Engineering Metamorphic Chemokine Lymphotactin/XCL1 into the GAG-Binding, HIV-Inhibitory Dimer Conformation

Abstract

Unlike other chemokines, XCL1 undergoes a distinct metamorphic interconversion between a canonical monomeric chemokine fold and a unique β-sandwich dimer. The monomeric conformation binds and activates the receptor XCR1, while the dimer binds extracellular matrix glycosaminoglycans and has been associated with anti-human immunodeficiency virus (HIV) activity. Functional studies of WT-XCL1 are complex as both conformations are populated in solution. To overcome this limitation, we engineered a stabilized dimeric variant of XCL1 designated CC5. This variant features a neo-disulfide bond (A46C-A49C) that prevents structural interconversion by locking the chemokine into the β-sandwich dimeric conformation, as demonstrated by NMR structural analysis and hydrogen-deuterium exchange experiments. Functional studies analyzing glycosaminoglycan binding demonstrate that CC5 binds with high affinity to heparin. In addition, CC5 exhibits potent inhibition of HIV-1 activity in primary peripheral blood mononuclear cells (PBMCs), demonstrating the importance of the dimer in blocking viral infection. Conformational variants like CC5 are valuable tools for elucidating the biological relevance of the XCL1 native-state interconversion and will assist in future anti-viral and functional studies.

Transferring the C-terminus of the chemokine CCL21 to CCL19 confers enhanced heparin binding

“…[26, 27] and others [28, 29]. This method is utilized to elucidate whether affinity is a result of a specific binding affinity to heparin or nonspecific electrostatic interactions.…”

“…However, it is necessary to further interrogate the nature of CCL21’s heparin interaction. Using a SP-sepharose cation exchange column and a heparin affinity column helps distinguish between specific heparin interactions and affinity resulting from general electrostatic interactions [26–29]. Figure 2D shows that the chimera elutes at similar retention times from both the heparin and SP-sepharose columns, this is shown in Table 1 in terms of NaCl concentrations at elution.…”

“…Heparin binding may be due to electrostatic interaction between the basic CCL21 C-terminus and negatively charged heparin, specificity for heparin, or a combination of both. Comparison of elution profiles from heparin and cation exchange sepharose columns has been used to address specific heparin binding versus non-specific electrostatic interactions [26–29]. Comparing the elution profile for CCL21 reveals a difference in NaCl concentration at elution of 105 mM from heparin and cation exchange columns (Table 1).…”

“…[26, 27] and others [28, 29]. Roughly 50 µg of chemokine was loaded onto either a 1 mL HiTrap® heparin HP or 1 mL HiTrap® SP-sepharose HP column (GE Healthcare) and eluted with a 0 to 1.3 M NaCl gradient in 20 mM sodium phosphate at pH 7 over 39 minutes using a Shimadzu LC-20 HPLC system.…”

Cytokines Elevated in HIV Elite Controllers Reduce HIV Replication In Vitro and Modulate HIV Restriction Factor Expression

“…In fact, the R&D Systems XCL1 was tested recently and was shown to have only modest suppressive activity compared to that of other preparations (53). If XCL1 were to be utilized for anti-HIV therapy, it would need to be formulated in the ␤-dimer form, and a variant with a disulfide bond that stabilizes the all-␤ conformation has been engineered (54).…”