An integrated computational pipeline for designing high-affinity nanobodies with expanded genetic codes
An integrated computational pipeline for designing high-affinity nanobodies with expanded genetic codes
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用于设计具有扩展遗传密码的高亲和力纳米体的集成计算管道
DOI：
10.1093/bib/bbab338
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发表时间：
2021
影响因子：
9.5
通讯作者：
Kam Y J Zhang
中科院分区：
文献类型：
--
作者：
Aditya K Padhi;Ashutosh Kumar;Ken-ichi Haruna;Haruna Sato;Hiroko Tamura;Satoru Nagatoishi;Kouhei Tsumoto;Atushi Yamaguchi;Fumie Iraha;Mihoko Takahashi;Kensaku Sakamoto;Kam Y J Zhang
采用20种标准氨基酸的蛋白质工程和设计原理已被广泛用于获得稳定的蛋白质支架并提供其特定活性。虽然这赋予了一些优势，但它通常限制了蛋白质的序列，化学空间，并最终限制了蛋白质的功能多样性。此外，尽管非天然氨基酸（nnAA）的位点特异性掺入已被证明是蛋白质工程和治疗学开发中的有价值的策略，但其在纳米抗体的亲和力成熟中的效用尚未得到充分探索。此外，目前的实验方法并没有常规地采用nnAA，由于其巨大的库大小和无限的组合。为了解决这个问题，我们开发了一个集成的计算管道，采用基于结构的蛋白质设计方法，分子动力学模拟和自由能计算，用于预测nnAA掺入的纳米抗体对其靶标的结合亲和力和选择有效的结合剂。我们表明，通过掺入卤代酪氨酸，9 G8纳米抗体的亲和力可以提高对表皮生长因子受体（EGFR），一个关键的癌症靶点。表面等离子体共振（SPR）测定显示，几种3-氯-1-酪氨酸（3 MY）掺入的纳米抗体的结合被提高高达6倍至皮摩尔范围，并且计算估计的结合亲和力与SPR结果共享0.87的Pearson'srf。发现改善的亲和力是由于关键的3 MY-近端纳米抗体残基与EGFR的增强的货车范德华相互作用，以及纳米抗体的结构稳定性的总体增加。总之，我们表明，我们的方法可以促进筛选大型文库，并预测针对关键疾病靶标的有效位点特异性nnAA掺入纳米抗体结合剂。
Protein engineering and design principles employing the 20 standard amino acids have been extensively used to achieve stable protein scaffolds and deliver their specific activities. Although this confers some advantages, it often restricts the sequence, chemical space, and ultimately the functional diversity of proteins. Moreover, although site-specific incorporation of non-natural amino acids (nnAAs) has been proven to be a valuable strategy in protein engineering and therapeutics development, its utility in the affinity-maturation of nanobodies is not fully explored. Besides, current experimental methods do not routinely employ nnAAs due to their enormous library size and infinite combinations. To address this, we have developed an integrated computational pipeline employing structure-based protein design methodologies, molecular dynamics simulations and free energy calculations, for the binding affinity prediction of an nnAA-incorporated nanobody toward its target and selection of potent binders. We show that by incorporating halogenated tyrosines, the affinity of 9G8 nanobody can be improved toward epidermal growth factor receptor (EGFR), a crucial cancer target. Surface plasmon resonance (SPR) assays showed that the binding of several 3-chloro-l-tyrosine (3MY)-incorporated nanobodies were improved up to 6-fold into a picomolar range, and the computationally estimated binding affinities shared a Pearson’srof 0.87 with SPR results. The improved affinity was found to be due to enhanced van der Waals interactions of key 3MY-proximate nanobody residues with EGFR, and an overall increase in the nanobody’s structural stability. In conclusion, we show that our method can facilitate screening large libraries and predict potent site-specific nnAA-incorporated nanobody binders against crucial disease-targets.
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大肠杆菌中 3-碘-L-酪氨酸的遗传编码，用于蛋白质晶体学中的单波长异常色散定相
DOI：
--
发表时间：
2009
期刊：
Structure 17
影响因子：
--
作者：
Sakamoto;K.;Murayama;K.;Oki;K.;Iraha;F.;Kato-Murayama;M.;Takahashi;M.;Ohtake;K.;Kobayashi;T.;Kuramitsu;S.;Shirouzu;M.;Yokoyama S.
通讯作者：
Yokoyama S.
影响因子：
15
作者：
Kim CH;Axup JY;Dubrovska A;Kazane SA;Hutchins BA;Wold ED;Smider VV;Schultz PG
通讯作者：
Schultz PG
影响因子：
5.6
作者：
Wang, W;Kollman, PA
通讯作者：
Kollman, PA
影响因子：
5.7
作者：
Schmitz, Karl R.;Bagchi, Atrish;Roovers, Rob C.;Henegouwen, Paul M. P. van Bergen En;Ferguson, Kathryn M.
通讯作者：
Ferguson, Kathryn M.
影响因子：
4.6
作者：
Ohtake K;Yamaguchi A;Mukai T;Kashimura H;Hirano N;Haruki M;Kohashi S;Yamagishi K;Murayama K;Tomabechi Y;Itagaki T;Akasaka R;Kawazoe M;Takemoto C;Shirouzu M;Yokoyama S;Sakamoto K
通讯作者：
Sakamoto K