Li M, Dyda F, Benhar I, Pastan I, Davies D R
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Proc Natl Acad Sci U S A. 1996 Jul 9;93(14):6902-6. doi: 10.1073/pnas.93.14.6902.
The catalytic, or third domain of Pseudomonas exotoxin A (PEIII) catalyzes the transfer of ADP ribose from nicotinamide adenine dinucleotide (NAD) to elongation factor-2 in eukaryotic cells, inhibiting protein synthesis. We have determined the structure of PEIII crystallized in the presence of NAD to define the site of binding and mechanism of activation. However, NAD undergoes a slow hydrolysis and the crystal structure revealed only the hydrolysis products, AMP and nicotinamide, bound to the enzyme. To better define the site of NAD binding, we have now crystallized PEIII in the presence of a less hydrolyzable NAD analog, beta-methylene-thiazole-4-carboxamide adenine dinucleotide (beta-TAD), and refined the complex structure at 2.3 angstroms resolution. There are two independent molecules of PEIII in the crystal, and the conformations of beta-TAD show some differences in the two binding sites. The beta-TAD attached to molecule 2 appears to have been hydrolyzed between the pyrophosphate and the nicotinamide ribose. However molecule 1 binds to an intact beta-TAD and has no crystal packing contacts in the vicinity of the binding site, so that the observed conformation and interaction with the PEIII most likely resembles that of NAD bound to PEIII in solution. We have compared this complex with the catalytic domains of diphtheria toxin, heat labile enterotoxin, and pertussis toxin, all three of which it closely resembles.
铜绿假单胞菌外毒素A的催化结构域（PEIII）可催化烟酰胺腺嘌呤二核苷酸（NAD）中的ADP核糖转移至真核细胞中的延伸因子-2，从而抑制蛋白质合成。我们已确定在NAD存在下结晶的PEIII的结构，以确定其结合位点和激活机制。然而，NAD会发生缓慢水解，晶体结构仅显示了与该酶结合的水解产物AMP和烟酰胺。为了更好地确定NAD的结合位点，我们现在在一种较难水解的NAD类似物β-亚甲基-噻唑-4-甲酰胺腺嘌呤二核苷酸（β-TAD）存在下使PEIII结晶，并在2.3埃分辨率下优化了复合物结构。晶体中有两个独立的PEIII分子，β-TAD在两个结合位点的构象存在一些差异。与分子2相连的β-TAD似乎在焦磷酸和烟酰胺核糖之间发生了水解。然而，分子1与完整的β-TAD结合，且在结合位点附近没有晶体堆积接触，因此观察到的构象以及与PEIII的相互作用很可能类似于溶液中与PEIII结合的NAD的构象和相互作用。我们已将此复合物与白喉毒素、不耐热肠毒素和百日咳毒素的催化结构域进行了比较，发现它与这三者都非常相似。
