The inter-ligand Overhauser effect: A powerful new NMR approach for mapping structural relationships of macromolecular ligands

NMR experiments that transfer conformational information from the bound to the uncomplexed state via exchange have been utilized for many years. It is demonstrated here that inter-ligand NOEs (ILOEs), which exist in ternary complexes with enzymes or other macromolecular receptors, can be transferred via exchange to pairs of uncomplexed ligands. This approach is illustrated by studies of glycolate + NAD⁺ in the presence of porcine heart lactate dehydrogenase, and by glucose-6-phosphate + NADPH in the presence of L. mesenteroides glucose-6-phosphate dehydrogenase. This strategy opens up a general methodology for exploring the active sites of enzymes and for the development of artificial ligands which can function as inhibitors, or more generally as modifiers of protein function.     

Stereochemistry of ATP and GTP bound to fish haemoglobins. A transferred nuclear overhauser enhancement, 31P-nuclear magnetic resonance, oxygen equilibrium and molecular modelling study

This study was undertaken in order to test the models of ATP and GTP binding to carp deoxyhaemoglobin proposed by Perutz & Brunori (1982) and to find out why GTP is a more potent allosteric effector than ATP. We have determined the conformations of both nucleoside triphosphates by nuclear magnetic resonance studies and found them to be the same. The purines are in anti conformation about the glycosidic bond that links them to the ribose; the pentose ring is 3'-endo; the P-O5'-C5'-C4' torsion angle lies in the trans domain (180 degrees +/- 20 degrees); the P alpha-O-P beta and P beta-O-P gamma angles are as in the free nucleotides, i.e. the trinucleotide chain is fully extended. Models having this conformation were fitted, first manually and then by energy refinement, to the effector site of an atomic model of human deoxyhaemoglobin in which the side-chains in the NA, EF and H segments had been replaced by those of carp. The results showed the location of the polar groups in carp haemoglobin to be such that (PO4) gamma can accept hydrogen bonds from Val NA1 beta 2 and from Arg H21 beta 1, while (PO4) beta and (PO4) alpha can accept hydrogen bonds from Lys EF6 beta 1 and beta 2. In ATP, the 6-amino group of the purine can donate a hydrogen bond to Glu NA2 beta 1. In GTP, the 2-amino group can donate a hydrogen bond to Glu NA2 beta 1; in addition, Val Na1 beta 1 can donate a hydrogen bond to O2' of the ribose. This additional hydrogen bond may explain why in carp haemoglobin GTP is a stronger allosteric effector than ATP. We have found the influence of the two allosteric effectors on the oxygen affinity of trout IV haemoglobin to be the same, even though the only difference in the lining of the allosteric effector sites lies in the replacement of Glu Na2 beta in carp by Asp in trout IV haemoglobin. Model building then showed that formation of a hydrogen bond between Asp Na2 beta and the 2-amino group of guanine precludes formation of a hydrogen bond between Val NA1 beta and O2' of the ribose or vice versa, which makes the number of hydrogen bonds formed between trout IV haemoglobin and GTP the same as those formed with ATP.     

Time-Resolved Binding of Azithromycin to Escherichia coli Ribosomes

Azithromycin is a semisynthetic derivative of erythromycin that inhibits bacterial protein synthesis by binding within the peptide exit tunnel of the 50S ribosomal subunit. Nevertheless, there is still debate over what localization is primarily responsible for azithromycin binding and as to how many molecules of the drug actually bind per ribosome. In the present study, kinetic methods and footprinting analysis are coupled together to provide time-resolved details of the azithromycin binding process. It is shown that azithromycin binds to Escherichia coli ribosomes in a two-step process: The first-step involves recognition of azithromycin by the ribosomal machinery and places the drug in a low-affinity site located in the upper part of the exit tunnel. The second step corresponds to the slow formation of a final complex that is both much tighter and more potent in hindering the progression of the nascent peptide through the exit tunnel. Substitution of uracil by cytosine at nucleoside 2609 of 23S rRNA, a base implicated in the high-affinity site, facilitates the shift of azithromycin to this site. In contrast, mutation U754A hardly affects the binding process. Binding of azithromycin to both sites is hindered by high concentrations of Mg²⁺ ions. Unlike Mg²⁺ ions, polyamines do not significantly affect drug binding to the low-affinity site but attenuate the formation of the final complex. The low- and high-affinity sites of azithromycin binding are mutually exclusive, which means that one molecule of the drug binds per E. coli ribosome at a time. In contrast, kinetic and binding data indicate that in Deinococcus radiodurans, two molecules of azithromycin bind cooperatively to the ribosome. This finding confirms previous crystallographic results and supports the notion that species-specific structural differences may primarily account for the apparent discrepancies between the antibiotic binding modes obtained for different organisms.     

Conformation of NAD+ bound to yeast and horse liver alcohol dehydrogenase in solution. The use of the proton-proton transferred nuclear Overhauser enhancement

Changes in reduced viscosity of nuclear lysates from rat liver cells have been studied, in conditions of very low shear stress by the use of an oscillating viscometer, as a function of incubation time in alkaline (pH 12.5) and neutral (pH 8.0) solutions. In non-denaturing conditions, nuclear DNA showed a stepwise, time-dependent increase of reduced viscosity, which suggests that it behaves as a single hydrodynamic unit that progressively changes its radius and viscoelastic properties because of a very slow unfolding, through discrete successive transitions, from a highly superpacked structure toward a linear relaxed B-form fiber. Experimental conditions shown to reduce chromatin-DNA superpacking without changing DNA length (e.g. G₁ cycling versus G₀ non-cycling liver cells, or young versus old rat liver cells) dramatically increased the initial value of reduced viscosity and its time-dependent increment. Conversely, in denaturing conditions, reduced viscosity increased in the initial phase (probably because DNA unfolding prevails on DNA unwinding), then exhibited a plateau level (when unfolding balances unwinding), and subsequently decreased progressively to the value of sheared DNA (when unwinding becomes more rapid due to the progressive breakage of phosphodiester bridges in alkali). Experimental conditions known to induce DNA single- or double-strand breaks (i.e. the use of liver cells from rats treated with dimethylnitrosamine or 2-acetylaminofluorene, or of liver cells exposed to X-rays) caused in both neutral and alkaline solution an increment in the initial reduced viscosity and in the slope of its time-dependent increase, which may be related to a reduction of chromatin-DNA superpacking. Moreover, it became evident in denaturing conditions that a decrease of the maximum viscosity and of the time taken to reach it both related to a reduced DNA length. These viscoelastic properties are constantly correlated with independent DNA structural measurements on the same nuclear lysates, to discriminate the effect due to mere aggregation and disaggregation.