GXXXG‐Mediated Parallel and Antiparallel Dimerization of Transmembrane Helices and Its Inhibition by Cholesterol: Single‐Pair FRET and 2D IR Studies

Abstract

Small-residue-mediated interhelical packings are ubiquitously found in helical membrane proteins, although their interaction dynamics and lipid dependence remain mostly uncharacterized. We used a single-pair FRET technique to examine the effect of a GXXXG motif on the association of denovo designed (AALALAA) 3 helices in liposomes. Dimerization occurred with sub-second lifetimes, which was abolished by cholesterol. Utilizing the nearly instantaneous time-resolution of 2D IR spectroscopy, parallel and antiparallel helix associations were identified by vibrational couplings across helices at their interface. Taken together, the data illustrate that the GXXXG motif controls helix packing but still allows for a dynamic and lipid-regulated oligomeric state. HHS Public Access AbstractUnique interaction inside membrane: Model transmembrane helices containing a GXXXG motif were synthesized to explore interplay of the motif and lipid composition on the intramembrane helix associations. Single-pair FRET with controlled association topology and isotope-edited 2D-IR experiments revealed the dynamic nature, distinct interfaces, and high cholesterol sensitivity of the GXXXG-mediated interactions, which can actively regulate the functions of membrane proteins.

All-Atom Molecular Dynamics Elucidating Molecular Mechanisms of Single-Transmembrane Model Peptide Dimerization in a Lipid Bilayer

“… 6 − 8 In contrast, our simulation with the GXXXG motif revealed a tightly packed dimer with a small crossing angle ( Figure 4 ), in agreement with the findings of the single-pair FRET experiments. 17 In addition, only a few hydrogen bonds were observed in the GXXXG dimer ( Figure 3 ). This difference in the mechanisms of dimerization by the GXXXG motif is likely caused by the peptide sequences.…”

“…In the models of the GXXXG parallel dimers, our simulations showed that introducing cholesterols reduced the interpeptide contacts, which agrees with the results of the single-pair FRET experiments. 17 Because the cholesterols thickened the membrane ( Figure 5 ), the tilt and crossing angles decreased to minimize the hydrophobic mismatch ( Figure 4 ). Tighter packing of the two helices sterically requires a certain crossing angle, and thus a decrease in the crossing angle weakens the peptide–peptide packing.…”

“…This discrepancy may originate from the differences in the treatment of lateral pressure. Yano et al 17 discussed that it is difficult to release lateral pressure for the GXXXG peptides because of the flexibility of the Gly backbone and this effect can inhibit dimerization by cholesterol. Our simulation may not appropriately reproduce the macro properties of the membrane.…”

“…Although cholesterols enhanced the binding of the “host” peptide without the GXXXG motif, they inhibited the binding of the GXXXG peptide. 16 , 17 …”

“…Although cholesterols enhanced the binding of the "host" peptide without the GXXXG motif, they inhibited the binding of the GXXXG peptide. 16,17 The molecular mechanisms of this phenomenon are not fully understood at the atomic level. The atomic details of interactions, such as those in highly complicated, heterogeneous, and dynamical molecular systems, cannot be easily observed through current experimental techniques.…”