Insights into the key determinants of membrane protein topology enable the identification of new monotopic folds

Abstract

Monotopic membrane proteins integrate into the lipid bilayer via reentrant hydrophobic domains that enter and exit on a single face of the membrane. Whereas many membrane-spanning proteins have been structurally characterized and transmembrane topologies can be predicted computationally, relatively little is known about the determinants of membrane topology in monotopic proteins. Recently, we reported the X-ray structure determination of PglC, a full-length monotopic membrane protein with phosphoglycosyl transferase (PGT) activity. The definition of this unique structure has prompted in vivo, biochemical, and computational analyses to understand and define key motifs that contribute to the membrane topology and to provide insight into the dynamics of the enzyme in a lipid bilayer environment. Using the new information gained from studies on the PGT superfamily we demonstrate that two motifs exemplify principles of topology determination that can be applied to the identification of reentrant domains among diverse monotopic proteins of interest.

Monotopic Membrane Proteins Join the Fold

“…The mode of membrane association for the fold cannot be inferred from the sole reported structure, in which the N-terminal domain protrudes from the soluble domain as a helix-break-helix motif. However, recent biochemical and bioinformatics analyses of LpxM from E. coli now suggest a monotopic topology with the soluble domain anchored on the cytoplasmic face of the membrane by a reentrant helix [57].…”

Investigation of the conserved reentrant membrane helix in the monotopic phosphoglycosyl transferase superfamily supports key molecular interactions with polyprenol phosphate substrates

“…PglC from C. jejuni strain 11168 was cloned into the pET24a vector to insert a C-terminal His6tag [37] or into the pE-SUMO vector [34]. P24A variants of both constructs were generated using QuikChange II Site-Directed Mutagenesis (Agilent Technologies, Santa Clara, CA) as described previously [37].…”

“…Activity assays were performed using the UMP/CMP-Glo assay (Promega, Madison, WI) on a 10 μL scale using UndP as the PrenP acceptor, as described previously [37]. Data were plotted using Graphpad Prism 8 (GraphPad Software).…”

“…Additionally, a conserved proline (Pro24 in C. concisus PglC), located at the break in the reentrant helix domain has been shown to be important for PglC activity (Fig. 2) [34,37]. Based on the position of Pro24 within the only membrane-resident domain of PglC, and on the known conservation of prolines in polyisoprenol-recognition sequences (PIRSs) in diverse proteins [19,24,38], it has been hypothesized that Pro24 may play a key role in recognition and binding of PrenP substrates in the membrane, both in the context of PglC and in other members of the monotopic PGT superfamily [27,34].…”

Lipid Head Group Parameterization for GROMOS 54A8: A Consistent Approach with Protein Force Field Description

“… 9 In addition, many transmembrane proteins tessellate the cell surface, promoting signaling pathways and influencing the membrane’s structural and mechanical properties. 10 , 11 Phospholipid bilayers and micelles have been investigated, in particular, as these lipids represent the main components of the eukaryotic and the inner bacterial membranes. Both have been modeled selecting specific phospholipids to emulate the appropriate surface charge or to reproduce the human cell membrane fluidity by introducing, for example, cholesterol.…”