Molecular ortheses for the crystallization of membrane proteins. Application to multi-drug efflux pumps. – X-Or

Membrane proteins have widely diverse, essential functions and constitute a major class of molecular targets for therapeutic research. However, membrane proteins remain challenging for structural studies as all steps, from expression to structural studies, are potentially complicated by their obligatory association with a hydrophobic environment. In spite of a wealth of information regarding the handling of membrane proteins, their structure determination remains a very difficult task and, indeed, they are still strongly under-represented in the Protein Database. It was therefore proposed that the critical step of membrane protein crystallization could be considerably facilitated by using membrane proteins in complex with a specific and tight-binding protein partner such as a specific antibody. The rationale is that interactions with the antibody stabilize the membrane protein and reduce its conformational heterogeneity. Moreover, the antibody fragment increases the polar part of the protein from which crystal packing intermolecular interactions originates.
However, antibodies have important drawbacks for application in structural biology of membrane proteins since most antibodies or antibody fragments are very poorly expressed in simple and efficient recombinant system.
We propose to focus on a recently introduced technology that directly addresses this problem and offers creative solutions with potentially large applicability: the design and production of a new set of synthetic scaffolds (dubbed alpha-reps for “artificial alpha repeat protein”) as membrane protein stabilizers and crystallization ortheses. Such interactants will be selected through in vitro screening of the membrane proteins targets. The stabilization and immobilization of native membrane proteins is an absolute prerequisite in order to generate binders truly specific for the “native” membrane protein. We will make use of amphipathic polymers (amphipols) to stabilize and immobilize membrane protein targets onto solid supports so that the library of possible interactants can be screened.
We have selected the bacterial efflux pumps from Pseudomonas aeruginosa to apply the new strategy to the problem of bacterial resistance. Among the various mechanisms developed by the bacteria to counter to the effect of antibiotics, active efflux is on the front line. In Gram-negative bacteria, which are protected by an outer membrane, some of the efflux transporters are organized as multicomponent systems, in which the efflux pump located in the inner membrane works in conjunction with a periplasmic protein and an outer membrane protein. It has been demonstrated that inhibition of such pumps increases antibiotic susceptibility and reduces the probability of emergence of antibiotic-resistant mutants but many questions remain on the actual mechanism of transport of the pump. The aim of this project is to explore methodological developments aiming at giving a significant impetus in the understanding of the architecture of efflux pumps.