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The function and structure of the metal coordination sites within the glucocorticoid receptor DNA binding domain 总被引:54,自引:0,他引:54
L P Freedman B F Luisi Z R Korszun R Basavappa P B Sigler K R Yamamoto 《Nature》1988,334(6182):543-546
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The expression of beta-globin in Escherichia coli has enabled us to study the functional role of individual amino-acid residues in haemoglobin (Hb) by site-directed mutagenesis. In contrast to mammalian Hbs, some teleost fish haemoglobins show a drastic lowering of oxygen affinity and cooperativity at low pH, a phenomenon known as the Root effect. We have produced the two mutant haemoglobins Hb Nymphéas [Cys(F9)93 beta----Ser] and Hb Daphne [His(H21)143 beta----Arg, Cys(F9)93 beta----Ser] to investigate this allosteric property. Although these substitutions were thought to be responsible for the Root effect, Hb Nymphéas and Hb Daphne show an increased oxygen affinity and a reduced effect of pH on oxygen affinity. Our X-ray crystallographic studies show that the hydroxyl group of Ser 93 beta forms a hydrogen bond with Asp 94 beta which is in equilibrium with the salt bridge between Asp 94 beta and His 146 beta. The oxygen-binding properties of Hbs Nymphéas and Daphne are accounted for by the partial disruption of the salt bridge. 相似文献
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Thiamine is an essential cofactor that is required for processes of general metabolism amongst all organisms, and it is likely to have played a role in the earliest stages of the evolution of life. Here, we review from a structural perspective the enzymatic mechanisms that involve this cofactor. We explore asymmetry within homodimeric thiamine diphosphate (ThDP)-dependent enzyme structures and discuss how this may be correlated with the kinetic properties of half-of-the-sites reactivity, and negative cooperativity. It is likely these structural and kinetic hallmarks may arise through reciprocal coupling of active sites. This mode of communication between distant active sites is not unique to ThDP-dependent enzymes, but is widespread in other classes of oligomeric enzyme. Thus, it appears likely to be a general phenomenon reflecting a powerful mechanism of accelerating the rate of a chemical pathway. Finally, we speculate on the early evolutionary history of the cofactor and its ancient association with protein and RNA. 相似文献
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Crystal structure of trp repressor/operator complex at atomic resolution 总被引:98,自引:0,他引:98
Z Otwinowski R W Schevitz R G Zhang C L Lawson A Joachimiak R Q Marmorstein B F Luisi P B Sigler 《Nature》1988,335(6188):321-329
The crystal structure of the trp repressor/operator complex shows an extensive contact surface, including 24 direct and 6 solvent-mediated hydrogen bonds to the phosphate groups of the DNA. There are no direct hydrogen bonds or non-polar contacts to the bases that can explain the repressor's specificity for the operator sequence. Rather, the sequence seems to be recognized indirectly through its effects on the geometry of the phosphate backbone, which in turn permits the formation of a stable interface. Water-mediated polar contacts to the bases also appear to contribute part of the specificity. 相似文献
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Distal residues in the oxygen binding site of haemoglobin studied by protein engineering 总被引:4,自引:0,他引:4
K Nagai B Luisi D Shih G Miyazaki K Imai C Poyart A De Young L Kwiatkowsky R W Noble S H Lin 《Nature》1987,329(6142):858-860
The geometries of the Fe-O2 and Fe-CO bonds in myoglobin and haemoglobin differ significantly from those in free porphyrin model compounds. It has been suggested that steric hindrance by Val-E11 and His-E7 and a hydrogen bond between His-E7 and oxygen affect the geometry and electronic state of the Fe-ligand bond, and that these interactions may be important in controlling oxygen affinity. We have produced mutant haemoglobins in E. coli having Val(67 beta)E11 replaced by Ala, Met, Leu or Ile and His(58 beta)E7 by Gln, Val or Gly. We have studied the effect of these mutations on the equilibrium and kinetics of ligand binding. The conformation of the new side chains and their effect on the protein structure have been examined by X-ray crystallography, and the vibrational properties of the Fe-CO bond observed by resonance Raman spectroscopy. We found that the steric hindrance of ligand binding by the E11 residue and the polarity of the E7 residue in the beta subunit are critical for fine-tuning ligand affinity. 相似文献
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Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export 总被引:64,自引:0,他引:64
Diverse molecules, from small antibacterial drugs to large protein toxins, are exported directly across both cell membranes of gram-negative bacteria. This export is brought about by the reversible interaction of substrate-specific inner-membrane proteins with an outer-membrane protein of the TolC family, thus bypassing the intervening periplasm. Here we report the 2.1-A crystal structure of TolC from Escherichia coli, revealing a distinctive and previously unknown fold. Three TolC protomers assemble to form a continuous, solvent-accessible conduit--a 'channel-tunnel' over 140 A long that spans both the outer membrane and periplasmic space. The periplasmic or proximal end of the tunnel is sealed by sets of coiled helices. We suggest these could be untwisted by an allosteric mechanism, mediated by protein-protein interactions, to open the tunnel. The structure provides an explanation of how the cell cytosol is connected to the external environment during export, and suggests a general mechanism for the action of bacterial efflux pumps. 相似文献
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Synthesizing life 总被引:14,自引:0,他引:14
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