Tracking lipid-induced effects on membrane protein structure in real time.

Abstract

Lipids in biological membranes can act as allosteric modulators of membrane protein function. P-type ATPase proteins carry out ATP-dependent transport of mostly ions across several different types of membranes. It has been shown that both general properties such as membrane thickness and interactions to lipids at specific protein sites dictate P-type ATPase activity. We have developed a methodology to track P-type ATPase domain rearrangements in real time triggered by laser-induced release of ATP and subsequent monitoring by synchrotron X-ray pulses. This time-resolved X-ray solution scattering (TR-XSS) technique was used to capture the dynamics of an equilibrium state of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA1a), and transient states at 1.5 and 13 milliseconds. The 13-millisecond state showed a previously unresolved actuator (A) domain arrangement that exposed the ADP-binding site after phosphorylation. To identify the effects of protein-lipid interactions, we have now used the TR-XSS technique to register kinetics and structural dynamics in a Ca2+-transporting P-type ATPase from Listeria monocytogenes (LMCA1) in nanodiscs at different lipid compositions. In negatively charged POPG lipids, the LMCA1 reaction kinetics and structural dynamics were reminiscent of the SERCA1a isoform, which is in agreement with the bacterial membranes being predominantly negatively charged. In uncharged POPC nanodiscs, the LMCA1 reaction was significantly different with slow buildup of the rate-limiting state. The results enable a mechanistic view of how protein-lipid interactions regulate the P-type ATPase reaction cycle. The TR-XSS technique presents a unique possibility to simultaneously determine kinetics and structural rearrangements in P-type ATPases to better understand the transport reactions and their regulation mechanisms.