Abstract
Membrane proteins are an important class of macromolecules found in all living organisms and many of them serve as important drug targets. In order to understand their biological and biochemical functions and to exploit them for structure-based drug design, high-resolution and accurate structures of membrane proteins are needed, but are still rarely available, e.g., predominantly from X-ray crystallography, and more recently from single particle cryo-EM — an increasingly powerful tool for membrane protein structure determination. However, while protein-lipid interactions play crucial roles for the structural and functional integrity of membrane proteins, for historical reasons and due to technological limitations, until recently, the primary method for membrane protein crystallization has relied on detergents. Bicelle and lipid cubic phase (LCP) methods have also been used for membrane protein crystallization, but the first step requires detergent extraction of the protein from its native cell membrane. The resulting, crystal structures have been occasionally questioned, but such concerns were generally dismissed as accidents or ignored. However, even a hint of controversy indicates that methodological drawbacks in such structural research may exist. In the absence of caution, structures determined using these methods are often assumed to be correct, which has led to surprising hypotheses for their mechanisms of action. In this communication, several examples of structural studies on membrane proteins or complexes will be discussed: Resistance-Nodulation-Division (RND) family transporters, microbial rhodopsins, Tryptophan-rich Sensory Proteins (TSPO), and Energy-Coupling Factor (ECF) type ABC transporters. These analyses should focus the attention of membrane protein structural biologists on the potential problems in structure determination relying on detergent-based methods. Furthermore, careful examination of membrane proteins in their native cell environments by biochemical and biophysical techniques is warranted, and completely detergent-free systems for membrane protein research are crucially needed.
Highlights
Cellular membrane systems are crucial for all living organisms
Some of the most important biological reactions are conducted by membrane proteins; for example, photosynthesis is conducted by the photosynthetic reaction centers, rhythmic heartbeats depend on the coherent functions of membrane protein channels, and all activities of the nervous systems—from simple non-conscious reactions to human consciousness—depend on the function of numerous membrane proteins channels, transporters, enzymes, and receptors
The cryo-EM-based observations, where the proteins are in more native conformations, are distinctly different from the results obtained with the X-ray crystal structures of AcrB, where the detergent n-dodecyl-β-D-maltopyranoside (DDM) was used in extraction and crystallization
Summary
Cellular membrane systems are crucial for all living organisms. They actively maintain the homeostasis of the cell and the body of a living organism. Cholesterol has been known to affect the binding affinity of agonists for GPCRs and activation of Ca2+ ATPase is found to be diacylglycerol dependent [7,8] These detergent-based methods for membrane protein structural biology have significant and intrinsic drawbacks because they are based on the potentially flawed assumption that membrane proteins extracted from the lipid bilayer will be structurally and functionally the same, or at least similar to how they are on the cell membrane. Regardless of whether or not the structural information obtained from membrane protein crystallography is generated with benign disregard for the importance of protein-lipid interactions, such data could be misleading In this communication, my goal is to alert the readers of this widespread problem and offer supporting evidence with several analyses of previously published membrane protein structures where the protein-lipid interactions were clearly significant, and their absence affects our understanding of their inherent biology.
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