Charged residues next to transmembrane regions revisited: “Positive-inside rule” is complemented by the “negative inside depletion/outside enrichment rule”

Abstract

BackgroundTransmembrane helices (TMHs) frequently occur amongst protein architectures as means for proteins to attach to or embed into biological membranes. Physical constraints such as the membrane’s hydrophobicity and electrostatic potential apply uniform requirements to TMHs and their flanking regions; consequently, they are mirrored in their sequence patterns (in addition to TMHs being a span of generally hydrophobic residues) on top of variations enforced by the specific protein’s biological functions.ResultsWith statistics derived from a large body of protein sequences, we demonstrate that, in addition to the positive charge preference at the cytoplasmic inside (positive-inside rule), negatively charged residues preferentially occur or are even enriched at the non-cytoplasmic flank or, at least, they are suppressed at the cytoplasmic flank (negative-not-inside/negative-outside (NNI/NO) rule). As negative residues are generally rare within or near TMHs, the statistical significance is sensitive with regard to details of TMH alignment and residue frequency normalisation and also to dataset size; therefore, this trend was obscured in previous work. We observe variations amongst taxa as well as for organelles along the secretory pathway. The effect is most pronounced for TMHs from single-pass transmembrane (bitopic) proteins compared to those with multiple TMHs (polytopic proteins) and especially for the class of simple TMHs that evolved for the sole role as membrane anchors.ConclusionsThe charged-residue flank bias is only one of the TMH sequence features with a role in the anchorage mechanisms, others apparently being the leucine intra-helix propensity skew towards the cytoplasmic side, tryptophan flanking as well as the cysteine and tyrosine inside preference. These observations will stimulate new prediction methods for TMHs and protein topology from a sequence as well as new engineering designs for artificial membrane proteins.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-017-0404-4) contains supplementary material, which is available to authorized users.

Warfarin and vitamin K epoxide reductase: a molecular accounting for observed inhibition

“…This pocket is consistent with the hydrophobic characteristics of the enzyme's substrate/ inhibitor. Additionally, in the 3-TM model, the distribution of the charged residue flanking TM1 agrees with the "positiveinside rule" [48][49][50][51] ( Figure 7A-B), a dominant factor that affects the orientation of a given TM helix of membrane proteins. Furthermore, 4 tryptophan residues and 1 cytoplasmic lysine residue are located at the membrane's bilayer interface of the 3-TM model of VKOR ( Figure 7C), serving as anchoring residues to fix the TM helix within the lipid bilayer.…”

The ΔF508 CFTR defect: molecular mechanism of suppressor mutation V510D and the contribution of transmembrane helix unraveling

“…The replacement of either Q1042 or P1050 with acidic residues Asp and Glu did not appear to improve trafficking more so than V510D alone, suggesting that the insertion of a negative charge, while not potentially destabilizing, provided no suppression of helical instability. This is in keeping with the "positive inside rule," which states that positively charged residues (Arg and Lys) will frequently be located at the cytoplasmic edge of transmembrane helices, (46,47), with the opposite being true for negatively charged residues (48), a trend that is supported by extensive statistical observations for most membrane proteins (48,49). The proposed utility of this "charged-residue flanking bias" (48) is that positively charged amino acids bordering hydrophobic helices might be involved in TM orientation within the lipid bilayer (47), as well as helix-helix interactions (47,50,51).…”

“…This is in keeping with the "positive inside rule," which states that positively charged residues (Arg and Lys) will frequently be located at the cytoplasmic edge of transmembrane helices, (46,47), with the opposite being true for negatively charged residues (48), a trend that is supported by extensive statistical observations for most membrane proteins (48,49). The proposed utility of this "charged-residue flanking bias" (48) is that positively charged amino acids bordering hydrophobic helices might be involved in TM orientation within the lipid bilayer (47), as well as helix-helix interactions (47,50,51). With regard to L1096, we must consider that leucine is highly hydrophobic, and is known to play a critical role in both helical stabilization and TM helix-helix interactions (47,48,52).…”

“…The proposed utility of this "charged-residue flanking bias" (48) is that positively charged amino acids bordering hydrophobic helices might be involved in TM orientation within the lipid bilayer (47), as well as helix-helix interactions (47,50,51). With regard to L1096, we must consider that leucine is highly hydrophobic, and is known to play a critical role in both helical stabilization and TM helix-helix interactions (47,48,52). Unsurprisingly, modifying this residue did not improve ΔF508-CFTR trafficking as shown by western blot (Figure 8), and in the case of the L1096D mutation, abolished the V510D stabilizing effect.…”

Parvovirus B19 and Human Parvovirus 4 Encode a Homologous "X Protein" in a Reading Frame Overlapping the VP1 Capsid Gene

“…The erythroparvovirus X protein contains a predicted central transmembrane segment ( Fig 5A). It is followed by a positively charged region, predicted to be inside the cytosol ("positive-inside rule" [24]). Therefore, the N-terminus of X, which must be on the other side of the transmembrane segment, is necessarily extra-cytosolic ( Fig 5A).…”