RESUMEN
TTHA1846 is a conserved hypothetical protein from Thermus thermophilus HB8 with a molecular mass of 15.1 kDa that belongs to the thioesterase superfamily (Pfam 03061). Here, the 1.9 A resolution crystal structure of TTHA1846 from T. thermophilus is reported. The crystal structure is a dimer of dimers. Each subunit adopts the so-called hot-dog fold composed of five antiparallel beta-strands flanked on one side by a rather long alpha-helix and shares structural similarity to a number of thioesterases. Unexpectedly, TTHA1846 binds one metal ion and one ligand per subunit. The ligand density was modelled as coenzyme A (CoA). Its structure was confirmed by MALDI-TOF mass spectrometry and electron-density mapping. X-ray absorption fine-structure (XAFS) measurement of the crystal unambiguously characterized the metal ion as zinc. The zinc ion is tetrahedrally coordinated by the side chains of Asp18, His22 and Glu50 and the CoA thiol group. This is the first structural report of the interaction of CoA with a zinc ion. From structural and database analyses, it was speculated that the zinc ion may play an inhibitory role in the enzymatic activity.
Asunto(s)
Coenzima A/química , Iones/química , Complejos Multiproteicos/química , Palmitoil-CoA Hidrolasa/química , Thermus thermophilus/enzimología , Zinc/química , Coenzima A/metabolismo , Cristalización , Cristalografía por Rayos X , Bases de Datos de Proteínas , Dimerización , Iones/metabolismo , Modelos Químicos , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Palmitoil-CoA Hidrolasa/genética , Palmitoil-CoA Hidrolasa/metabolismo , Unión Proteica , Conformación Proteica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Compuestos de Sulfhidrilo/química , Thermus thermophilus/genética , Zinc/metabolismoRESUMEN
The Sto12a protein, from the thermoacidophilic archaeon Sulfolobus tokodaii, has been identified as a small putative DNA-binding protein. Most of the proteins with a high level of amino acid sequence homology to this protein are derived from members of the Sulfolobaceae family, including a transcriptional regulator. We determined the crystal structure of Sto12a at 2.05 A resolution by multiple-wavelength anomalous dispersion phasing from the selenomethionine-containing protein crystal. This is the first structure of a member of this family of DNA-binding proteins. The Sto12a protein forms a homodimer, and the structure is composed of an N-terminal alpha-helix, a winged-helix-turn-helix domain, and a C-terminal alpha-helix that forms an interchain antiparallel coiled coil. The two winged-helix domains are located at both ends of the coiled coil, with putative DNA-recognition helices separated by approximately 34 A. A structural homology search indicated that the winged-helix domain shared a high level of homology with those found in B-DNA- or Z-DNA-binding proteins from various species, including archaea, bacteria, and human, despite a low level of sequence similarity. The unique structural features of the Sto12a protein include intrachain and interchain disulfide bonds, which stabilize the chain and homodimer structures. There are three cysteine residues: Cys15 and Cys16 in the N-terminal alpha-helix, and Cys100 in the C-terminal alpha-helix. Cys15 is involved in an interchain disulfide bridge with the other Cys15, and Cys16 forms an intrachain disulfide bridge with Cys100. This is a novel fold among winged-helix DNA-binding proteins. Possible DNA-binding interactions of the Sto12a protein are discussed based on the crystal structure of Sto12a and comparisons to other winged-helix DNA-binding proteins.
Asunto(s)
Proteínas Arqueales/química , Proteínas de Unión al ADN/química , Disulfuros/química , Sulfolobus/química , Algoritmos , Secuencia de Aminoácidos , Proteínas Arqueales/genética , Cristalografía por Rayos X , Proteínas de Unión al ADN/genética , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
We analyzed the effect of nine 'rare' codons (AGA, AGG, AUA, CCC, CGA, CGG, CUA, GGA, and UUA) on gene expression in an Escherichia coli coupled transcription/translation cell-free system, in comparison with a cell-based expression system. Each reporter gene contained five consecutive repeats of a rare codon, or in some experiments, three consecutive repeats. The cell-free expression of the genes bearing the codons CGA, CUA, GGA, and UUA was not affected, although these codons, except for GGA, were inefficiently translated in E. coli cells. Translation of the remaining five codons (AGA, AGG, AUA, CCC, and CGG) was severely reduced in both systems, and was remarkably facilitated in the cell-free system based on an S30 extract from the E. coli cells overproducing 'minor' tRNAs for these codons.