Molecular stabilizers of membrane proteins for structural studies in solution – CLAMP2

Membrane proteins (MPs) account for 25-30% of proteomes. Their key role in signaling and transport make them the target of 70% of the current drugs and -estimated- 80% of the future ones . Knowing their 3D-structure is a mandatory step for rational drug design, requiring the extraction of MPs from their native lipid environment and their purification in aqueous solution. Detergents are key players in that process but, more exchangeable than lipids, they commonly destabilize the native structure of MPs. This instability makes structure determination particularly difficult, Membrane proteins only account for 2% of available protein structures.
To tackle this problem, the French partners P1 and P2 have recently developed a new concept of detergents named Clamps, characterized by an enhanced capacity of interaction with MPs. Among them, a series of dicarboxylate-oside detergents, DCODs, increased the thermostability by 20-30 °C of native versions of BmrA, a prokaryotic ABC transporter and of A2AR, the human adenosine G-Protein coupled receptor. They also promoted crystallization and crystal diffraction of BmrA from 6 to 3.7-4 Å (Angew Chem Int Ed 57, 2948-52, 2018). Partnership with the Swedish partner, IP3 allowed us to collect cryo electron microscopy (cryoEM) data of a BmrA[ATPMg (hydrolyzed substrate)-rhodamine 6G (transported substrate)] complex, corresponding to a so far unknown conformation.
As a continuation, the 1st aim of CLAMP2 will be to scale up the synthesis of the 6 best Clamps/DCODs and test them during the full extraction/purification process including detergent exchange, and looking for the improvement of the structural information on BmrA. Such tests and structural assays will be extended to another ABC pump, the human ABCG2 for which P1 and P2 have developed a potent inhibitor acting in vivo (Oncotarget. 2014, 5(23):11957) and aim at getting the structure of the corresponding complex in a drug-design perspective. Partnership with P3 will allow to carry out the same tests and structural assays on Wzz, a MP of the lipo-polysaccharide (LPS) biosynthesis Wz machinery. Here the aim will be to improve the current resolution (5.5 Å from crystals and better by cryoEM) and stabilize and resolve the structure of a Wzz[LPS] complex.
A 2nd aim of CLAMP2 will be to design and synthesize a second generation of Clamps, exploring new concepts for replacing the oside of the DCODs as H-bond generator by PEG and amine groups; preliminary results with PEGs have already given promising results in terms of thermostabilization.
Oligomeric and multiprotein complexes of MPs can be destabilized during the extraction step with detergents. The ABCG2 and Wz proteins included in this study exemplify such cases. ABCG2 for which the recent x-ray structure shows a homodimer (Nature 2017 546-7659:504) while previous EM data from P1 suggested a tetramer (BBA 2010 1798-11:2094). The LPS machinery Wz complex is supposed to be made of at least 3 proteins, Wzx which flips lipid-bound trisaccharides from the inner to the outer leaflets of the membrane, Wzy that polymerizes the trisaccharides and Wzz that modulates the length of the polymers. Recent results (references above) have shown that long aliphatic tail DCODs stabilize MPs oligomers, a 3rd aim of CLAMP2 will be to design, synthesize and test a new type of dimeric and tetrameric Clamps, conceived to stabilize large/weak MPs complexes.
CLAMP2 gathers 3 teams chosen to cover the fields of MP structural biology and Chemistry. P1 and P2 have successfully worked together, leading to the 1st Clamp generation. The novelty of the consortium points to the Swedish team IP3 who broadened the structural MP aspect by working on MPs forming large complexes and bringing a deep knowledge in cryoEM. P1, P2 and IP3 have already started to collaborate in 2017 leading to the BmrA cryoEM data mentioned above.