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- Hansson, Sebastian, et al.
(författare)
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Structural insights into fusidic acid resistance and sensitivity in EF-G
- 2005
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 348:4, s. 939-949
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Tidskriftsartikel (refereegranskat)abstract
- Fusidic acid (FA) is a steroid antibiotic commonly used against Gram positive bacterial infections. It inhibits protein synthesis by stalling elongation factor G (EF-G) on the ribosome after translocation. A significant number of the mutations conferring strong FA resistance have been mapped at the interfaces between domains G, III and V of EF-G. However, direct information on how such mutations affect the structure has hitherto not been available. Here we present the crystal structures of two mutants of Thermus thermophilus EF-G, G16V and T84A, which exhibit FA hypersensitivity and resistance in vitro, respectively. These mutants also have higher and lower affinity for GTP respectively than wild-type EF-G. The mutations cause significant conformational changes in the switch 11 loop that have opposite effects on the position of a key residue, Phe90, which undergoes large conformational changes. This correlates with the importance of Phe90 in FA sensitivity reported in previous studies. These structures substantiate the importance of the domain G/domain III/domain V interfaces as a key component of the FA binding site. The mutations also cause subtle changes in the environment of the "P-loop lysine", Lys25. This led us to examine the conformation of the equivalent residue in all structures of translational GTPases, which revealed that EF-G and eEF2 form a group separate from the others and suggested that the role of Lys25 may be different in the two groups. (c) 2005 Elsevier Ltd. All rights reserved.
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| 4. |
- Pozzo, Tania, et al.
(författare)
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Structural and Functional Analyses of beta-Glucosidase 3B from Thermotoga neapolitana: A Thermostable Three-Domain Representative of Glycoside Hydrolase 3.
- 2010
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 397, s. 724-739
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Tidskriftsartikel (refereegranskat)abstract
- Based on sequence and phylogenetic analyses, glycoside hydrolase (GH) 3 can be divided into several clusters that differ in the length of their primary sequences. However, structural data on representatives of GH3 are still scarce, since only three of their structures are known and only one of them has been thoroughly characterized-that of an exohydrolase from barley. To allow a deeper structural understanding of the GH3 family, we have determined the crystal structure of the thermostable beta-glucosidase from Thermotoga neapolitana, which has potentially important applications in environmentally friendly industrial biosynthesis at a resolution of 2.05 A. Selected active-site mutants have been characterized kinetically, and the structure of the mutant D242A is presented at 2.1 A resolution. Bgl3B from Thermot. neapolitana is the first example of a GH3 glucosidase with a three-domain structure. It is composed of an (alpha/beta)(8) domain similar to a triose phosphate isomerase barrel, a five-stranded alpha/beta sandwich domain (both of which are important for active-site organization), and a C-terminal fibronectin type III domain of unknown function. Remarkably, the direction of the second beta-strand of the triose phosphate isomerase barrel domain is reversed, which has implications for the active-site shape. The active site, at the interface of domains 1 and 2, is much more open to solvent than the corresponding site in the structurally homologous enzyme from barley, and only the -1 site is well defined. The structures, in combination with kinetic studies of active-site variants, allow the identification of essential catalytic residues (the nucleophile D242 and the acid/base E458), as well as other residues at the -1 subsite, including D58 and W243, which, by mutagenesis, are shown to be important for substrate accommodation/interaction. The position of the fibronectin type III domain excludes a direct participation of this domain in the recognition of small substrates, although it may be involved in the anchoring of the enzyme on large polymeric substrates and in thermostability.
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| 5. |
- Wang, Ellen, et al.
(författare)
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Small-angle X-ray Scattering Study of a Rex Family Repressor: Conformational Response to NADH and NAD(+) Binding in Solution.
- 2011
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 408, s. 670-683
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Tidskriftsartikel (refereegranskat)abstract
- The transcriptional repressor Rex is a sensor of the intracellular NADH/NAD(+) redox state through direct binding of NADH or NAD(+). Homodimeric Rex protein from Thermus aquaticus (T-Rex) and Bacillus subtilis (B-Rex) exists in several different conformations. In both organisms, Rex in complex with NADH has the DNA binding domains packed together at the dimer interface, whereas in the apo form of B-Rex the linkers connecting these domains to the core are flexible. The crystal structures of the apo forms of B-Rex and a mutated variant of T-Rex are radically different. We describe the solution structures of B-Rex in complex with NAD(+) or NADH and in its apo form, on the basis of small-angle X-ray scattering (SAXS) measurements. This study addresses to what extent the unusual orientation of the DNA recognition domains of the crystal structure of apo B-Rex is owing to stabilization by crystal packing. Low-resolution ab initio solution structures were obtained for apo B-Rex, B-Rex:NADH and B-Rex:NAD(+). Models giving a more detailed picture of these three solution structures were obtained also by rigid body fitting of the crystallographic domains. The SAXS data confirm the elongated and flexible nature of apo-B-Rex and the existence of two distinct and more rigid conformations for the complexes with NADH and NAD(+). The models emerging from this study indicate a reaction mechanism for B-Rex in which the recognition domains are rotated upon binding to NADH.
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