Marginally hydrophobic transmembrane α‐helices shaping membrane protein folding

Cells have developed an incredible machinery to facilitate the insertion of membrane proteins into the membrane. While we have a fairly good understanding of the mechanism and determinants of membrane integration, more data is needed to understand the insertion of membrane proteins with more complex insertion and folding pathways. This review will focus on marginally hydrophobic transmembrane helices and their influence on membrane protein folding. These weakly hydrophobic transmembrane segments are by themselves not recognized by the translocon and therefore rely on local sequence context for membrane integration. How can such segments reside within the membrane? We will discuss this in the light of features found in the protein itself as well as the environment it resides in. Several characteristics in proteins have been described to influence the insertion of marginally hydrophobic helices. Additionally, the influence of biological membranes is significant. To begin with, the actual cost for having polar groups within the membrane may not be as high as expected; the presence of proteins in the membrane as well as characteristics of some amino acids may enable a transmembrane helix to harbor a charged residue. The lipid environment has also been shown to directly influence the topology as well as membrane boundaries of transmembrane helices—implying a dynamic relationship between membrane proteins and their environment.     

Functional architecture of the CFTR chloride channel

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ATP-binding cassette (ABC) family of membrane transport proteins. CFTR is unique among ABC proteins in that it functions not as an active transporter but as an ATP-gated Cl^? channel. As an ion channel, the function of the CFTR transmembrane channel pore that mediates Cl^? movement has been studied in great detail. On the other hand, only low resolution structural data is available on the transmembrane parts of the protein. The structure of the channel pore has, however, been modeled on the known structure of active transporter ABC proteins. Currently, significant barriers exist to building a unified view of CFTR pore structure and function. Reconciling functional data on the channel with indirect structural data based on other proteins with very different transport functions and substrates has proven problematic. This review summarizes current structural and functional models of the CFTR Cl^? channel pore, including a comprehensive review of previous electrophysiological investigations of channel structure and function. In addition, functional data on the three-dimensional arrangement of pore-lining helices, as well as contemporary hypotheses concerning conformational changes in the pore that occur during channel opening and closing, are discussed. Important similarities and differences between different models of the pore highlight current gaps in our knowledge of CFTR structure and function. In order to fill these gaps, structural and functional models of the membrane-spanning pore need to become better integrated.     

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR): CLOSED AND OPEN STATE CHANNEL MODELS*

The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily. CFTR controls the flow of anions through the apical membrane of epithelia. Dysfunctional CFTR causes the common lethal genetic disease cystic fibrosis. Transitions between open and closed states of CFTR are regulated by ATP binding and hydrolysis on the cytosolic nucleotide binding domains, which are coupled with the transmembrane (TM) domains forming the pathway for anion permeation. Lack of structural data hampers a global understanding of CFTR and thus the development of “rational” approaches directly targeting defective CFTR. In this work, we explored possible conformational states of the CFTR gating cycle by means of homology modeling. As templates, we used structures of homologous ABC transporters, namely TM(287–288), ABC-B10, McjD, and Sav1866. In the light of published experimental results, structural analysis of the transmembrane cavity suggests that the TM(287–288)-based CFTR model could correspond to a commonly occupied closed state, whereas the McjD-based model could represent an open state. The models capture the important role played by Phe-337 as a filter/gating residue and provide structural information on the conformational transition from closed to open channel.     

Large Tilts in Transmembrane Helices Can Be Induced during Tertiary Structure Formation

While early structural models of helix-bundle integral membrane proteins posited that the transmembrane α-helices [transmembrane helices (TMHs)] were orientated more or less perpendicular to the membrane plane, there is now ample evidence from high-resolution structures that many TMHs have significant tilt angles relative to the membrane. Here, we address the question whether the tilt is an intrinsic property of the TMH in question or if it is imparted on the TMH during folding of the protein. Using a glycosylation mapping technique, we show that four highly tilted helices found in multi-spanning membrane proteins all have much shorter membrane-embedded segments when inserted by themselves into the membrane than seen in the high-resolution structures. This suggests that tilting can be induced by tertiary packing interactions within the protein, subsequent to the initial membrane-insertion step.     

Reversible silencing of CFTR chloride channels by glutathionylation

The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation- and ATP-dependent chloride channel that modulates salt and water transport across lung and gut epithelia. The relationship between CFTR and oxidized forms of glutathione is of potential interest because reactive glutathione species are produced in inflamed epithelia where they may be modulators or substrates of CFTR. Here we show that CFTR channel activity in excised membrane patches is markedly inhibited by several oxidized forms of glutathione (i.e., GSSG, GSNO, and glutathione treated with diamide, a strong thiol oxidizer). Three lines of evidence indicate that the likely mechanism for this inhibitory effect is glutathionylation of a CFTR cysteine (i.e., formation of a mixed disulfide with glutathione): (a) channels could be protected from inhibition by pretreating the patch with NEM (a thiol alkylating agent) or by lowering the bath pH; (b) inhibited channels could be rescued by reducing agents (e.g., DTT) or by purified glutaredoxins (Grxs; thiol disulfide oxidoreductases) including a mutant Grx that specifically reduces mixed disulfides between glutathione and cysteines within proteins; and (c) reversible glutathionylation of CFTR polypeptides in microsomes could be detected biochemically under the same conditions. At the single channel level, the primary effect of reactive glutathione species was to markedly inhibit the opening rates of individual CFTR channels. CFTR channel inhibition was not obviously dependent on phosphorylation state but was markedly slowed when channels were first "locked open" by a poorly hydrolyzable ATP analogue (AMP-PNP). Consistent with the latter finding, we show that the major site of inhibition is cys-1344, a poorly conserved cysteine that lies proximal to the signature sequence in the second nucleotide binding domain (NBD2) of human CFTR. This region is predicted to participate in ATP-dependent channel opening and to be occluded in the nucleotide-bound state of the channel based on structural comparisons to related ATP binding cassette transporters. Our results demonstrate that human CFTR channels are reversibly inhibited by reactive glutathione species, and support an important role of the region proximal to the NBD2 signature sequence in ATP-dependent channel opening.     

Converting a marginally hydrophobic soluble protein into a membrane protein

δ-Helices are marginally hydrophobic α-helical segments in soluble proteins that exhibit certain sequence characteristics of transmembrane (TM) helices [Cunningham, F., Rath, A., Johnson, R. M. & Deber, C. M. (2009). Distinctions between hydrophobic helices in globular proteins and TM segments as factors in protein sorting. J. Biol. Chem., 284, 5395-402]. In order to better understand the difference between δ-helices and TM helices, we have studied the insertion of five TM-like δ-helices into dog pancreas microsomal membranes. Using model constructs in which an isolated δ-helix is engineered into a bona fide membrane protein, we find that, for two δ-helices originating from secreted proteins, at least three single-nucleotide mutations are necessary to obtain efficient membrane insertion, whereas one mutation is sufficient in a δ-helix from the cytosolic protein P450BM-3. We further find that only when the entire upstream region of the mutated δ-helix in the intact cytochrome P450BM-3 is deleted does a small fraction of the truncated protein insert into microsomes. Our results suggest that upstream portions of the polypeptide, as well as embedded charged residues, protect δ-helices in globular proteins from being recognized by the signal recognition particle-Sec61 endoplasmic-reticulum-targeting machinery and that δ-helices in secreted proteins are mutationally more distant from TM helices than δ-helices in cytosolic proteins.     

Membrane Insertion of Marginally Hydrophobic Transmembrane Helices Depends on Sequence Context

In mammalian cells, most integral membrane proteins are initially inserted into the endoplasmic reticulum membrane by the so-called Sec61 translocon. However, recent predictions suggest that many transmembrane helices (TMHs) in multispanning membrane proteins are not sufficiently hydrophobic to be recognized as such by the translocon. In this study, we have screened 16 marginally hydrophobic TMHs from membrane proteins of known three-dimensional structure. Indeed, most of these TMHs do not insert efficiently into the endoplasmic reticulum membrane by themselves. To test if loops or TMHs immediately upstream or downstream of a marginally hydrophobic helix might influence the insertion efficiency, insertion of marginally hydrophobic helices was also studied in the presence of their neighboring loops and helices. The results show that flanking loops and nearest-neighbor TMHs are sufficient to ensure the insertion of many marginally hydrophobic helices. However, for at least two of the marginally hydrophobic helices, the local interactions are not enough, indicating that post-insertional rearrangements are involved in the folding of these proteins.     

Traffic pattern of cystic fibrosis transmembrane regulator through the early exocytic pathway

The pathway of transport of the cystic fibrosis transmembrane regulator (CFTR) through the early exocytic pathway has not been examined. In contrast to most membrane proteins that are concentrated during export from the ER and therefore readily detectable at elevated levels in pre-Golgi intermediates and Golgi compartments, wild-type CFTR could not be detected in these compartments using deconvolution immunofluorescence microscopy. To determine the basis for this unusual feature, we analyzed CFTR localization using quantitative immunoelectron microscopy (IEM). We found that wild-type CFTR is present in pre-Golgi compartments and peripheral tubular elements associated with the cis and trans faces of the Golgi stack, albeit at a concentration 2-fold lower than that found in the endoplasmic reticulum (ER). delta F508 CFTR, a mutant form that is not efficiently delivered to the cell surface and the most common mutation in cystic fibrosis, could also be detected at a reduced concentration in pre-Golgi intermediates and peripheral cis Golgi elements, but not in post-Golgi compartments. Our results suggest that the low level of wild-type CFTR in the Golgi region reflects a limiting step in selective recruitment by the ER export machinery, an event that is largely deficient in delta F508. We raise the possibility that novel modes of selective anterograde and retrograde traffic between the ER and the Golgi may serve to regulate CFTR function in the early secretory compartments.     

Targets for cystic fibrosis therapy: proteomic analysis and correction of mutant cystic fibrosis transmembrane conductance regulator

Proteomic analysis has proved to be an important tool for understanding the complex nature of genetic disorders, such as cystic fibrosis (CF), by defining the cellular protein environment (proteome) associated with wild-type and mutant proteins. Proteomic screens identified the proteome of CF transmembrane conductance regulator (CFTR), and provided fundamental information to studies designed for understanding the crucial components of physiological CFTR function. Simultaneously, high-throughput screens for small-molecular correctors of CFTR mutants provided promising candidates for therapy. The majority of CF cases are caused by nucleotide deletions (ΔF508 CFTR; >75%), resulting in CFTR misfolding, or insertion of premature termination codons (～10%), leading to unstable mRNA and reduced levels of truncated dysfunctional CFTR. In this article, we review recent results of proteomic screens, developments in identifying correctors for the most frequent CFTR mutants, and comment on how integration of the knowledge gained from these studies may aid in finding a cure for CF and a number of other genetic disorders.     

Orientational preferences of neighboring helices can drive ER insertion of a marginally hydrophobic transmembrane helix

α-helical integral membrane proteins critically depend on the correct insertion of their transmembrane α helices into the lipid bilayer for proper folding, yet a surprisingly large fraction of the transmembrane α helices in multispanning integral membrane proteins are not sufficiently hydrophobic to insert into the target membrane by themselves. How can such marginally hydrophobic segments nevertheless form transmembrane helices in the folded structure? Here, we show that a transmembrane helix with a strong orientational preference (N(cyt)-C(lum) or N(lum)-C(cyt)) can both increase and decrease the hydrophobicity threshold for membrane insertion of a neighboring, marginally hydrophobic helix. This effect helps explain the "missing hydrophobicity" in polytopic membrane proteins.     

Membrane insertion and topology of the translocating chain-associating membrane protein (TRAM)

The translocating chain-associating membrane protein (TRAM) is a glycoprotein involved in the translocation of secreted proteins into the endoplasmic reticulum (ER) lumen and in the insertion of integral membrane proteins into the lipid bilayer. As a major step toward elucidating the structure of the functional ER translocation/insertion machinery, we have characterized the membrane integration mechanism and the transmembrane topology of TRAM using two approaches: photocross-linking and truncated C-terminal reporter tag fusions. Our data indicate that TRAM is recognized by the signal recognition particle and translocon components, and suggest a membrane topology with eight transmembrane segments, including several poorly hydrophobic segments. Furthermore, we studied the membrane insertion capacity of these poorly hydrophobic segments into the ER membrane by themselves. Finally, we confirmed the main features of the proposed membrane topology in mammalian cells expressing full-length TRAM.     

CFTR haplotypes in northern Iranian population

Background

Cystic fibrosis (CF) is a multiorganic autosomal recessive disorder, caused by mutation in cystic fibrosis transmembrane conductance regulator (CFTR). CF is highly heterogeneous in Iranian population and molecular diagnosis based on direct identification of mutations is not completely efficient. The use of polymorphic intragenic markers not only can facilitate phenotype prediction in prenatal diagnosis by gene tracking, but also can lead to the demonstration of possible associations between haplotypes and specific mutations.

Methods

60 CF patients and 53 fertile normal subjects originating from North of Iran were analyzed for F508del mutation and c.1210-12T(5_9), c.1408A>G and c.744-33GATT(6_8) polymorphisms.

Results

c.1210-12T[7] is the most prevalent allele in normal individuals and CF non-F508del patients with 87.7%and 86.7% frequencies respectively. c.1408A>G survey showed that frequency of allele G and A is nearly equal in both non-F508del CF patients and normal individuals. c.744-33GATT(6_8) study showed that 7 repeat is the most prevalent allele in normal individuals and non-F508del CF patients with 80.2% and 82.1% frequencies respectively. The [c.1408A; c.1210-12T[9]; c.744-33GATT[6]] haplotype was only associated with mutant alleles including F508del.

Conclusions

The allelic distribution and heterozygosity results suggest that c.1408A>G, c.1210-12T(5_9) and c.744-33GATT(6_8) can contribute to carrier detection and prenatal diagnosis of CF in Iranian families with previous history of the disease.     

CFTR型氯离子通道研究进展

囊性纤维化跨膜传导调节因子（CFTR）是一种重要的氯离子通道,突变易引起囊性纤维化病变,故得名。一系列研究表明,CFTR由5个结构域组成：两个跨膜结构域形成氯离子通道;两个核苷酸结合结构域调节通道的开闭;一个调节结构域主要影响氯通道的活动。这些结构域通过协同作用共同控制了氯离子的跨膜流动,而一些突变可以影响细胞功能而导致囊性纤维化的发生。本文通过介绍CFTR基本结构、调节机制、与囊性纤维化病变的关系及针对CFTR的治疗而对CFTR型氯离子通道有一个的全面的理解。     

Introduction of the most common cystic fibrosis mutation (Delta F508) into human P-glycoprotein disrupts packing of the transmembrane segments

The most common mutation in cystic fibrosis (deletion of phenylalanine 508 (DeltaF508) in the cystic fibrosis conductance transmembrane regulator (CFTR) gene) causes defective synthesis of CFTR protein. To understand how this deletion interferes with protein folding, we made the equivalent deletion (DeltaY490) in P-glycoprotein (P-gp). A Cys-less P-gp with cysteines in transmembrane (TM) 4 or TM5 can be cross-linked with a cysteine in TM12. Deleting Tyr(490) in P-gp resulted in an inactive and defectively processed mutant in which no cross-linking between TM4 or TM5 and TM12 was detected. Expression of the DeltaY490 mutant in the presence of a chemical chaperone corrected the processing defect and yielded active P-gp mutants that could be cross-linked between TM4 or TM5 and TM12. Cross-linking between TM4 or TM5 and TM12 was also detected when residues (483)TIAENIRYG(491) in P-gp were replaced with residues (501)TIKENIIFG(509) from CFTR (P-gp/CFTR). Deleting Phe(508) in the P-gp/CFTR chimera, however, caused defective processing of the mutant protein and no detectable cross-linking between TM4 or TM5 and TM12. The processing defect was corrected with a chemical chaperone and yielded active P-gp/CFTR mutant proteins that could be cross-linked. These results show that deletion at residue 490 disrupts packing of the TM segments possibly by affecting interaction between the first nucleotide-binding domain (Tyr(490)) and the first cytoplasmic loop (Glu(184)).     

Evidence that the translocon may function as a hydropathy partitioning filter

Developing a greater understanding of the function of the translocon-and the source of its selectivity for transmembrane helix insertion-are important steps toward deciphering the role of disease-causing mutations in membrane regions. To address these phenomena, we have prepared a library of helix-loop-helix (“hairpin”) constructs derived from helices 3 and 4 of the first membrane domain of CFTR, in which position 232 was mutated individually to each of the 20 commonly-occurring amino acids. Using retention times on a reverse phase-HPLC C18 column to mimic the process of hairpin partitioning, we have quantitatively determined a hydropathy scale in the context of a bona fide membrane protein fragment that correlates to an in vivo hydropathy scale with r = −0.78—a value that rises to r = −0.92 when Asp and Glu are excluded due to protonation effects. Our results provide evidence that the translocon may act as a facilitator in the insertion selection process, effectively allowing the bilayer to “decide” through favorable non-polar solvation whether or not to allow a translocating helix to enter the membrane.     

囊性纤维化跨膜转运调节体氯离子通道——跨上皮离子转运的多功能引擎

囊性纤维化跨膜转运调节体（cystic fibrosis transmembrane conductance regulator,CFTR）是ATP结合转运体超家族（ATP-binding cassette transporter superfamily）的一名特殊成员,因为它是一个具有相当复杂调控机制的氯离子通道。CFTR由五个结构域（domain）组成：两个跨膜结构域（membrane-spanning domains,MSDs）,两个核苷酸结合域（nucleotide-binding domains．NBDs）和一个特殊的调控域（regulatory domain,RD）。MSDs构成一个低电导（6-12pS）的阴离子选择性孔道（pore）,其形状如同不对称的沙漏,胞外小胞内大,狭窄部分为离子筛。两个NBDs组成头尾相对的二聚体,在二聚体之间的接触面上有两个能和ATP结合的位点（位点1和位点2）。CFTR的门控机制是：ATP分子与位点1和2相互作用促使NBD二聚体的结合与解离,从而引起MSDs的构象发生变化进而使通道孔打开和关闭。RD具有多样化的结构,它含有多个磷酸化共有位点（consensus　phosphorylation　sites）。RD的磷酸化促进NBDs与ATP的结合,从而使CFTR得以激活。CFTR通过支架蛋白与其它膜受体以及蛋白激酶、磷酸酶形成大分子信号复合体。在复杂的细胞信号系统参与下,CFTR的功能活动在时间和空间上得到精确的调控。此外,CFTR的活动与细胞代谢有紧密联系：CFTR与代谢酶形成大分子复合体,当细胞能量需求增加时,CFTR活动会受到抑制而使细胞能量得以保存。CFTR广泛分布于机体上皮组织,它通过促进水盐转运而控制上皮细胞分泌物的量与组成。值得注意的是,在呼吸道,CFTR还对机体的防御机制起重要作用。CFTR功能失常严重影响跨上皮离子转运,进而引起或加重某些疾病。     

The cystic fibrosis transmembrane conductance regulator's expanding SNARE interactome

The cystic fibrosis transmembrane conductance regulator (CFTR) interacts with multiple N-ethylmaleimide sensitive factor attachment protein (SNARE) molecules largely via its N-terminal cytoplasmic domain. The earliest known among these SNAREs are the cognate Q-SNARE pair of Syntaxin 1A (STX1A) and SNAP23 on the plasma membrane. These SNAREs affect CFTR chloride channel gating. CFTR exocytosis/recycling in intestinal epithelial cells is dependent on another SNARE located in the apical plasma membrane, STX3. Members of the STX8/STX7/vesicle transport through interaction with t-SNAREs homolog 1b/VAMP8 SNARE complex, which function in early to late endosome/lysosome traffic, are all known to interact with CFTR. Two SNAREs, STX6 and STX16 that function at the trans-Golgi network (TGN), have now been revealed as members of the CFTR SNARE interactome. We summarize here the SNAREs that interact with CFTR and discuss the roles of these SNAREs in the intracellular trafficking of CFTR and CFTR-associated pathophysiology.     

The membrane insertase YidC

The membrane insertases YidC–Oxa1–Alb3 provide a simple cellular system that catalyzes the transmembrane topology of newly synthesized membrane proteins. The insertases are composed of a single protein with 5 to 6 transmembrane (TM) helices that contact hydrophobic segments of the substrate proteins. Since YidC also cooperates with the Sec translocase it is widely involved in the assembly of many different membrane proteins including proteins that obtain complex membrane topologies. Homologues found in mitochondria (Oxa1) and thylakoids (Alb3) point to a common evolutionary origin and also demonstrate the general importance of this cellular process. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Sequence and structural homology among membrane-associated domains of CFTR and certain transporter proteins

A sequence comparison of the two membrane-associated (MA) domains of the cystic fibrosis transmembrane conductance regulator (CFTR), multidrug resistance transporter (MDR), and -factor pheromone export system (STE6) proteins, each of which are believed to contain a total of 12 transmembrane (TM) segments, reveals significant amino acid homology and length conservation in the loop regions that connect individual TM sequences. Similar structural homology is observed between these proteins, hemolysin B (HLYB) and the major histocompatibility-linked peptide transporter, HAM1, the latter two which contain a single MA domain composed of six TM segments. In addition, there are specific sequences that are conserved within the TM segments of the five different membrane proteins. This observation suggests that the folding topologies of the MA domains of MDR, STE6, and CFTR in the plasma membrane are likely to be very similar. The sequence analysis also reveals that there are three characteristic motifs (a pair of aromatic residues, LTLXXXXXXP and GXXL) that are conserved in MDR, STE6, HLYB, HAM1, but not in CFTR. We propose that although CFTR may be evolutionarily related to these other membrane proteins, it belongs to a separate subclass.     

Intra-Helical Salt Bridge Contribution to Membrane Protein Insertion

Salt bridges between negatively (D, E) and positively charged (K, R, H) amino acids play an important role in protein stabilization. This has a more prevalent effect in membrane proteins where polar amino acids are exposed to a hydrophobic environment. In transmembrane (TM) helices the presence of charged residues can hinder the insertion of the helices into the membrane. It is possible that the formation of salt bridges could decrease the cost of membrane integration. However, the presence of intra-helical salt bridges in TM domains and their effect on insertion has not been properly studied yet. In this work, we show that potentially salt-bridge forming pairs are statistically over-represented in TM-helices. We then selected some candidates to experimentally determine the contribution of these electrostatic interactions to the translocon-assisted membrane insertion process. Using both in vitro and whole cell systems, we confirm the presence of intra-helical salt bridges in TM segments during biogenesis and determined that they contribute ～0.5 kcal/mol to the apparent free energy of membrane insertion (ΔG_app). Our observations suggest that salt bridge interactions can be stabilized during translocon-mediated insertion and thus could be relevant to consider for the future development of membrane protein prediction software.