Critical role in CXCR4 signaling and internalization of the polypeptide main chain in the amino terminus of SDF-1α probed by novel N-methylated synthetically and modularly modified chemokine analogues.

The replication of human immunodeficiency virus type 1 (HIV-1) can be profoundly inhibited by the natural ligands of two major HIV-1 coreceptors, CXCR4 and CCR5. Stromal cell-derived factor-1 alpha (SDF-1 alpha) is a natural ligand of CXCR4. We have recently developed a synthetic biology approach of using synthetically and modularly modified (SMM)-chemokines to dissect various aspects of the structure-function relationship of chemokines and their receptors. Here, we used this approach to design novel SMM-SDF-1 alpha analogues containing unnatural N-methylated residues in the amino terminus to investigate whether the polypeptide main chain amide bonds in the N-terminus of SDF-1 alpha play a role in SDF-1 alpha signaling via CXCR4 and/or receptor internalization. The results show that SDF-1 alpha analogues with a modified N-methylated main chain at position 2, 3, or 5 retain significant CXCR4 binding and yet completely lose signaling activities. Furthermore, a representative N-methylated analogue has been shown to be incapable of causing CXCR4 internalization. These results suggest that the ability of SDF-1 alpha to activate CXCR4 signaling and internalization is dependent upon the main chain amide bonds in the N-terminus of SDF-1 alpha. This study demonstrates the feasibility and value of applying a synthetic biology approach to chemically engineer natural proteins and peptide ligands as probes of important biological functions that are not addressed by other biological techniques.