Prentice G A, Merrill A R
Department of Chemistry and Biochemistry, University of Guelph, Guelph, Ontario, N1G 2W1, Canada.
Anal Biochem. 1999 Aug 1;272(2):216-23. doi: 10.1006/abio.1999.4188.
The X-ray structure of the catalytic domain of Pseudomonas aeruginosa exotoxin A (PE24) has recently been solved to high resolution, facilitating studies on the interaction of PE24 with its target substrate, eukaryotic elongation factor-2 (eEF-2). PE24 exhibits mono-ADP-ribosyltransferase (ADPRT) activity in a mechanism that has been proposed to feature a nucleophilic attack by the diphthamide residue (nucleophile) of eEF-2 on the C-1 of the nicotinamide ribose of NAD(+). The interaction of wheat germ eEF-2 with PE24 was studied by employing an enzyme-linked immunosorbent assay (ELISA), devised to assess protein-protein interactions. It was shown that the proteins associate with each other only in the presence of the enzyme's nucleotide substrate, NAD(+), and exhibit a dose-dependent association that is saturable. The apparent dissociation constant (K(d)) for this protein-protein interaction is 50 nM and is salt-dependent. The association is maximal at low ionic strength and is progressively weaker at higher salt concentrations, which corroborates previous findings on the salt dependence of ADPRT activity for this toxin. This finding suggests that the sensitivity of ADPRT activity toward high salt resides in the interaction between the catalytic domain of the toxin and eEF-2. A major product of the glycohydrolase activity of PE24, nicotinamide, inhibits the binding between PE24 and eEF-2 with an ID(50) of 20 microM. The naturally occurring, noncatalytic mutant of PE24, H426Y, did not bind eEF-2 in the ELISA, verifying that His 426 is located at the center of the eEF-2 binding site within ETA.
铜绿假单胞菌外毒素A（PE24）催化结构域的X射线结构最近已被解析至高分辨率，这有助于研究PE24与其靶底物真核延伸因子-2（eEF-2）之间的相互作用。PE24在一种机制中表现出单ADP-核糖基转移酶（ADPRT）活性，该机制被认为其特征在于eEF-2的双氢乳清酸酰胺残基（亲核试剂）对NAD(+)烟酰胺核糖C-1位的亲核攻击。通过采用一种设计用于评估蛋白质-蛋白质相互作用的酶联免疫吸附测定（ELISA），研究了小麦胚芽eEF-2与PE24的相互作用。结果表明，这些蛋白质仅在酶的核苷酸底物NAD(+)存在时相互结合，并表现出剂量依赖性的、可饱和的结合。这种蛋白质-蛋白质相互作用的表观解离常数（K(d)）为50 nM，且依赖于盐。这种结合在低离子强度下最大，在较高盐浓度下逐渐减弱，这证实了先前关于该毒素ADPRT活性对盐依赖性的研究结果。这一发现表明，ADPRT活性对高盐的敏感性存在于毒素催化结构域与eEF-2之间的相互作用中。PE24糖水解酶活性的一种主要产物烟酰胺以20 microM的半数抑制浓度（ID(50)）抑制PE24与eEF-2之间的结合。PE24天然存在的非催化突变体H426Y在ELISA中不与eEF-2结合，证实His 426位于ETA内eEF-2结合位点的中心。
