Host Lipid Screen Strategy for the Lipid Cubic Phase Crystallization of the Integral Membrane Glycerol 3-Phosphate Acyltransferase with Substrates

Lipid cubic phase (LCP) is a bicontiuous material consisting a continuous lipid bilayer which separates two continuous water channels. LCP has proven to be a promising method in crystallization of membrane proteins. To date, the method has been responsible for providing crystals for over 430 membrane protein PDB entries. Like any other crystallization method, optimization of crystal quality is often required for X-ray diffraction experiments. Recently, the lipid composition of LCP has attracted considerable attentions in the optimization process.

 

In a recent Crystal Growth & Design issue (https://pubs.acs.org/doi/abs/10.1021/acs.cgd.7b01678), a research team led by Dr. Dianfan Li, a principle investigator in Shanghai Institute of Biochemistry & Cell Biology, Chinese Academy of Sciences, report the successful application of the “host lipid screening” strategy in the LCP crystallization of PlsY, a seven-transmembrane glycerol 3-phosphate (G3P) acyltransferase that catalyzes the committed step in bacterial phospholipid biosynthesis. Two monoacylglycerols (MAGs) with different chain lengths, namely, the 9.9 MAG and 7.8 MAG, were the two main host lipids used in the study. The two lipids share the same glycerol head group but differ in the length and the position of the cis-double bond in the acyl chain. The 9.9 MAG contains 18 carbons in the fatty chain with the double bond in the middle, whereas the 7.8 MAG contains 15 carbon with the double bond in between carbon 7 and 8. The native PlsY crystals growing in 9.9 MAG LCP diffracted to high resolution (1.62 Å). However, the SeMet crystals growing in the 9.9 MAG under similar precipitant conditions only diffracted to 4.3 Å. Switching 9.9 MAG to 7.8 MAG yielded larger crystals that diffracted to 2.0 Å, and provided strong anomalous signal which was used to solve the structure. The 7.8 MAG was also critical for the cocrystallization of PlsY with its unstable lipid substrate acyl phosphate. Interestingly, however, 7.8 MAG was found to be unsuitable for PlsY-G3P cocrystallization; multiple crystal forms obtained from a broad screening did not reveal electron density for this water-soluble substrate. In contrast, co-crystallization of PlsY-G3P in 9.9 MAG yielded a 2.37 Å data set, revealing the details of G3P binding. The authors hypothesize that, under crystallization conditions, G3P binding which occurs at the membrane boundary favors a more rigid LCP lipid bilayer formed by the longer-chain 9.9 MAG.