Creating Paramagnetically-Labeled PF4 Mutants to Evaluate Interactions with Mac-1 in NMR

PF4 (CXCL4) is a cationic platelet chemokine that has been identified as a ligand for the integrin Mac-1 (αMβ2). The interaction between PF4 and Mac-1 has been shown to cause leukocyte migration, improve phagocytosis, and trigger the up-regulation of Mac-1 expression in leukocytes, thereby increasing leukocytic adhesion. Though Mac-1 is known to serve as the site of interaction between PF4 and the leukocyte, the PF4 binding site of Mac-1 remains unknown. 1H-15N HSQC NMR spectroscopy of the interaction between PF4 and Mac-1’s binding site, the αMI domain, can provide this data. This project seeks to create PF4 mutants with site-directed spin labels to enhance the sensitivity of NMR for future experiments that seek to locate the PF4-Mac-1 binding site. It was hypothesized that the mutants created would adopt the native conformation and accept an MTSL label. Two mutants were successfully created and harvested, PF4 S17C and PF4 S26C. Both were soluble and the Sanger sequencing results show that primary structure was conserved except for the substitutions of structurally similar residues indicating the protein folds and likely adopts native conformation. PF4 S26C was labeled with MTSL, and 1H-15N HSQC NMR spectroscopy was performed on unlabeled PF4 S26C (at pH 3.40), MTSL-labeled PF4 S26C (at pH 3.15), and MTSL-labeled PF4 S26C exposed to ascorbic acid (at pH 3.15) to evaluate if the mutant accepts the label and, resultantly, experiences reduced signal intensity. Significant change in signal intensity occurred without change in location of the peaks between the unlabeled and labeled spectra, showing that PF4 S26C accepts the spin label without changing the protein structure and that the label works as expected; however, no change occurred after reducing the spin label with ascorbic acid, preventing confirmation that signal changes were exclusively caused by the MTSL-label. Therefore, though these mutants show potential for future titration with the αMI domain and the hypothesis is supported, a future attempt to reduce MTSL-labeled PF4 S26C at a higher pH (approximately pH 5) is required. Additionally, PF4 S17C should also be evaluated with the methodology used to assess PF4 S26C before its employment in future projects.