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Substrate-binding Domains (substrate-binding + domain)
Selected AbstractsDirect inter-subdomain interactions switch between the closed and open forms of the Hsp70 nucleotide-binding domain in the nucleotide-free stateACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2010Meiri Shida The 70,kDa heat-shock proteins (Hsp70s) are highly conserved chaperones that are involved in several cellular processes, such as protein folding, disaggregation and translocation. In this study, the crystal structures of the human Hsp70 nucleotide-binding domain (NBD) fragment were determined in the nucleotide-free state and in complex with adenosine 5,-(,,,-imido)triphosphate (AMPPNP). The structure of the nucleotide-free NBD fragment is similar to that of the AMPPNP-bound NBD fragment and is designated as the `closed form'. In the nucleotide-free NBD fragment the closed form is intrinsically supported through interactions between Tyr15, Lys56 and Glu268 which connect subdomains IA, IB and IIB at the centre of the protein. Interaction with the substrate-binding domain (SBD) of Hsp70 or the BAG domain of BAG1 impairs this subdomain connection and triggers the rotation of subdomain IIA around a hydrophobic helix from subdomain IA. The subdomain rotation is limited by Asp199 and Asp206 from subdomain IIA and clearly defines the open form of the NBD. The open form is further stabilized by a new interaction between Gly230 from subdomain IIB and Ser340 from subdomain IIA. The structure of the NBD in the nucleotide-free state is determined by switching of the inter-subdomain interactions. [source] Crystallization of selenomethionyl exo-,-1,3-galactanase from the basidiomycete Phanerochaete chrysosporiumACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 12 2009Takuya Ishida Exo-,-1,3-galactanase from Phanerochaete chrysosporium (Pc1,3Gal43A) consists of a glycoside hydrolase family 43 catalytic domain and a substrate-binding domain that belongs to carbohydrate-binding module family 35. It catalyzes the hydrolysis of ,-1,3-galactan, which is the backbone of the arabinogalactan proteins; the C-terminal carbohydrate-binding module family 35 domain increases the local concentration of the enzyme around ,-1,3-galactan by its high affinity for the substrate. To enable phase determination using the multiwavelength anomalous dispersion method, selenomethionyl Pc1,3Gal43A was crystallized at 298,K using the hanging-drop vapour-diffusion method. The presence of selenium in the crystals was confirmed from the X-ray absorption spectrum. The crystals belonged to space group P21 and diffracted to 1.8,Å resolution. [source] Bivalent phenethylamines as novel dopamine transporter inhibitors: evidence for multiple substrate-binding sites in a single transporterJOURNAL OF NEUROCHEMISTRY, Issue 6 2010Kyle C. Schmitt J. Neurochem. (2010) 112, 1605,1618. Abstract Bivalent ligands , compounds incorporating two receptor-interacting moieties linked by a flexible chain , often exhibit profoundly enhanced binding affinity compared with their monovalent components, implying concurrent binding to multiple sites on the target protein. It is generally assumed that neurotransmitter sodium symporter (NSS) proteins, such as the dopamine transporter (DAT), contain a single domain responsible for recognition of substrate molecules. In this report, we show that molecules possessing two substrate-like phenylalkylamine moieties linked by a progressively longer aliphatic spacer act as progressively more potent DAT inhibitors (rather than substrates). One compound bearing two dopamine (DA)-like pharmacophoric ,heads' separated by an 8-carbon linker achieved an 82-fold gain in inhibition of [3H] 2,-carbomethoxy-3,-(4-fluorophenyl)-tropane (CFT) binding compared with DA itself; bivalent compounds with a 6-carbon linker and heterologous combinations of DA-, amphetamine- and ,-phenethylamine-like heads all resulted in considerable and comparable gains in DAT affinity. A series of short-chain bivalent-like compounds with a single N -linkage was also identified, the most potent of which displayed a 74-fold gain in binding affinity. Computational modelling of the DAT protein and docking of the two most potent bivalent (-like) ligands suggested simultaneous occupancy of two discrete substrate-binding domains. Assays with the DAT mutants W84L and D313N , previously employed by our laboratory to probe conformation-specific binding of different structural classes of DAT inhibitors , indicated a bias of the bivalent ligands for inward-facing transporters. Our results strongly indicate the existence of multiple DAT substrate-interaction sites, implying that it is possible to design novel types of DAT inhibitors based upon the ,multivalent ligand' strategy. [source] Structure of the N-terminal fragment of Escherichia coli Lon proteaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2010Mi Li The structure of a recombinant construct consisting of residues 1,245 of Escherichia coli Lon protease, the prototypical member of the A-type Lon family, is reported. This construct encompasses all or most of the N-terminal domain of the enzyme. The structure was solved by SeMet SAD to 2.6,Å resolution utilizing trigonal crystals that contained one molecule in the asymmetric unit. The molecule consists of two compact subdomains and a very long C-terminal ,-helix. The structure of the first subdomain (residues 1,117), which consists mostly of ,-strands, is similar to that of the shorter fragment previously expressed and crystallized, whereas the second subdomain is almost entirely helical. The fold and spatial relationship of the two subdomains, with the exception of the C-terminal helix, closely resemble the structure of BPP1347, a 203-amino-acid protein of unknown function from Bordetella parapertussis, and more distantly several other proteins. It was not possible to refine the structure to satisfactory convergence; however, since almost all of the Se atoms could be located on the basis of their anomalous scattering the correctness of the overall structure is not in question. The structure reported here was also compared with the structures of the putative substrate-binding domains of several proteins, showing topological similarities that should help in defining the binding sites used by Lon substrates. [source] Crystallization of the proline-rich-peptide binding domain of human type I collagen prolyl 4-hydroxylaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2003Mira Pekkala Collagen prolyl 4-hydroxylases catalyze the hydroxylation of -X -Pro-Gly- sequences and play an essential role in the synthesis of all collagens. They require Fe2+, 2-oxoglutarate, molecular oxygen and ascorbate, and all vertebrate collagen prolyl 4-hydroxylases are ,2,2 tetramers. The ,-subunits contain separate catalytic and peptide substrate-binding domains. Here, the crystallization of the peptide substrate-binding domain consisting of residues 144,244 of the 517-residue human ,(I) subunit is described. The crystals are well ordered and diffract to at least 3,Å. The space group is P31 or P32 and the asymmetric unit most probably contains a dimer. [source] | ||||||||||