Interplay between hydrophobicity and the positive-inside rule in determining membrane-protein topology

Abstract

The energetics of membrane-protein interactions determine protein topology and structure: hydrophobicity drives the insertion of helical segments into the membrane, and positive charges orient the protein with respect to the membrane plane according to the positive-inside rule. Until recently, however, quantifying these contributions met with difficulty, precluding systematic analysis of the energetic basis for membrane-protein topology. We recently developed the dsTβL method, which uses deep sequencing and in vitro selection of segments inserted into the bacterial plasma membrane to infer insertion-energy profiles for each amino acid residue across the membrane, and quantified the insertion contribution from hydrophobicity and the positive-inside rule. Here, we present a topology-prediction algorithm called TopGraph, which is based on a sequence search for minimum dsTβL insertion energy. Whereas the average insertion energy assigned by previous experimental scales was positive (unfavorable), the average assigned by TopGraph in a nonredundant set is −6.9 kcal/mol. By quantifying contributions from both hydrophobicity and the positive-inside rule we further find that in about half of large membrane proteins polar segments are inserted into the membrane to position more positive charges in the cytoplasm, suggesting an interplay between these two energy contributions. Because membrane-embedded polar residues are crucial for substrate binding and conformational change, the results implicate the positive-inside rule in determining the architectures of membrane-protein functional sites. This insight may aid structure prediction, engineering, and design of membrane proteins. TopGraph is available online (topgraph.weizmann.ac.il).membrane insertion | topology prediction | positive-inside rule | Bellman-Ford search T he plasma membrane is a complex physical environment comprising a hydrophobic core and a polar exterior, which is more negatively charged on its cytoplasmic side (1). Two hallmarks of membrane proteins are hydrophobic segments that span the membrane core, and positive charges at the membrane-cytoplasm interface (the positive-inside rule; ref. 2); these features drive insertion and orient segments relative to the membrane plane, respectively. Furthermore, recent work has shown that positive charges placed close to engineered segments can drive membrane insertion even of marginally polar segments (3, 4), suggesting a role for the positive-inside rule in insertion, and emphasizing the importance of accurate models of membraneprotein energetics for protein engineering and for understanding the physical basis of membrane-protein topology.Topology prediction is a stringent test of our models of membraneprotein energetics. The most parsimonious membrane-topology predictor would locate the membrane-spanning segments and determine their orientations by a sequence search for minimum insertion energy. To achieve that, however, the insertion energy scale must at a minimum exhibit two properties: (i) to drive membrane in...

Molecular dynamics of the cryo-EM CFTR structure

“…However, electrostatic properties of the transmembrane region indicate a valid arrangement of this L0/Lasso segment in the structure, since it is attached to a hydrophobic spot interrupting the positive ring of the transmembrane domains, located at the depth of the inner bilayer boundary. This ring is formed by positively charged amino acids at the interface region of transmembrane helices according to the positive-inside rule [31,32] and is observable in the CFTR homology models, but not well-defined in the experimental structure (Fig. 2).…”

The importance of the membrane interface as the reference state for membrane protein stability

“…Unfortunately, due to hydrophobic aggregation, no equivalent direct experimental measurements of peptide insertion currently exist, and even the most recent experimental techniques employ very different systems [59]. However, full-peptide autonomous insertion calculations, such as shown here, are critical for our understanding of antimicrobial peptides, cell-penetrating peptides, toxins, fusion peptides, and other membrane active peptides.…”

A lipophilicity-based energy function for membrane-protein modelling and design

“…Membrane-insertion profiles. The original dsTβL insertion profiles 15 were modified to generate smooth and symmetric functions 22 . The polar and charged residues Asp, Glu, Gln and Asn, which exhibited few counts in the deep sequencing analysis, were averaged such that the insertion energy at the membrane core (-10 to 10 Å; negative values correspond to the inner membrane leaflet and positive values to the outer leaflet) was applied uniformly to the entire membrane span.…”

“…While this preference, known as the "positive-inside" rule, was revealed based on sequence analysis 30 years ago [19][20][21] , the dsTβL assay was the first to indicate a large energy gap favouring positively charged residues in the intracellular relative to the extracellular membrane leaflet. The accuracy and generality of the dsTβL apparent transfer energies were partly verified by demonstrating that they correctly predicted the locations and orientations of membrane spans directly from sequence even in several large and complex eukaryotic transporters 22 . Taken together, these results provided reassurance that the dsTβL apparent insertion energies correctly balanced essential aspects of membrane-protein solvation.…”