Mapping Interaction Sites on Human Chemokine Receptors by Deep Mutational Scanning.
Mapping Interaction Sites on Human Chemokine Receptors by Deep Mutational Scanning.
复制标题
DOI：
10.4049/jimmunol.1800343
复制
发表时间：
2018-06-01
期刊：
影响因子：
--
通讯作者：
Procko E
中科院分区：
文献类型：
--
作者：
Heredia JD;Park J;Brubaker RJ;Szymanski SK;Gill KS;Procko E
趋化因子受体CXCR 4和CCR 5调节白色血细胞运输，并在感染期间与HIV-1包膜糖蛋白gp 120接合。我们将联合收割机选择包含几乎所有~ 7，000个单一氨基酸取代的人CXCR 4和CCR 5文库与深度测序相结合，以定义表面表达和配体相互作用的序列活性景观。考虑到表面表达的序列限制后，在抗体结合的文库分选后，将与HIV-1阻断抗体的已知相互作用位点适当鉴定为保守残基，验证了使用深度突变扫描来绘制G蛋白偶联受体中的功能性相互作用位点。发现趋化因子CXCL 12与不对称延伸到CXCR 4配体结合腔中的残基相互作用，类似于先前在晶体学上观察到的拮抗性病毒趋化因子所识别的CXCR 4的结合表面。鉴定了远离趋化因子结合位点的CXCR 4突变，其增强趋化因子识别。这包括G蛋白偶联位点的破坏性突变，其减少了钙动员，以及膜暴露位点（CXCR 4残基H792.45和W1614.50）的保守突变，其增加了配体结合而不损失信号传导。与CXCR 4-CXCL 12相互作用相比，对于gp 120（HIV-1 BaL株）相互作用保守的CCR 5残基映射到更广阔的表面，模拟同源趋化因子CCL 5如何在整个CCR 5结合腔中进行接触。CCR 5 N-末端和细胞外环中的酸性取代增强了gp 120结合。这项研究展示了如何全面的突变扫描可以定义受体上的功能相互作用位点，同时可以发现增强受体活性的新突变。
Chemokine receptors CXCR4 and CCR5 regulate white blood cell trafficking, and are engaged by the HIV-1 envelope glycoprotein gp120 during infection. We combine selection of human CXCR4 and CCR5 libraries comprising nearly all ~7,000 single amino acid substitutions with deep sequencing to define sequence-activity landscapes for surface expression and ligand interactions. After consideration of sequence constraints for surface expression, known interaction sites with HIV-1-blocking antibodies were appropriately identified as conserved residues following library sorting for antibody binding, validating the use of deep mutational scanning to map functional interaction sites in G protein-coupled receptors. Chemokine CXCL12 was found to interact with residues extending asymmetrically into the CXCR4 ligand-binding cavity, similar to the binding surface of CXCR4 recognized by an antagonistic viral chemokine previously observed crystallographically. CXCR4 mutations distal from the chemokine binding site were identified that enhance chemokine recognition. This included disruptive mutations in the G protein-coupling site that diminished calcium mobilization, as well as conservative mutations to a membrane-exposed site (CXCR4 residues H792.45 and W1614.50) that increased ligand binding without loss of signaling. Compared to CXCR4-CXCL12 interactions, CCR5 residues conserved for gp120 (HIV-1 BaL strain) interactions map to a more expansive surface, mimicking how the cognate chemokine CCL5 makes contacts across the entire CCR5 binding cavity. Acidic substitutions in the CCR5 N-terminus and extracellular loops enhanced gp120 binding. This study demonstrates how comprehensive mutational scanning can define functional interaction sites on receptors, while simultaneously novel mutations that enhance receptor activities can be found.
登录
查看更多内容
影响因子：
56.9
作者：
Feng, Y;Broder, CC;Berger, EA
通讯作者：
Berger, EA
影响因子：
5.7
作者：
Harris, Daniel T.;Singh, Nishant K.;Baker, Brian M.
通讯作者：
Baker, Brian M.
影响因子：
9.2
作者：
Haas, J;Park, EC;Seed, B
通讯作者：
Seed, B
影响因子：
6.7
作者：
Haddox HK;Dingens AS;Bloom JD
通讯作者：
Bloom JD
影响因子：
4.8
作者：
Garcia-Perez, Javier;Rueda, Patricia;Kellenberger, Esther
通讯作者：
Kellenberger, Esther