Activation mechanism of a protein water channel by cardiolipin: implications for the fabrication of block copolymer nanosheets

Optimization of biomimetic membranes that incorporate membrane proteins for their use in desalination, filtration, dialysis or drug discovery applications is still challenging. In these systems, amphiphilic block copolymers (BCPs) are often used instead of lipids, because of BCP’s far higher chemical and mechanical stability. For example, high water flux has been successfully demonstrated in BCP nanosheets incorporating a bacterial porin and other similar architectures (Tu et al. Nature Materials, 2020). Despite this artificial environment, many membrane proteins also require tightly bound native lipids for optimal function. Tetrameric aquaporin Z (AqpZ) from E. coli has been used as a workhorse for water purification/desalination systems for the last 15 years. Binding of the lipid cardiolipin (CDL) to AqpZ more than doubles water permeability and increases stability (Laganowsky et al., Nature, 2014), and we found recently that this activation by CDL binding may be highly cooperative (Tan, C.L.J. and Torres, J., BBA-Mol Cell Biol Lipids 2021). Although in some proteins lipid-binding can allosterically modulate binding affinity of other lipids, our model suggests that in AqpZ, CDL binding induces a concerted conformational change that affects not one, but the four monomers in the AqpZ homo-tetramer. Also, the efficacy and compatibility of these tightly bound lipids (CDL) in BCP environments has not been addressed at the molecular level. Herein we will address these two issues by exploring the conformational changes induced by CDL binding, and its enhancing effect on AqpZ activity and stability, in the presence of both lipid or BCP environments. If CDL activation is cooperative, only two observable types of tetrameric structure will be observed, instead of gradually varying conformations with increasing CDL. We will use single particle cryo-electron microscopy (nanodiscs) and solid-state NMR, to extract this information in both lipidic and BCP environments. The AqpZ activation promoted by CDL will be tested in the context of AqpZ-based BCPs polymersomes (liposome-like particles made of BCPs)and also planar nanosheets, following published protocols. The conclusions of this project will be key to optimize membrane biomimetics when applied to AqpZ and other MPs that also use lipid modulators. (i) intellectual merit. It is known that tightly bound lipids can allosterically modulate the binding affinity for other lipids (e.g., in the E. coli ammonia channel, AmtB), but this allostery was not observed for AqpZ [1]. Lipid-binding events can also modify protein-protein interaction affinity [2], but the conformational change induced by CDL on AqpZ, responsible for its increased activity and stability is not known. Thus our aim is to determine how CDL is able to change protein structure, in native and synthetic lipidic environments, and also in a polymeric context (BCPs) that is relevant for bioengineering applications. These answers will therefore merit publication in a high IF journal. (ii) broader impact. The role of lipid modulators of membrane proteins is an emerging field, with implications in bioengineering, storage and transport, biotechnology and drug discovery. Especially, the role of these modulators in a synthetic polymeric lipid-like context has not been addressed, even less at the molecular level. Our research will address this issue head-on to establish the role of these lipids in controling membrane protein function. The techniques used allow exploration of even subtle protein conformational changes in native lipids, synthetic lipids and block copolymers. These technologies and expertise may be applied to other important membrane proteins. Lastly, some aquaporins reside in CDL-containing membranes; since aquaporins are physiologically and medically important, our results may suggest novel approaches for modulation of function. Overall, the two questions we want to answer are: • What are the structural changes mediated by CDL binding to AqpZ that enhance water permeability and stability in lipidic environments? • What is the effect of CDL in the context of AqpZ-based biomimetic membranes made with lipid-mimic block copolymers?