T4 lysozyme-engineered human chemokine receptor ccr5 mutants exhibit differing effects on hiv-1 co-receptor function and ligand binding

Qingwen Jin, Qi Niu, Wei Zhang, LiandongZhao, Hong Chen, XingxiaWang, QingchenXu, Lin Ji, Huijuan Wang, GuanyuLi, Xiaofan Yang and Xiaohui Ji

Background: The insertion of T4 lysozyme (T4L) to replace portions of G protein-coupled receptors has proved a successful approach to enhance protein stability and crystallization. However, T4L engineering alters the protein sequence and raises uncertainties regarding the details ofreceptor activationmodels that are based on the structures of receptor-T4L fusion proteins.
Methodology/Principal Findings: we generated six T4L-CCR5 variants containing a replacement at the first, second, or third intracellular loop or at different C-terminal regions and compared their functions. We demonstrated that only two of the constructs, T4L-CCR5-C352 and T4L-CCR5-ICL2, retain sufficient HIV-1 co-receptor function when expressed in HEK-293 cells. However, other constructs preserve the ability to bind and initiate signaling in response to chemokine CCL4 although T4L-CCR5-ICL1, T4L-CCR5-ICL3, and T4L-CCR5-C310 mutant receptors are defective in their abilities to activate G-proteins.
Conclusions/Significance: T4L replacement at the second intracellular loop retains the structural features of wild-type CCR5, whereas replacements at other positions may change the conformation of the N-terminus or the extracellular loops of CCR5.

Download PDF: