Abstract
Squalene Monooxygenase (SM) is the 2nd rate-limiting enzyme in the cholesterol synthesis pathway. Recent years of research has classified SM as an emerging drug target covering high cholesterol, cancer, and fungal skin infections. However, SM is a membrane protein residing in the membrane of the Endoplasmatic Reticulum (ER), thus rendering this enzyme difficult to study experimentally. Despite the extensive efforts that have been put into unraveling the mechanistic detail of SM activity, the molecular basis of the enzymatic reaction remains elusive.
Recently, Padyana et al. (2019) published the first crystal structures of the catalytic domain of human SM, which was recombinantly expressed in Escherichia coli. Their work covered three structures of SM; one structure in its apoform and two structures with two different potent human inhibitors. However, the substrate binding site could not be experimentally verified and thus remains speculative. SM has been computationally predicted to comprise two C-terminal transmembrane helices. However, the topology of SM in the ER membrane is unknown. Likewise, how the hydrophobic substrate, squalene, moves from the ER membrane into the active site is still puzzling.
We aimed to determine high-resolution crystal structures of SM bound to the substrate, substrate mimics, product, or product mimics in an effort to elucidate the mechanism of action, which can ultimately assist in the design of new SM inhibitors.
This thesis work reports the complexity of expressing and purifying sufficient amounts of high-quality protein in a detergent-based system. Human and fungal variants of SM were recombinantly expressed in the eukaryotic expression host Saccharomyces cerevisiae. We show for the first time the successful purification of fungal SM from Trichopython rubrum, a fungal species causing almost half of all cases of foot fungus, in amounts suitable for crystallization screening. We further report two crystal structures of the catalytic domain of human SM in its apoform. Our highest-resolution structure at 2.6 Å accommodates the same SM dimer conformation as previously published, but represents the highest- resolution structure of the apoform reported to date. Finally, we present a novel crystal conformation at 3.0 Å, which suggests that SM is anchored to one side of the membrane rather than fully spanning the lipid bilayer as currently suggested in literature – a finding that could be relevant in future design of SM inhibitors.
Recently, Padyana et al. (2019) published the first crystal structures of the catalytic domain of human SM, which was recombinantly expressed in Escherichia coli. Their work covered three structures of SM; one structure in its apoform and two structures with two different potent human inhibitors. However, the substrate binding site could not be experimentally verified and thus remains speculative. SM has been computationally predicted to comprise two C-terminal transmembrane helices. However, the topology of SM in the ER membrane is unknown. Likewise, how the hydrophobic substrate, squalene, moves from the ER membrane into the active site is still puzzling.
We aimed to determine high-resolution crystal structures of SM bound to the substrate, substrate mimics, product, or product mimics in an effort to elucidate the mechanism of action, which can ultimately assist in the design of new SM inhibitors.
This thesis work reports the complexity of expressing and purifying sufficient amounts of high-quality protein in a detergent-based system. Human and fungal variants of SM were recombinantly expressed in the eukaryotic expression host Saccharomyces cerevisiae. We show for the first time the successful purification of fungal SM from Trichopython rubrum, a fungal species causing almost half of all cases of foot fungus, in amounts suitable for crystallization screening. We further report two crystal structures of the catalytic domain of human SM in its apoform. Our highest-resolution structure at 2.6 Å accommodates the same SM dimer conformation as previously published, but represents the highest- resolution structure of the apoform reported to date. Finally, we present a novel crystal conformation at 3.0 Å, which suggests that SM is anchored to one side of the membrane rather than fully spanning the lipid bilayer as currently suggested in literature – a finding that could be relevant in future design of SM inhibitors.
| Original language | English |
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| Place of Publication | Kgs. Lyngby, Denmark |
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| Publisher | DTU Bioengineering |
| Number of pages | 120 |
| Publication status | Published - 2022 |
