Biology subjects

Schäfer, J.-H.

Publications and source records attributed to Schäfer, J.-H..

3 recordsLinked to original sources

bioRxiv2026

Structural Plasticity and Ligand Promiscuity of CYP3A4 Revealed by Cryo-EM

Cytochrome P450 3A4 (CYP3A4) metabolizes roughly half of all marketed drugs, and its inhibition can cause clinically significant drug-drug interactions. The enzyme accommodates chemically diverse ligands, making binding modes and metabolic outcomes difficult to predict. Previous X-ray crystallography efforts have leveraged a truncated construct without the N-terminal segment that tethers CYP3A4 to the membrane. Here we show that the same construct assembles into a symmetric trimer that can be resolved by cryo-EM and determine structures of both unliganded and ligand-bound CYP3A4. Multiple ligands are resolved with density consistent with several mutually exclusive conformations. Protein remodeling to reshape the binding pocket is concentrated in the F/G loop, which is poorly resolved and unmodeled in many X-ray structures. These features likely underlie the poor predictive performance of co-folding methods on this target. The routine use of cryo-EM to resolve CYP3A4 ligand-bound complexes will provide the ground truth data needed to make predictive models of drug metabolism useful in practice.

biophysics↗

bioRxiv2024

The structure of the Orm2-containing serine palmitoyltransferase complex reveals distinct inhibitory potentials of yeast Orm proteins.

The levels of sphingolipids are crucial determinants of neurodegenerative disorders. Therefore, sphingolipid levels have to be tightly regulated. The Orm protein family and ceramides act as inhibitors in the rate-limiting step of sphingolipid biosynthesis - the condensation of L-serine and palmitoyl-CoA. The two yeast isoforms Orm1 and Orm2 form a complex with the serine palmitoyltransferase (SPT). While the sequences of the Orm proteins are highly similar, however, Orm1 and Orm2 are differentially regulated in yeast cells. The mechanistic details of the differential regulation remain elusive. To elucidate the regulatory mechanism, we determined the cryo-electron microscopy structure of the SPT complex containing Orm2. Through in vitro activity assays and a newly developed plasmid coverage assay combined with targeted lipidomics, we demonstrate that the SPT complex with Orm2 exhibits lower activity in comparison to the complex containing Orm1. This collectively suggests a higher inhibitory potential of Orm2, despite the remarkably similar structures of the Orm1- and Orm2 containing complexes. The high conservation of the SPT from yeast to man implies that the three human ORMDL isoforms could also have different regulatory capacities, which might be essential to understanding their role in sphingolipid-mediated neurodegenerative disorders. HighlightsO_LICryo-EM structures of serine palmitoyltransferase with Orm2 from S. cerevisiae C_LIO_LICeramide-mediated inhibition on SPT activity is conserved amongst SPTs C_LIO_LIThe two yeast Orm isoforms have different regulatory capacity C_LI

biochemistry↗

bioRxiv2023

Structure of the yeast ceramide synthase

Ceramides play a pivotal role as essential lipids, serving as foundational components for complex sphingolipids and potent signaling molecules. Ceramides are the products of the N-acylation of a sphingoid base and a CoA-activated fatty acid. This reaction is catalyzed by the enzymes of the evolutionarily conserved ceramide synthase (CerS) family. Yet, the precise structural details and catalytic mechanisms of CerSs have remained elusive. Here, we employed cryo-EM single particle analysis to unravel the structure of the yeast ceramide synthase complex in both an active and a fumonisin B1 inhibited state. Our findings shed light on the complexs architecture, revealing a dimer of Lip1 subunits bound to the two catalytic subunits, Lag1 and Lac1. Each catalytic subunit forms a hydrophobic crevice that is accessible from both the cytosolic site as well as from the intermembrane space of the endoplasmic reticulum (ER). Within this cavity, we identify amino acids forming the active center and a sphingoid base, one of the substrates of the complex. Together, this suggests a pre-loaded state of the CerS. Additionally, the fumonisin B1 bound structure reveals the inhibitory mechanism by blocking the cytosolic acyl-CoA binding site.

biochemistry↗