A general chemical crosslinking strategy for structural analyses of weakly interacting proteins applied to preTCR-pMHC complexes.
A general chemical crosslinking strategy for structural analyses of weakly interacting proteins applied to preTCR-pMHC complexes.
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DOI：
10.1016/j.jbc.2021.100255
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发表时间：
2021-01
期刊：
影响因子：
--
通讯作者：
Mallis RJ
中科院分区：
文献类型：
--
作者：
Mizsei R;Li X;Chen WN;Szabo M;Wang JH;Wagner G;Reinherz EL;Mallis RJ
T淋巴细胞通过T细胞受体介导的对由细胞表面表达的主要组织相容性复合体分子（MHC）结合和呈递的肽的识别来区分健康细胞和感染细胞或癌细胞。胸腺细胞上的前T细胞受体（preTCR）通过其β链与胸腺上皮上展示自身肽的MHC相互作用促进αβT淋巴细胞的发育。preTCR与肽-MHC复合物（pMHC）的特异性结合先前已被鉴定为形成具有与成熟αβTCR不同的界面的弱亲和力复合物。然而，缺乏适当的工具，限制了以前的努力，调查这一独特的接口。在这里，我们设计了一个小规模的连锁筛选协议，使用双马来酰亚胺接头，用于确定在溶液中瞬时相互作用的蛋白质对之间的残基特异性距离限制。采用键距约束指导的分子模拟方法，我们报道了preTCRβ-pMHC相互作用的定向溶液对接几何。用未连接蛋白混合物的顺磁假接触化学位移（PCS）NMR独立验证了preTCRβ-pMHC复合物的连接模型。使用连接屏幕，我们表明，前TCR结合不同的亲和力，由MHC在溶液中提出的肽。此外，C-末端肽段是preTCR-pMHC识别的关键决定因素。我们还描述了未来大规模生产和纯化的NMR，X射线晶体学和单分子电子显微镜研究的连接结构的过程。
T lymphocytes discriminate between healthy and infected or cancerous cells via T-cell receptor-mediated recognition of peptides bound and presented by cell-surface-expressed major histocompatibility complex molecules (MHCs). Pre-T-cell receptors (preTCRs) on thymocytes foster development of αβT lymphocytes through their β chain interaction with MHC displaying self-peptides on thymic epithelia. The specific binding of a preTCR with a peptide–MHC complex (pMHC) has been identified previously as forming a weak affinity complex with a distinct interface from that of mature αβTCR. However, a lack of appropriate tools has limited prior efforts to investigate this unique interface. Here we designed a small-scale linkage screening protocol using bismaleimide linkers for determining residue-specific distance constraints between transiently interacting protein pairs in solution. Employing linkage distance restraint-guided molecular modeling, we report the oriented solution docking geometry of a preTCRβ–pMHC interaction. The linkage model of preTCRβ–pMHC complex was independently verified with paramagnetic pseudocontact chemical shift (PCS) NMR of the unlinked protein mixtures. Using linkage screens, we show that the preTCR binds with differing affinities to peptides presented by MHC in solution. Moreover, the C-terminal peptide segment is a key determinant in preTCR–pMHC recognition. We also describe the process for future large-scale production and purification of the linked constructs for NMR, X-ray crystallography, and single-molecule electron microscopy studies.
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影响因子：
7.3
作者：
Brazin KN;Mallis RJ;Das DK;Feng Y;Hwang W;Wang JH;Wagner G;Lang MJ;Reinherz EL
通讯作者：
Reinherz EL
影响因子：
16.1
作者：
Chen, Xiaoying;Zaro, Jennica L.;Shen, Wei-Chiang
通讯作者：
Shen, Wei-Chiang
影响因子：
7.3
作者：
Natarajan K;Jiang J;May NA;Mage MG;Boyd LF;McShan AC;Sgourakis NG;Bax A;Margulies DH
通讯作者：
Margulies DH
DOI：
10.1073/pnas.2005899117
发表时间：
2020-09-01
影响因子：
11.1
作者：
Hwang, Wonmuk;Mallis, Robert J.;Reinherz, Ellis L.
通讯作者：
Reinherz, Ellis L.
DOI：
10.1016/j.pnmrs.2011.05.001
发表时间：
2011-11
影响因子：
6.1
作者：
Koehler, Julia;Meiler, Jens
通讯作者：
Meiler, Jens