Combining in-situ proteolysis and microseed matrix screening to promote crystallization of PrPc-nanobody complexes
Combining in-situ proteolysis and microseed matrix screening to promote crystallization of PrPc-nanobody complexes
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DOI：
10.1093/protein/gzr017
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发表时间：
2011-09-01
影响因子：
2.4
通讯作者：
Wohlkonig, Alexandre
中科院分区：
文献类型：
--
作者：
Abskharon, Romany N. N.;Soror, Sameh H.;Wohlkonig, Alexandre
朊病毒蛋白（PrPs）很难结晶，可能是由于其固有的灵活性。几个PrPs结构已解决了核磁共振（NMR）技术;然而，只有三个结构解决了X射线晶体学。在这里，我们结合原位蛋白水解与自动微种子基质筛选（MMS）结晶两种不同的PrPC-纳米抗体（Nb）复合物。纳米抗体是来源于骆驼科动物的仅重链抗体的单结构域抗体。使用与纳米抗体（Nb_PrP_01）复合的小鼠朊病毒（23 - 230）的原位蛋白水解的初始结晶筛选条件得到薄的针状聚集体，其具有差的衍射质量。接下来，我们使用这些微晶作为自动MMS的成核剂。从小鼠PrP（89 - 230）/Nb_PrP_01中获得了高质量的晶体，属于单斜空间群P1211，晶胞参数a59.13，B 63.80，c69.79，101.96，用同步辐射衍射可达2.1分辨率。人PrP（90 - 231）/Nb_PrP_01晶体属单斜晶系，空间群C_2，晶胞参数a为131.86，B为45.78，c为45.09，96.23。这种组合策略受益于MMS技术的力量，而不遭受原位蛋白水解的缺点。这被证明是一个成功的策略，结晶PrP-纳米抗体复合物，并可用于其他困难的抗原抗体复合物的结晶。
Prion proteins (PrPs) are difficult to crystallize, probably due to their inherent flexibility. Several PrPs structures have been solved by nuclear magnetic resonance (NMR) techniques; however, only three structures were solved by X-ray crystallography. Here we combined in-situ proteolysis with automated microseed matrix screening (MMS) to crystallize two different PrPC-nanobody (Nb) complexes. Nanobodies are single-domain antibodies derived from heavy-chain-only antibodies of camelids. Initial crystallization screening conditions using in-situ proteolysis of mouse prion (23-230) in complex with a nanobody (Nb_PrP_01) gave thin needle aggregates, which were of poor diffraction quality. Next, we used these microcrystals as nucleants for automated MMS. Good-quality crystals were obtained from mouse PrP (89-230)/Nb_PrP_01, belonged to the monoclinic space group P 1 21 1, with unit-cell parameters a 59.13, b 63.80, c 69.79 , 101.96 and diffracted to 2.1 resolution using synchrotron radiation. Human PrP (90-231)/Nb_PrP_01 crystals belonged to the monoclinic space group C2, with unit-cell parameters a 131.86, b 45.78, c 45.09 , 96.23 and diffracted to 1.5 resolution. This combined strategy benefits from the power of the MMS technique without suffering from the drawbacks of the in-situ proteolysis. It proved to be a successful strategy to crystallize PrP-nanobodies complexes and could be exploited for the crystallization of other difficult antigenantibody complexes.