BINDING OF HISACTOPHILIN-I AND HISACTOPHILIN-II TO LIPID-MEMBRANES ISCONTROLLED BY A PH-DEPENDENT MYRISTOYL-HISTIDINE SWITCH

The interaction of the two N-terminally myristoylated isoforms of Dict yostelium hisactophilin with lipid model membranes was investigated by means of the monolayer expansion method and high-sensitivity titratio n calorimetry. The two isoforms, hisactophilin I and hisactophilin II, were found to insert with their N-terminal myristoyl residue into an electrically neutral POPC monolayer corresponding in its lateral packi ng density to that of a lipid bilayer. The partition coefficient for t his insertion process was K-p = (1.1 +/- 0.2) x 10(4) M(-1). The area requirement of the protein in the lipid membrane was estimated as 44 /- 6 Angstrom(2) which corresponds to the cross sectional area of the myristoyl moiety with an additional small contribution from amino acid side chains, The interaction of hisactophilin I (hisactophilin II) wi th negatively charged membrane surfaces is modulated in a pH-dependent manner by charged amino acid residues clustered around the myristoyl moiety. The electrostatic binding site consists of three lysine (one a rginine and two lysine), seven (nine) histidine, and four (four) gluta mic acid residues and has an isoelectric point of 6.9 (7.1), For small unilamellar POPC/POPG (75/25 mole/mole) vesicles, an apparent binding constant, K-app = (8 +/- 1) x 10(5) M(-1), was measured at pH 6.0 by means of high-sensitivity titration calorimetry. Electrostatic interac tions hence increase the binding constant by about 2 orders of magnitu de compared to hydrophobic binding alone, With increasing pH, the elec trostatic attraction decreases and turns into an electrostatic repulsi on at pH > 7.0 +/- 0.1, The area occupied by the cluster of charged re sidues constituting the membrane binding region was 280 +/- 20 Angstro m(2) as derived from monolayer measurements in close agreement with mo lecular modeling data derived from the NMR structure of hisactophilin I [Habazettl et al, (1992) Nature 359, 855-858].