Transmembrane Segments Form Tertiary Hairpins in the Folding Vestibule of the Ribosome
LiWeiTu
Department of Physiology, University of Pennsylvania, Philadelphia., , PA 19104-6085, USA
⁎Corresponding author.
Edited by R. Ruben L. Gonzalez J Mol Biol 2013
Abstract
Folding of membrane proteins begins in the ribosome as the peptide is elongated. During this process, the nascent peptide navigates along 100 Å 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.
AbbreviationsERendoplasmic reticulumPTCpeptidyl transferase centerVSDvoltage sensor domainPEG-MALpolyethylene glycol maleimidePDMphenyldimaleimideBMHbismaleimide hexaneLDSlithium dodecyl sulfatePBSphosphate-buffered saline

Keywordsfolding of nascent peptidespotassium channel voltage sensor biogenesistransmembrane helical hairpinPEG-MALintramolecular crosslinking

Discussion
Hairpin interactions may be characterized as an equilibrium between folded and unfolded species. The equilibrium constant (affinity) will be different for different peptide sequences at different locations within and without the ribosomal tunnel. If the affinity is high, then the dwell time of the peptide in the hairpin configuration will be longer, leading to a higher intra-hairpin Pxlink. Additionally, conformational flexibility and length of the intervening hairpin loop may be factors in hairpin formation in the vestibule. An entropic window, in which a nascent peptide explores conformational space and samples potential tertiary partners, has been described for some cytosolic hairpin interactions in the ribosomal folding vestibule [17]. We now show that transmembrane helical hairpins can also form in this vestibule. The hairpin investigated herein is part of the voltage sensor (S1–-S4), a modular and functionally critical unit of voltage-sensing proteins [24,25,43].

Secondary structure of VSD segments
The C-terminal region of S4, in the folding vestibule, is compact and likely helical, even in the absence of S3 and all other upstream elements of Kv1.3. Both accessibility and crosslinking results support this conclusion. Transmembrane segments S1, S2, S3, and S4 each acquire their individual secondary structures before completely emerging from the ribosomal exit and likely retain this structure in the translocon [3,8] (this paper). These preformed secondary structures may facilitate tertiary folding of the VSD, whether inside the vestibule or in a more distal compartment such as the translocon or lipid bilayer, assisting membrane insertion. Moreover, such folding capacity might underlie coupled secondary–-tertiary folding for helical hairpins. Vectorial (N-terminus to C-terminus) secondary folding of an entire transmembrane segment occurs in the absence of microsomal membranes when C-terminal residues reside in the folding vestibule [3] (this paper). Upon emergence from the vestibule, a transmembrane helix has been suggested to be stabilized by the signal recognition particleSRP, even in the absence of microsomal membranes [15].

Tertiary interactions of VSD segments
S3–-S4 hairpin interactions in the Kv monomer are detected in the folding vestibule, that isi.e., at an early stage in Kv biogenesis. Although precedents exist, both experimental and theoretical, for tertiary interactions within soluble domains in the vestibule [17–20], our results are the first demonstrating that transmembrane tertiary interactions can occur in the vestibule. To do this, we used both accessibility and crosslinking assays. In the latter case, three important considerations should be noted: (1) apparent side-chain distances between cysteines on opposite hairpin arms are distinct from backbone distances between the hairpin arms, (2) a hairpin may be either parallel, or skewed in a crossed configuration, and (3) a lack of crosslinking with PDM (6 Å) may be due to the rigidity of the probe, as well as its short linker length. Crosslinking will depend on the position of the hairpin backbones (arms), the orientations of the cysteine side chains, and consequently on the linker length and flexibility of the bifunctional maleimide. Whereas PDM does not crosslink residues within S3–-S4, BMH, a longer (13 Å) and more flexible molecule, can crosslink specific residues within S3–-S4. We therefore suggest that the helix–-turn–-helix has distance constraints for the paired cysteine side chains that limit the use of PDM. Four cysteine pairs, 279C/323C, 280C/324C, 289C/310C, and 290C/311C, crosslink, which is not consistent with predictions derived from a parallel orientation of the S3–-S4 hairpin and may implicate a skewed hairpin arrangement. Bimodal crosslinking implies two populations, one that is crosslinked and one that is not. Each could contain several conformational states. Based on the high efficiency of maleimide labeling (Ppeg, Table 1, Ppeg), 0.4–-0.5 crosslinking from a bimodal distribution indicates that 40–-50% of the peptide is in a conformation(s) characterized by near proximity of the specified cysteine pair, as in a hairpin conformation. This hairpin in the vestibule resembles the conformation in the ER membrane (Fig. 7), suggesting that the vestibule hairpin is a bona fide conformation along the biogenic pathway; , however, the precise orientation of such a hairpin may eventually be fine-tuned in the context of the fully assembled tetramer. The cysteine pairs I289C/I310C and D280C/K324C in Kv1.3 (≥ 0.4 Pxlink) correspond to Shaker residues that are crosslinkable in the closed state of the mature channel in the plasma membrane [5] and produce functional consequences when mutated [42]. Thus, the nascent Kv paddle structure in the vestibule may represent a physiologically relevant functional state of the mature channel.
Although S3–-S4 have an intrinsic affinity and crosslink in the absence of S2 (Fig. 6), S2 may nonetheless serve a role in biogenesis of the VSD. At least one important role is targeting the Kv nascent peptide to the ER membrane [40]. However, But S2 may serve additional roles. First, S2 may interact with chaperones in the vicinity of the tunnel's exit port and/or translocon to align the nascent VSD for efficient integration. Facilitation of VSD insertion/integration into the bilayer could also be mediated by S2's sequence and consequent integration propensity. S2 integrates well into the ER membrane [40]. In contrast, the isolated S3 segment is a secretory peptide and does not integrate into the membrane [40]. S4 manifests only 50% integration efficiency [40,44] and S3–-S4 does not integrate efficiently [40]. Thus, a strong integrator such as S2 would confer the required driving force for insertion of the VSD.
Yet another possible role for S2 is as a translocon retention signal [28–30]. An increased dwell time of S2 in the translocon would allow the C-terminal segments of the VSD (S3–-S4) to partner correctly (prefold) and form a cooperative biogenic unit (S2-S3-S4) for efficient membrane integration of Kv VSD transmembrane helices [40,45,46]. It also participates in electrostatic interactions between charged side chains within S2, S3, and S4 that drive the gating of mature Kv channels at the plasma membrane in response to membrane potential [4,42,47,48]. While S2 plays a role in both these functions, the roles need not be the same. These salt bridges may not be critical for promotion of initial folding of the hairpin in the ribosome–-nascent chain complex, as charge-neutralizing (e.g., D280S, R321C, R260A) and charge-reversing (e.g., D280R) mutations failed to alter Fpeg (Tu and Deutsch, data not shown). Nonetheless, salt bridges within the VSD may exist later in the translocon and, if so, their formation could be facilitated by prior hairpin formation in the adjacent folding vestibule of the ribosome.

Why does early hairpin biogenesis matter?
Hairpin formation in the tunnel raises an interesting conundrum, namely, how will such a bulky unit proceed into the translocon and thereafter into the lipid bilayer of the ER? Moreover, we may ask if there is an advantage for the nascent peptide to acquire a helix–-turn–-helix motif at this stage of biogenesis. The cytosolic entryway to the translocon (20–-25 Å; Ref. [49]) appears to be dynamic and large enough to accommodate a hairpin. It widens upon engagement of the translocon with a ribosome–-nascent chain complex [50,51]. This expansion also includes the narrowest passage (5–-8 Å), a ring of bulky hydrophobic residues. Secondary folds are maintained as a nascent Kv chain transits from folding vestibule to translocon [3], and specific crosslinking of S3–-S4 is similar in the folding vestibule and ER membrane. Thus, we suggest that, for a nascent chain entering the upper cavity of the translocon, between the putative canaliculus and the ribosomal exit port, the prefolded hairpin signals expansion of the canaliculus. This could accommodate efficient passage of the nascent chain through the translocon. As such, the folding vestibule would constitute an operational antechamber where reorientation and refolding can be optimized for efficient integration. Moreover, successive transmembrane segments, for examplee.g., S2, might serve to keep the translocon canaliculus patent throughout the peptide's journey from translocon to bilayer. These provocative hypotheses require further experimental investigation.
Efficient protein folding and packing is key to understanding not only biogenesis but also trafficking mechanisms, because ER export and degradation signals are modulated by how the protein is folded and packed and will alter levels of expression of Kv channels at the cell surface, which leads to pathology. In addition to defining fundamental steps in cotranslational folding, these studies begin to address folding events in Kv formation of a critical region of the voltage sensor.