Role of surface lysine residues of adipocyte fatty acid-binding protein infatty acid transfer to phospholipid vesicles

The tertiary structure of murine adipocyte fatty acid-binding protein (AFAB P) is a flattened 10-stranded beta -barrel capped by a helix-turn-helix seg ment. This helical domain is hypothesized to behave as a "lid" or portal fo r ligand entry into and exit from the binding cavity. Previously, we demons trated that anthroyloxy-labeled fatty acid (AOFA) transfer from AFABP to ph ospholipid membranes occurs by a collisional process, in which ionic intera ctions between positively charged lysine residues on the protein surface an d negatively charged phospholipid headgroups are involved. In the present s tudy, the role of specific lysine residues located in the portal and other regions of AFABP was directly examined using site-directed mutagenesis. The results showed that isoleucine replacement for lysine in the portal region , including the alphaI- and alpha II-helices and the beta C-D turn, resulte d in much slower 2-(9-anthroyloxy)palmitate (2AP) transfer rates to acidic membranes than those of native AFABP. An additive effect was found for muta nt K22,59I, displaying the slowest rates of FA transfer. Rates of 2AP trans fer from "nonportal" mutants on the beta -G and I strands were affected onl y moderately; however, a lysine --> isoleucine mutation in the nonportal be ta -A strand decreased the 2AP transfer rate. These studies suggest that ly sines in the helical cap domain are important for governing ionic interacti ons between AFABP and membranes. Furthermore, it appears that more than one distinct region, including the alphaI-helix, alpha II-helix, beta C-D turn , and the beta -A strand, is involved in these charge-charge interactions.