Examination of the structure of Escherichia coli heat-labile enterotoxin in the AB5 complex at a resolution of 2.3A reveals that the doughnut-shaped B pentamer binds the enzymatic A subunit using a hairpin of the A2 fragment, through a highly charged central pore. Putative ganglioside GM1-binding sites on the B subunits are more than 20A removed from the membrane-crossing A1 subunit. This ADP-ribosylating (A1) fragment of the toxin has structural homology with the catalytic region of exotoxin A and hence also to diphtheria toxin.
A renal-specific controlled release of an active drug may enable a reduction of the required dose and may provide a reduction of extra-renal toxicity. To achieve renal specific targeting of the NSAID naproxen, the low-molecular-weight protein (LMWP) lysozyme was employed as carrier since it is mainly taken up and catabolized in the proximal tubules of the kidney. A conjugate was synthesized with an average coupling degree of 2 mol naproxen per 1 mol lysozyme in which the drug was directly coupled to the protein via a peptide bond. First, we investigated whether naproxen conjugation affects the renal disposition of lysozyme. As native lysozyme, the conjugate was predominantly and rapidly (within 20 min) taken up by the kidney. The subsequent decrease in renal content reflecting the renal degradation of the conjugated lysozyme molecules appeared also to be similar to that of native lysozyme with a half life of four hours. Second, the effect of lysozyme conjugation on the body distribution of naproxen was studied. An important observation with regard to the aimed reduction in extra-renal side effects was that no detectable amounts of free naproxen were present in the plasma after administration of conjugate. Conjugation of naproxen to lysozyme resulted in a pronounced (70-fold) increase of naproxen accumulation in the kidney. In agreement with the protein disposition study, the conjugate was rapidly taken up by the kidney and subsequently degraded. In conclusion, renal selective targeting of the NSAID naproxen can be obtained by conjugation with the LMWP lysozyme. This concept of drug delivery to the kidney has the potential to improve drug efficacy and safety.
A new native crystal form of heat-labile enterotoxin (LT) has two ABs complexes in the asymmetric unit with different orientations of the A subunit with respect to the B pentamer. Comparison with other crystal forms of LT shows that there is considerable conformational freedom for orientating the A subunit with respect to the B pentamer. The rotations erA in different crystal forms do not follow one sp~fl¢ axis, but most ofthem share a hinge point, clos~ to the main interaction area between A and Bs. Analysis of the two high-resolution structures available shows that these rotations cause very little change in the actual interactions between A and B vHeat-labile enterotoxin; Toxin; Conformational change
Enzyme IImtl catalyzes the concomitant transport and phosphorylation of the hexitol mannitol.Here we demonstrate that the heptitol perseitol is not phosphorylated and not transported by the enzyme.However, the enzyme binds perseitol with an affinity comparable to the affinity for mannitol. Apparent affinities of the phosphorylated enzyme for perseitol were inferred from the inhibition by perseitol of mannitol phosphorylation and uptake. Apparent affinities of the unphosphorylated enzyme follow from * To whom correspondence should be addressed.
The crystal structure dclermination of heat labile cntcroloxin (LT) bound to LIYO different lanthanidc ions, crbium and samarium, rcvcaled two distinct ion binding sites in the interface of lhc A subunit and ihc 6 pcntamcr or ihc toxin. One or Ihc inicrkc sites is conserved in the very similar cholera toxin scquuncc. These sites may bc polemial calcium binding siies. Erbium and samarium binding causes a change in the siruciurc of LT: a rotation oi' the Al subunit of up to two dcgrccs relative to the B penlamer.i-kdl labile cnleroloxin; Cholera toxin; Calcium; Lanthanide ion: Conformalional change
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