Metal ion cofactors modulate integral enzyme activity by varying differential membrane curvature stress

Abstract

Metal ions are well-known cofactors of protein function and stability. In the case of the integral membrane enzyme OmpLA (outer membrane phospholipase A) the active dimer is stabilized by calcium ions. We studied the lipid hydrolysis kinetics of OmpLA in charge-neutral and charged membranes with symmetric or asymmetric transbilayer lipid distributions. In charge-neutral membranes, OmpLA was more active in symmetric bilayers due to the lower differential curvature stress between membrane leaflets. Strikingly, this behavior was completely reversed in charged bilayers. Measurements revealed intrinsic molecular shape changes in the charged lipids upon addition of calcium. This effectively reduces the differential curvature stress in charged asymmetric membranes leading to increased protein activity. This conclusion is further supported by similar effects observed upon the addition of sodium ions, which also alter the shape of the lipids, but do not specifically interact with the protein. Additional lipid-protein interactions likely contribute to this phenomenon. Our findings demonstrate that ion cofactors not only interact directly with membrane proteins but also modulate protein activity indirectly by altering the effective molecular shape of charged lipid species.

Fig. 1. Effects of lipid asymmetry on the activity of OmpLA in plain buffer (basal) and upon the addition of 200 mM NaCl, or 20 mM CaCl₂. Panel (a): Analysis of POPE hydrolysis kinetics of asymmetric PE/PG and PE/PC/PG proteoliposomes; see Table 1 for composition. Solid lines show fits according to eqn (1), and dashed lines indicate deviations from the initial hydrolysis kinetics. Panel (b): Normalized hydrolysis rates in asymmetric (top) and symmetric (middle) proteoliposomes, as well as the ratio r = k^asym/k^sym (bottom). Note the different ordinate scales for k^asym and k^sym; the horizontal dashed line at |k^asym| = |k^sym| = 0.1 s⁻¹ allows a tentative comparison. k^sym was multiplied by −1 for data presentation reasons. The blue arrow and number for PE/PC₂ indicate the observed hydrolysis rate upon adding of Na⁺ ions. Panel (c): Intrinsic lipid curvatures of POPE and POPG as a function of salt concentration. The insert shows the definition of c₀, for details see ref. .

Fig. 2. Allosteric modulation of OmpLA activity by curvature stress differences between symmetric and asymmetric membranes. The grey area includes a range of possible ΔG° values of −9 to −3k_BT (according to eqn (3)). Colored solid and dashed lines show how a shift of values by about −5k_BT (possibly additional electrostatic contribution) could represent the data for r > 1.

Fig. 3. Schematic of coupling between the ion-mediated change of POPG shape (grey lipid) in an asymmetric membrane containing an OmpLA dimer (Protein Data Bank code: 1QD6 (ref. 21)). The upper scheme depicts the system at basal conditions. POPG has a weakly expressed cone-like shape (c₀ ≳ 0), while POPE (yellow lipid) adopts an inverted conical shape (c₀ < 0). Imposing a packing into flat monolayer creates significant curvature stress in the POPE enriched leaflet, but not in the POPG-rich leaflet. This leads to differential curvature stress between the two leaflets as indicated by the asymmetric lateral pressure profile p(z). With POPG adopting a more inverted cone-like shape in the presence of Ca²⁺ or Na⁺ (lower panel) the curvature stress in the POPG-rich leaflet also increases. However, the differential lateral stress between the membrane leaflets is now partially alleviated (p(z) becomes more symmetric), leading to an increase of enzyme activity.