The fluid mosaic model of the cell membrane proposed nearly half a century ago is still largely appropriate. However, we now know that the mosaic of proteins is not formed by passive diffusion resulting in a homogeneous dispersion. Rather, it is the result of the ‘accretion’ of certain lipids and proteins forming microdomains called ‘lipid rafts’ in eukaryotes. More recently, such structures have been identified in prokaryotic cells, where they are referred to as functional membrane microdomains (FMMs) to distinguish them. Lipid rafts serve important functions in cells, linked to their proteins’ functioning, which in turn depends on the integrity of the microdomains. Disruptions are associated with neurodegenerative diseases and cardiovascular disease, among others. The detailed structure of the lipid rafts is largely unknown, and even less is known about the structure and function of their prokaryotic counterparts FMMs. Within the EU-funded RaftsStruc project and supported by a Marie Skłodowska-Curie Actions fellowship, Marta Ukleja set out to investigate the structure of FMMs, to validate their existence and provide insight into their potential functions.
One thing leads to another
A little more than a decade ago, Daniel Lopez, RaftsStruc project coordinator, identified FMMs and their lipids for the first time. The FMMs contained flotillin, a protein in eukaryotes found only in lipid rafts, increasing the interest in conserved structures and functions. Over the next decade, at the National Centre for Biotechnology (CNB-CSIC), Lopez’ lab added pieces to the FMM puzzle. Among them was an important link between FMMs, flotillin and a multidrug-resistant strain of Staphylococcus aureus. Methicillin-resistant S. aureus (MRSA) is the cause of life-threatening infections in hospitals worldwide. It first occurred within a few years of penicillin usage and has long been associated with the penicillin-binding protein 2a (PBP2a). PBP2a has lower affinity for penicillin and penicillin-like antibiotics (beta lactams) than other PBPs. Lopez’ team showed that flotillin plays a role in FMM formation and PBP2a polymerisation and that perturbation of FMM assembly interfered with PBP2a polymerisation and eliminated antibiotic resistance in MRSA. The pieces of the puzzle were accumulating, but a vital piece was missing.
Uncovering a key route to combatting multidrug-resistant staph infections
Ukleja joined Lopez’ lab in 2017 to study the 3D structure of flotillin and PBP2a with cutting-edge cryo-electron microscopy (cryoEM) and to characterise the interactions between them. Given what little was already known and the pressing public health issue of MRSA, any new results would be of value, making this a high-risk, high-gain proposition. “We successfully overexpressed and purified both flotillin and PBP2a and performed structural studies. Strikingly, we observed that the purified full-length PBP2a protein exists as a dimer, not a monomer as previously thought. Further, the dimer formation appears to be required for antibiotic resistance,” explains Ukleja. Ukleja and Lopez followed this serendipitous discovery leading to additional progress being finalised in two manuscripts. One describes PBP2a dimerisation and its influence on antibiotic resistance, and another describes the cryoEM structure and function of another flotillin protein partner. In recognition of the tremendous progress and value of Ukleja’s work, the Spanish National Research Council is funding the continuation of her research. Effective therapies for MRSA could be on the horizon.
RaftsStruc, FMM, PBP2a, flotillin, lipid rafts, MRSA, functional membrane microdomain, penicillin binding protein 2a, methicillin resistant S. aureus