Structural effects of extracellular loop mutations in CFTR helical hairpins.

Missense mutations constitute 40% of 2000 cystic fibrosis-phenotypic mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) database, yet the precise mechanism as to how a point mutation can render the entire 1480-residue CFTR protein dysfunctional is not well-understood. Here we investigate the structural effects of two CF-phenotypic mutations - glutamic acid to glycine at position 217 (E217G) and glutamine to arginine at position 220 (Q220R) - in the extracellular (ECL2) loop region of human CFTR using helical hairpin constructs derived from transmembrane (TM) helices 3 and 4 of the first membrane domain. We systematically replaced the wild type (WT) residues E217 and Q220 with the subset of missense mutations that could arise through a single nucleotide change in their respective codons. Circular dichroism spectra of E217G revealed that a significant increase in helicity vs. WT arises in the membrane-mimetic environment of sodium dodecylsulfate (SDS) micelles, while this mutant showed a similar gel shift to WT on SDS-PAGE gels. In contrast, the CF-mutant Q220R showed similar helicity but an increased gel shift vs. WT. These structural variations are compared with the maturation levels of the corresponding mutant full-length CFTRs, which we found are reduced to approx. 50% for E217G and 30% for Q220R vs. WT. The overall results with CFTR hairpins illustrate the range of impacts that single mutations can evoke in intramolecular protein-protein and/or protein-lipid interactions - and the levels to which corresponding mutations in full-length CFTR may be flagged by quality control mechanisms during biosynthesis.

Structural impact of proline mutations in the loop region of an ancestral membrane protein.

The sodium ion-translocating F0 F1 ATP synthase from the bacterium Ilyobacter tartaricus contains a highly stable rotor ring composed of 11 c subunits. The synthase subunit c-in effect an 89-residue peptide that folds into a helical hairpin consisting of two membrane-spanning helices and a cytoplasmic loop-was probed for the structural impact of a series of substitutions with the ß-turn-inducing proline-glycine couplet scanning the hairpin loop (residues 44-51) of the I. tartaricus sequence. We found that a Pro residue in other than the wild type position 47 alters the gross secondary structure of subunit c from α-helical to ß-sheet-like, as well as changing its oligomeric ring structure, and its stability toward heat and trichloroacetic acid treatment. Such a Pro-mediated structural switch in one of the first membrane proteins in life hints to a potential evolutionary connection between α-helical and ß-sheet membrane proteins.

Targeting the membrane fusion event of human respiratory syncytial virus with rationally designed α-helical hairpin traps.

AIMS: Rational design of protein scaffolds with specific biological functions/activities has attracted much attention over the past decades. In the present study, we systematically examine the trimer-of-hairpins (TOH) motif of human respiratory syncytial virus (RSV) F protein, which plays a central role in viral membrane fusion and is a coiled-coil six-helix bundle formed by the antiparallel intermolecular interaction between three N-terminal heptad-repeat (HRN) helices and three C-terminal heptad-repeat (HRC) helices. MAIN METHODS: A rational strategy that integrates dynamics simulation, thermodynamics calculation, fluorescence polarization and circular dichroism is proposed to design HRC-targeted α-helical hairpin traps based on the crystal template of HRN core. KEY FINDINGS: The designed hairpin traps possess a typical helix-turn-helix scaffold that can be stabilized by stapling a disulfide bridge across its helical arms, which are highly structured (helicity >60%) and can mimic the native spatial arrangement of HRN helices in TOH motif to trap the hotspot sites of HRC with effective affinity (Kd is up to 6.4 µM). SIGNIFICANCE: The designed α-helical hairpin traps can be used as lead entities for further developing TOH-disrupting agents to target RSV membrane fusion event and the proposed rational design strategy can be readily modified to apply for other type I viruses.

Tracking the Stepwise Movement of a Membrane-inserting Protein In Vivo.

Proper membrane insertion is crucial for the structure and function of membrane proteins in all cells. The YidC insertase plays an essential role in this process, but the molecular mechanism of YidC-mediated insertion remains unknown. Here we track the stepwise movement of Pf3 coat through YidC by obtaining a series of translational arrested intermediates, and investigate them by thiol cross-linking. We show that Pf3 is inserted as a helical hairpin, i.e., the prospective transmembrane segment moves along the YidC greasy slide comprised of TM3 and TM5, whereas the N-terminal tail transiently folds back into the hydrophilic groove of YidC located in the inner leaflet of the membrane until it is translocated to the periplasm in a subsequent step involving the electrochemical membrane potential. In addition to providing virtual insights about how YidC inserts single-spanning membrane proteins, our study also demonstrates a valuable in vivo tracking method that can be applied to study more complicated substrates or other translocases.

Computational design of the helical hairpin structure of membrane-active antibacterial peptides based on RSV glycoprotein epitope scaffold.

Peptides with helical hairpin conformation have been found to possess potent membrane activity and can be exploited as the structural scaffold of antibacterial peptides (ABPs). Here, we attempted to computationally design membrane-active ABPs based on the helical hairpin motif of respiratory syncytial virus (RSV) glycoprotein epitope. Dynamics simulations revealed that the epitope peptide Rfe (net charge = -1) cannot effectively interact with and permeabilize bacterial membrane due to the electrostatic repulsion between the negatively charged peptide and anionic membrane surface. The native Rfe can be modified to a cationic peptide Rfe-KKK (net charge = +6) by triple mutation of its positively charged residues Glu256, Asp263 and Asp269 to a basic lysine as well as by C-terminal amidation. As might be expected, the modified peptide was able to target membrane surface with a moderate antibacterial potency (MIC = 50-100 µg/ml). Next, a cyclized version of the linear Rfe-KKK was generated, termed as cycRfe-KKK, which was observed to have improved membrane activity and increased antibacterial potency (MIC < 50 µg/ml) by pre-stabilizing amphipathic hairpin conformation of the peptide.

Transmembrane segments form tertiary hairpins in the folding vestibule of the ribosome.

Folding of membrane proteins begins in the ribosome as the peptide is elongated. During this process, the nascent peptide navigates along 100Å of tunnel from the peptidyltransferase center to the exit port. Proximal to the exit port is a "folding vestibule" that permits the nascent peptide to compact and explore conformational space for potential tertiary folding partners. The latter occurs for cytosolic subdomains but has not yet been shown for transmembrane segments. We now demonstrate, using an accessibility assay and an improved intramolecular crosslinking assay, that the helical transmembrane S3b-S4 hairpin ("paddle") of a voltage-gated potassium (Kv) channel, a critical region of the Kv voltage sensor, forms in the vestibule. S3-S4 hairpin interactions are detected at an early stage of Kv biogenesis. Moreover, this vestibule hairpin is consistent with a closed-state conformation of the Kv channel in the plasma membrane.