Identifying and quantifying membrane interactions of the protein human cis ‐prenyltransferase

Abstract Prenyl chains come in multiple sizes, fulfilling unique and essential functions across all domains of life. Prenyl chains are synthesized by prenyltransferase proteins. Despite their structural similarity, prenyltransferases exhibit substantial functional diversity to create lipophilic products of varying lengths. Human cis ‐prenyltransferase (h‐ cis PT) is a tetrameric enzyme responsible for the synthesis of long prenyl chains, consisting of 20‐prenyl‐unit products that are essential to specific posttranslational modifications such as N‐glycosylation upon downstream processing. These long products are hypothesized to transfer from h‐ cis PT to the ER membrane, but the mechanism of this transfer is not known. We use molecular dynamics simulations to identify a consistent membrane binding pose for h‐ cis PT. By quantifying protein‐membrane contacts, we identify the aromatic amino acid residues in the conserved catalytic domain as critical to membrane binding. Determining relative protein‐membrane binding free energies through free energy perturbation highlights the importance of these residues for membrane association, as mutations lower membrane affinity by as much as 27 kcal/mol. These results are validated using FRET to demonstrate decreased catalytic activity and membrane binding in response to mutation. Together, our results suggest a possible mechanism for prenyl substrate transfer, where key aromatic residues facilitate h‐ cis PT binding to the ER membrane in an orientation that holds the substrate‐containing active site near the membrane surface. Molecular dynamics simulations of the mutant exhibiting lower FRET show greater orientational variability relative to wild type. This evidence for a specific orientation of h‐ cis PT provides a structural basis for isoprenoid association to the membrane during synthesis and prior to its release.