Tan Kemin, Zhou Min, Ahrendt Angela J, Duke Norma E C, Tabaja Nassif, Ball William J, Kirley Terence L, Norman Andrew B, Joachimiak Andrzej, Schiffer Marianne, Wilton Rosemarie, Pokkuluri P Raj
Structural Biology Center, X-ray Science Division, Argonne National Laboratory, Lemont, IL 60439, USA.
Biosciences Division, Argonne National Laboratory, Lemont, IL 60439, USA.
Acta Crystallogr F Struct Biol Commun. 2019 Nov 1;75(Pt 11):697-706. doi: 10.1107/S2053230X19013608. Epub 2019 Nov 5.
A high-affinity anti-cocaine monoclonal antibody, designated h2E2, is entering phase 1 clinical trials for cocaine abuse therapy. To gain insight into the molecular details of its structure that are important for binding cocaine and cocaine metabolites, the Fab fragment was generated and crystallized with and without ligand. Structures of the unliganded Fab and the Fab fragment bound to benzoylecgonine were determined, and were compared with each other and with other crystallized anti-cocaine antibodies. The affinity of the h2E2 antibody for cocaine is 4 nM, while that of the cocaine metabolite benzoylecgonine is 20 nM. Both are higher than the reported affinity for cocaine of the two previously crystallized anti-cocaine antibodies. Consistent with cocaine fluorescent quenching binding studies for the h2E2 mAb, four aromatic residues in the CDR regions of the Fab (TyrL32, TyrL96, TrpL91 and TrpH33) were found to be involved in ligand binding. The aromatic side chains surround and trap the tropane moiety of the ligand in the complex structure, forming significant van der Waals interactions which may account for the higher affinity observed for the h2E2 antibody. A water molecule mediates hydrogen bonding between the antibody and the carbonyl group of the benzoyl ester. The affinity of binding to h2E2 of benzoylecgonine differs only by a factor of five compared with that of cocaine; therefore, it is suggested that h2E2 would bind cocaine in the same way as observed in the Fab-benzoylecgonine complex, with minor rearrangements of some hypervariable segments of the antibody.
一种名为h2E2的高亲和力抗可卡因单克隆抗体正在进入用于可卡因滥用治疗的1期临床试验。为了深入了解其结构的分子细节，这些细节对于结合可卡因和可卡因代谢物很重要，我们制备了Fab片段，并在有配体和无配体的情况下进行结晶。测定了未结合配体的Fab和与苯甲酰芽子碱结合的Fab片段的结构，并将它们相互比较，以及与其他结晶的抗可卡因抗体进行比较。h2E2抗体对可卡因的亲和力为4 nM，而对可卡因代谢物苯甲酰芽子碱的亲和力为20 nM。两者均高于之前报道的两种结晶抗可卡因抗体对可卡因的亲和力。与h2E2单克隆抗体的可卡因荧光猝灭结合研究一致，发现Fab的互补决定区（CDR）中的四个芳香族残基（TyrL32、TyrL96、TrpL91和TrpH33）参与配体结合。在复合物结构中，芳香族侧链围绕并捕获配体的托烷部分，形成显著的范德华相互作用，这可能解释了h2E2抗体观察到的更高亲和力。一个水分子介导抗体与苯甲酰酯羰基之间的氢键。苯甲酰芽子碱与h2E2的结合亲和力与可卡因相比仅相差五倍；因此，有人提出h2E2将以与Fab-苯甲酰芽子碱复合物中观察到的相同方式结合可卡因，只是抗体的一些高变区会有轻微重排。
