Solution NMR characterization of WT CXCL8 monomer and dimer binding to CXCR1 N-terminal domain
Solution NMR characterization of WT CXCL8 monomer and dimer binding to CXCR1 N-terminal domain
复制标题
DOI：
10.1002/pro.2590
复制
发表时间：
2015-01-01
期刊：
影响因子：
8
通讯作者：
Rajarathnam, Krishna
中科院分区：
文献类型：
--
作者：
Joseph, Prem Raj B.;Rajarathnam, Krishna
趋化因子CXCL8及其受体CXCR1是抗感染的关键介质，也参与了慢性阻塞性肺疾病(COPD)和癌症等多种疾病的病理生理过程。CXCL8以单体和二聚体的形式存在，但单体单独与CXCR1结合的亲和力很高。CXCL8的功能涉及结合两个不同的CXCR1位点--N-末端结构域(Site-I)和细胞外/跨膜结构域(Site-II)。因此，较高的单体亲和力可能是由于在第一位点或第二位点或两者都有较强的结合。我们现在已经表征了人CXCR1 N-末端结构域多肽(HCXCR1Ndp)与WT CXCL8在同时以单体和二聚体存在的条件下的结合。我们发现WT单体以更高的亲和力结合CXCR1N-结构域，结合与二聚体解离耦合。我们还表征了两个CXCL8单体变体和一个捕获的二聚体与两个不同的hCXCR1Ndp结构的结合，观察到单体与hCXCR1Ndp的结合亲和力大约是二聚体的10-100倍。我们的研究还表明，单体和二聚体与受体多肽的结合常数和二聚体解离常数随pH和缓冲液的变化而显著变化，因此观察WT单体峰的能力严重依赖于核磁共振实验条件。我们的结论是，该单体是高亲和力的CXCR1激动剂，I位相互作用在决定单体与二聚体亲和力方面起主导作用，二聚体在调节单体功能方面起间接作用。
Chemokine CXCL8 and its receptor CXCR1 are key mediators in combating infection and have also been implicated in the pathophysiology of various diseases including chronic obstructive pulmonary disease (COPD) and cancer. CXCL8 exists as monomers and dimers but monomer alone binds CXCR1 with high affinity. CXCL8 function involves binding two distinct CXCR1 sites - the N-terminal domain (Site-I) and the extracellular/transmembrane domain (Site-II). Therefore, higher monomer affinity could be due to stronger binding at Site-I or Site-II or both. We have now characterized the binding of a human CXCR1 N-terminal domain peptide (hCXCR1Ndp) to WT CXCL8 under conditions where it exists as both monomers and dimers. We show that the WT monomer binds the CXCR1 N-domain with much higher affinity and that binding is coupled to dimer dissociation. We also characterized the binding of two CXCL8 monomer variants and a trapped dimer to two different hCXCR1Ndp constructs, and observe that the monomer binds with approximate to 10- to 100-fold higher affinity than the dimer. Our studies also show that the binding constants of monomer and dimer to the receptor peptides, and the dimer dissociation constant, can vary significantly as a function of pH and buffer, and so the ability to observe WT monomer peaks is critically dependent on NMR experimental conditions. We conclude that the monomer is the high affinity CXCR1 agonist, that Site-I interactions play a dominant role in determining monomer vs. dimer affinity, and that the dimer plays an indirect role in regulating monomer function.