remodeling. Previously we have revealed a five-helix structure of the N-terminal CUT domain, which is essentially the folded region in the MAR-binding domain, of human SATB1 by NMR. Here we determined crystal structure of the complex of the CUT domain and a MAR DNA, in which the third helix of the CUT domain deeply enters the major groove of DNA in the B-form. Bases of 5′-CTAATA-3′ sequence are contacted by this helix, through direct and water-mediated hydrogen bonds and apolar and van der Waals contacts. Mutations at conserved base-contacting residues, Gln402 and Gly403, reduced the DNA-binding activity, which confirmed the importance of the observed interactions involving these residues. A significant number of equivalent contacts are observed also for typically four-helix POU-specific domains of POU-homologous proteins, indicating that these domains share a common framework of the DNA-binding mode, recognizing partially similar DNA sequences

Role of Phe283 in enzymatic reaction of cyclodextrin glycosyltransferase from alkalophilic Bacillus sp.1011: Substrate binding and arrangement of the catalytic site

Cyclodextrin glycosyltransferase (CGTase) belonging to the α-amylase family mainly catalyzes transglycosylation and produces cyclodextrins from starch and related α-1,4-glucans. The catalytic site of CGTase specifically conserves four aromatic residues, Phe183, Tyr195, Phe259, and Phe283, which are not found in α-amylase. To elucidate the structural role of Phe283, we determined the crystal structures of native and acarbose-complexed mutant CGTases in which Phe283 was replaced with leucine (F283L) or tyrosine (F283Y). The temperature factors of the region 259–269 in native F283L increased >10 Å2 compared with the wild type. The complex formation with acarbose not only increased the temperature factors (>10 Å2) but also changed the structure of the region 257–267. This region is stabilized by interactions of Phe283 with Phe259 and Leu260 and plays an important role in the cyclodextrin binding. The conformation of the side-chains of Glu257, Phe259, His327, and Asp328 in the catalytic site was altered by the mutation of Phe283 with leucine, and this indicates that Phe283 partly arranges the structure of the catalytic site through contacts with Glu257 and Phe259. The replacement of Phe283 with tyrosine decreased the enzymatic activity in the basic pH range. The hydroxyl group of Tyr283 forms hydrogen bonds with the carboxyl group of Glu257, and the pKa of Glu257 in F283Y may be lower than that in the wild type

Role of Trp140 at subsite −6 on the maltohexaose production of maltohexaose-producing amylase from alkalophilic Bacillus sp.707

Maltohexaose-producing amylase (G6-amylase) from alkalophilic Bacillus sp.707 predominantly produces maltohexaose (G6) in the yield of >30% of the total products from short-chain amylose (DP = 17). Our previous crystallographic study showed that G6-amylase has nine subsites, from −6 to +3, and pointed out the importance of the indole moiety of Trp140 in G6 production. G6-amylase has very low levels of hydrolytic activities for oligosaccharides shorter than maltoheptaose. To elucidate the mechanism underlying G6 production, we determined the crystal structures of the G6-amylase complexes with G6 and maltopentaose (G5). In the active site of the G6-amylase/G5 complex, G5 is bound to subsites −6 to −2, while G1 and G6 are found at subsites + 2 and −7 to −2, respectively, in the G6-amylase/G6 complex. In both structures, the glucosyl residue located at subsite −6 is stacked to the indole moiety of Trp140 within a distance of 4Å. The measurement of the activities of the mutant enzymes when Trp140 was replaced by leucine (W140L) or by tyrosine (W140Y) showed that the G6 production from short-chain amylose by W140L is lower than that by W140Y or wild-type enzyme. The face-to-face short contact between Trp140 and substrate sugars is suggested to regulate the disposition of the glucosyl residue at subsite −6 and to govern product specificity for G6 production