Reevaluation of hydropathy profiles of voltage-gated ionic channels

A reevaluation of the secondary structure of Na, Ca and K channel proteins led to the following results. Only three segments (S1, S5 and S6) of each repeat are sufficiently hydrophobic to be predicted as transmembrane helices, if a window of 19 amino acids is used. Some of the S2 and S3 segments show higher hydrophobic values when calculated with the window of 9 amino acids and can be predicted as short helices. S4 segments are strongly hydrophilic and cannot be predicted as transmembrane helices. Some of the S2, S3 and S4 segments have an amphipathic character; however, these helices do not span a membrane. A model is proposed where 12 hydrophobic transmembrane helices surround 12 shorter helices, forming a hydrophilic pore. In addition, a unique pattern for S4 segments of voltage-gated channel proteins is defined.     

The potassium channel KcsA and its interaction with the lipid bilayer

The crystal structure of the K⁺ channel KcsA explains many features of ion channel function. The selectivity filter corresponds to a narrow region about 12 Å long and 3 Å wide, lined by carbonyl groups of the peptide backbone, through which a K⁺ ion can only move in a dehydrated form. The selectivity filter opens into a central, water-filled cavity leading to a gating site on the intracellular side of the channel. The channel is tetrameric, each monomer containing two transmembrane a helices, M1 and M2. Helix M1 faces the lipid bilayer and helix M2 faces the central channel pore; the M2 helices participate in subunit-subunit interactions. Helices M1 and M2 in each subunit pack as a pair of antiparallel coils with a heptad repeat, but the M2 helices of neighbouring subunits show fewer interactions, crossing at an angle of about –40°. Trp residues at the ends of the transmembrane helices form clear girdles on the two faces of the membrane, which, together with girdles of charged residues, define a hydrophobic thickness of about 37 Å for the channel. Binding constants for phosphatidylcholines to KcsA vary with fatty acyl chain length, the optimum chain length being C22. A phosphatidylcholine with this chain length gives a bilayer of thickness about 34 Å in the liquid crystalline phase, matching the hydrophobic thickness of the protein. However, a typical biological membrane has a hydrophobic thickness of about 27 Å. Thus either the transmembrane a helices of KcsA are more tilted in the native membrane than they are in the crystal structure, or the channel is under stress in the native membrane. The efficiency of hydrophobic matching between KcsA and the surrounding lipid bilayer is high over the chain length range C10–C24. The channel requires the presence of some anionic lipids for function, and fluorescence quenching studies show the presence of two classes of lipid binding site on KcsA; at one class of site (nonannular sites) anionic phospholipids bind more strongly than phosphatidylcholine, whereas at the other class of site (annular sites) phosphatidylcholines and anionic phospholipids bind with equal affinity.     

Phosphatidylinositol-3,5-bisphosphate lipid-binding-induced activation of the human two-pore channel 2

Mammalian two-pore channels (TPCs) are activated by the low-abundance membrane lipid phosphatidyl-(3,5)-bisphosphate (PI(3,5)P₂) present in the endo-lysosomal system. Malfunction of human TPC1 or TPC2 (hTPC) results in severe organellar storage diseases and membrane trafficking defects. Here, we compared the lipid-binding characteristics of hTPC2 and of the PI(3,5)P₂-insensitive TPC1 from the model plant Arabidopsis thaliana. Combination of simulations with functional analysis of channel mutants revealed the presence of an hTPC2-specific lipid-binding pocket mutually formed by two channel regions exposed to the cytosolic side of the membrane. We showed that PI(3,5)P₂ is simultaneously stabilized by positively charged amino acids (K203, K204, and K207) in the linker between transmembrane helices S4 and S5 and by S322 in the cytosolic extension of S6. We suggest that PI(3,5)P₂ cross links two parts of the channel, enabling their coordinated movement during channel gating.     

Sterol carrier protein-2: structure reveals function

The multiple actions of sterol carrier protein-2 (SCP-2) in intracellular lipid circulation and metabolism originate from its gene and protein structure. The SCP-x/pro-SCP-2 gene is a fusion gene with separate initiation sites coding for 15-kDa pro-SCP-2 (no enzyme activity) and 58-kDa SCP-x (a 3-ketoacyl CoA thiolase). Both proteins share identical cDNA and amino acid sequences for 13-kDa SCP-2 at their C-termini. Cellular 13-kDa SCP-2 derives from complete, posttranslational cleavage of the 15-kDa pro-SCP-2 and from partial posttranslational cleavage of 58-kDa SCP-x. Putative physiological functions of SCP-2 have been proposed on the basis of enhancement of intermembrane lipid transfer (e.g., cholesterol, phospholipid) and activation of enzymes involved in fatty acyl CoA transacylation (cholesterol esters, phosphatidic acid) in vitro, in transfected cells, and in genetically manipulated animals. At least four important SCP-2 structural domains have been identified and related to specific functions. First, the 46-kDa N-terminal presequence present in 58-kDa SCP-x is a 3-ketoacyl-CoA thiolase specific for branched-chain acyl CoAs. Second, the N-terminal 20 amino acid presequence in 15-kDa pro-SCP-2 dramatically modulates the secondary and tertiary structure of SCP-2 as well as potentiating its intracellular targeting coded by the C-terminal peroxisomal targeting sequence. Third, the N-terminal 32 amino acids form an amphipathic a-helical region, one face of which represents a membrane-binding domain. Positively charged amino acid residues in one face of the amphipathic helices allow SCP-2 to bind to membrane surfaces containing anionic phospholipids. Fourth, the hydrophobic faces of the N-terminal amphipathic a helices along with beta strands 4, 5, and helix D form a ligand-binding cavity able to accommodate multiple types of lipids (e. g., fatty acids, fatty acyl CoAs, cholesterol, phospholipids, isoprenoids). Two-dimensional 1H-15N heteronuclear single quantum coherence spectra of both apo-SCP-2 and of the 1:1 oleate-SCP-2 complex, obtained at pH 6.7, demonstrated the homogenous formation of holo-SCP-2. While comparison of the apo- and holoprotein amide fingerprints revealed about 60% of the resonances remaining essentially unchanged, 12 assigned amide residues underwent significant chemical-shift changes upon oleic acid binding. These residues were localized in three regions: the juncture of helices A and B, the mid-section of the beta sheet, and the interface formed by the region of beta strands 4, 5, and helix D. Circular dichroism also showed that these chemical-shift changes, upon oleic acid binding, did not alter the secondary structure of SCP-2. The nuclear magnetic resonance chemical shift difference data, along with mapping of the nearby hydrophobic residues, showed the oleic acid-binding site to be comprised of a pocket created by the face of the beta sheet, helices A and B on one end, and residues associated with beta strands 4, 5, and helix D at the other end of the binding cavity. Furthermore, the hydrophobic nature of the previously ill-defined C-terminus suggested that these 20 amino acids may form a 'hydrophobic cap' which closes around the oleic acid upon binding. Thus, understanding the structural domains of the SCP-x/pro-SCP-2 gene and its respective posttranslationally processed proteins has provided new insights into their functions in intracellular targeting and metabolism of lipids.     

Membrane-traversing mechanism of thyroid hormone transport by monocarboxylate transporter 8

Monocarboxylate transporter 8 (MCT8) mediates thyroid hormone (TH) transport across the plasma membrane in many cell types. In order to better understand its mechanism, we have generated three new MCT8 homology models based on sugar transporters XylE in the intracellular opened (PDB ID: 4aj4) and the extracellular partly occluded (PDB ID: 4gby) conformations as well as FucP (PDB ID: 3o7q) and GLUT3 (PDB ID: 4zwc) in the fully extracellular opened conformation. T₃-docking studies from both sides revealed interactions with His192, His415, Arg445 and Asp498 as previously identified. Selected mutations revealed further transport-sensitive positions mainly at the discontinuous transmembrane helices TMH7 and 10. Lys418 is potentially involved in neutralising the charge of the TH substrate because it can be replaced by charged, but not by uncharged, amino acids. The side chain of Thr503 was hypothesised to stabilise a helix break at TMH10 that undergoes a prominent local shift during the transport cycle. A T503V mutation accordingly affected transport. The aromatic Tyr419, the polar Ser313 and Ser314 as well as the charged Glu422 and Glu423 lining the transport channel have been studied. Based on related sugar transporters, we suggest an alternating access mechanism for MCT8 involving a series of amino acid positions previously and newly identified as critical for transport.     

"Add-on" domains of<Emphasis Type="Italic"> Drosophila</Emphasis> β1,4-<Emphasis Type="Italic">N</Emphasis>-acetylgalactosaminyltransferase B in the stem region and its pilot protein

The glycolipid specific Drosophila melanogaster β1,4-N-acetylgalactosaminyltransferase B (β4GalNAcTB) depends on a zinc finger DHHC protein family member named GalNAcTB pilot (GABPI) for activity and translocation to the Golgi. The six-membrane spanning protein actually lacks the cysteine in the cytoplasmic DHHC motif, displaying DHHS instead. Here we show that the whole conserved region around the DHHS sequence, which is essential for palmitoylation in DHHC proteins, is not required for GABPI to interact with β4GalNAcTB. In contrast, the two luminal loops between transmembrane domain 3–4 and 5–6 contain conserved amino acids, which are crucial for activity. Besides the dependence on GABPI, β4GalNAcTB requires its exceptional short stem region for activity. A few hydrophobic amino acids positioned close to the transmembrane domain are essential for the interaction with GABPI. Along with its catalytic domain, β4GalNAcTB, thus, requires an area in its own stem region and two small luminal loops of GABPI as "add-on" domains. Moreover, some inactive GABPI mutants could be rescued by fusion with β4GalNAcTB, indicating their importance in direct GABPI-β4GalNAcTB interaction.     

Diversity of Cl− Channels

Cl⁻ channels are widely found anion pores that are regulated by a variety of signals and that play various roles. On the basis of molecular biologic findings, ligand-gated Cl⁻ channels in synapses, cystic fibrosis transmembrane conductors (CFTRs) and ClC channel types have been established, followed by bestrophin and possibly by tweety, which encode Ca²⁺-activated Cl⁻ channels. The ClC family has been shown to possess a variety of functions, including stabilization of membrane potential, excitation, cellvolume regulation, fluid transport, protein degradation in endosomal vesicles and possibly cell growth. The molecular structure of Cl⁻ channel types varies from 1 to 12 transmembrane segments. By means of computer-based prediction, functional Cl⁻ channels have been synthesized artificially, revealing that many possible ion pores are hidden in channel, transporter or unidentified hydrophobic membrane proteins. Thus, novel Cl⁻-conducting pores may be occasionally discovered, and evidence from molecular biologic studies will clarify their physiologic and pathophysiologic roles. Received 28 July 2005; received after revision 25 August 2005; accepted 21 September 2005     

Helix insertion into bilayers and the evolution of membrane proteins

Polytopic α-helical membrane proteins cannot spontaneously insert into lipid bilayers without assistance from polytopic α-helical membrane proteins that already reside in the membrane. This raises the question of how these proteins evolved. Our current knowledge of the insertion of α-helices into natural and model membranes is reviewed with the goal of gaining insight into the evolution of membrane proteins. Topics include: translocon-dependent membrane protein insertion, antibiotic peptides and proteins, in vitro insertion of membrane proteins, chaperone-mediated insertion of transmembrane helices, and C-terminal tail-anchored (TA) proteins. Analysis of the E. coli genome reveals several predicted C-terminal TA proteins that may be descendents of proteins involved in pre-cellular membrane protein insertion. Mechanisms of pre-translocon polytopic α-helical membrane protein insertion are discussed.     

Preferential hydrophobic interactions are responsible for a preference of D-amino acids in the aminoacylation of 5'-AMP with hydrophobic amino acids.

We have studied the chemistry of aminoacyl AMP to model reactions at the 3' terminus of aminoacyl tRNA for the purpose of understanding the origin of protein synthesis. The present studies relate to the D, L preference in the esterification of 5'-AMP. All N-acetyl amino acids we studied showed faster reaction of the D-isomer, with a generally decreasing preference for D-isomer as the hydrophobicity of the amino acid decreased. The beta-branched amino acids, Ile and Val, showed an extreme preference for D-isomer. Ac-Leu, the gamma-branched amino acid, showed a slightly low D/L ratio relative to its hydrophobicity. The molecular basis for these preferences for D-isomer is understandable in the light of our previous studies and seems to be due to preferential hydrophobic interaction of the D-isomer with adenine. The preference for hydrophobic D-amino acids can be decreased by addition of an organic solvent to the reaction medium. Conversely, peptidylation with Ac-PhePhe shows a preference for the LL isomer over the DD isomer.     

Conformational change of the extracellular parts of the CFTR protein during channel gating

Cystic fibrosis can be treated by potentiators, drugs that interact directly with the cystic fibrosis transmembrane conductance regulator (CFTR) Cl^? channel to increase its open probability. These substances likely target key conformational changes occurring during channel opening and closing, however, the molecular bases of these conformational changes, and their susceptibility to manipulation are poorly understood. We have used patch clamp recording to identify changes in the three-dimensional organization of the extracellularly accessible parts of the CFTR protein during channel opening and closing. State-dependent formation of both disulfide bonds and Cd²⁺ bridges occurred for pairs of cysteine side-chains introduced into the extreme extracellular ends of transmembrane helices (TMs) 1, 6, and 12. Between each of these three TMs, we found that both disulfide bonds and metal bridges formed preferentially or exclusively in the closed state and that these inter-TM cross-links stabilized the closed state. These results indicate that the extracellular ends of these TMs are close together when the channel is closed and that they separate from each other when the channel opens. These findings identify for the first time key conformational changes in the extracellular parts of the CFTR protein that can potentially be manipulated to control channel activity.     

Triple arginines in the cytoplasmic tail of endomannosidase are not essential for type II membrane topology and Golgi localization

Endomannosidase is a Golgi-localized endoglycosidase, which provides an alternate glucosidase-independent pathway of glucose trimming. Using a protease protection assay we demonstrated that Golgi-endomannosidase is a type II membrane protein. The first 25 amino acids of this protein, containing the cytoplasmic tail and the transmembrane domain, were sufficient for Golgi retention of fused reporter proteins alpha1-antitrypsin or green fluorescent protein. However, shortening or deletion of the transmembrane domain prevented Golgi localization, while lengthening it partially reduced Golgi retention of the enzyme. Substitution of the highly conserved positively charged amino acids within the cytoplasmic tail had neither an effect on type II topology nor on the inherent Golgi localization of the enzyme. In contrast, cytoplasmic tail-deleted rat endomannosidase possessed an inverted topology resulting in endoplasmic reticulum mislocalization. Thus, proper topology rather than the presence of positively charged amino acids in the cytoplasmic tail is critical for Golgi localization of rat endomannosidase.     

SecA,a remarkable nanomachine

Biological cells harbor a variety of molecular machines that carry out mechanical work at the nanoscale. One of these nanomachines is the bacterial motor protein SecA which translocates secretory proteins through the protein-conducting membrane channel SecYEG. SecA converts chemically stored energy in the form of ATP into a mechanical force to drive polypeptide transport through SecYEG and across the cytoplasmic membrane. In order to accommodate a translocating polypeptide chain and to release transmembrane segments of membrane proteins into the lipid bilayer, SecYEG needs to open its central channel and the lateral gate. Recent crystal structures provide a detailed insight into the rearrangements required for channel opening. Here, we review our current understanding of the mode of operation of the SecA motor protein in concert with the dynamic SecYEG channel. We conclude with a new model for SecA-mediated protein translocation that unifies previous conflicting data.     

Localization of Golgi-resident glycosyltransferases

For many glycosyltransferases, the information that instructs Golgi localization is located within a relatively short sequence of amino acids in the N-termini of these proteins comprising: the cytoplasmic tail, the transmembrane spanning region, and the stem region (CTS). Also, one enzyme may be more reliant on a particular region in the CTS for its localization than another. The predominance of these integral membrane proteins in the Golgi has seen these enzymes become central players in the development of membrane trafficking models of transport within this organelle. It is now understood that the means by which the characteristic distributions of glycosyltransferases arise within the subcompartments of the Golgi is inextricably linked to the mechanisms that cells employ to direct the flow of proteins and lipids within this organelle.     

Polysialic acids: potential in improving the stability and pharmacokinetics of proteins and other therapeutics

Naturally occurring polymers of N-acetylneuraminic acid (polysialic acids) are biodegradable, highly hydrophilic and have no known receptors in the body. Following intravenous injection, polysialic acids exhibit long half-lives in the blood circulation and have therefore been proposed as carriers of short-lived drugs and small peptides. In addition, shorter-chain polysialic acids can be used as a means to increase the circulatory half-life of proteins and thus serve as an alternative to the nonbiodegradable monomethoxypoly(ethylene glycol). Recent work has shown that covalent coupling of a low molecular weight polysialic acid (colominic acid) to catalase and asparaginase leads to a considerable increase of enzyme stability in the presence of proteolytic enzymes or blood plasma. Comparative studies in vivo with polysialylated and intact asparaginase revealed that polysialylation significantly increases the half-life of the enzyme. The highly hydrophilic and innocuous nature of polysialic acids renders them suitable as a means to prolong the circulation of peptides and proteins.     

Molecular mechanisms of BK channel activation

Large conductance, Ca²⁺-activated potassium (BK) channels are widely expressed throughout the animal kingdom and play important roles in many physiological processes, such as muscle contraction, neural transmission and hearing. These physiological roles derive from the ability of BK channels to be synergistically activated by membrane voltage, intracellular Ca²⁺ and other ligands. Similar to voltage-gated K⁺ channels, BK channels possess a pore-gate domain (S5–S6 transmembrane segments) and a voltage-sensor domain (S1–S4). In addition, BK channels contain a large cytoplasmic C-terminal domain that serves as the primary ligand sensor. The voltage sensor and the ligand sensor allosterically control K⁺ flux through the pore-gate domain in response to various stimuli, thereby linking cellular metabolism and membrane excitability. This review summarizes the current understanding of these structural domains and their mutual interactions in voltage-, Ca²⁺ - and Mg²⁺ -dependent activation of the channel. Received 25 September 2008; received after revision 23 October 2008; accepted 24 October 2008     

The Rh protein family: gene evolution, membrane biology, and disease association

The Rh (Rhesus) genes encode a family of conserved proteins that share a structural fold of 12 transmembrane helices with members of the major facilitator superfamily. Interest in this family has arisen from the discovery of Rh factor’s involvement in hemolytic disease in the fetus and newborn, and of its homologs widely expressed in epithelial tissues. The Rh factor and Rh-associated glycoprotein (RhAG), with epithelial cousins RhBG and RhCG, form four subgroups conferring upon vertebrates a genealogical commonality. The past decade has heralded significant advances in understanding the phylogenetics, allelic diversity, crystal structure, and biological function of Rh proteins. This review describes recent progress on this family and the molecular insights gleaned from its gene evolution, membrane biology, and disease association. The focus is on its long evolutionary history and surprising structural conservation from prokaryotes to humans, pointing to the importance of its functional role, related to but distinct from ammonium transport proteins.     

Nucleotide binding domains of human CFTR: a structural classification of critical residues and disease-causing mutations.

Defective function of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) causes CF, the most frequent lethal inherited disease among the Caucasian population. The structure of this chloride ion channel includes two nucleotide-binding domains (NBDs), whose ATPase activity controls channel gating. Recently, the experimental structures of mouse and human CFTR NBD1 and our model of the human CFTR NBD1/NBD2 heterodimer have provided new insights into specific structural features of the CFTR NBD dimer. In the present work, we provide a structural classification of CF-causing mutations which may complement the existing functional classification. Our analysis also identified amino acid residues which may play a critical role in interdomain interaction and are located at the NBD1-NBD2 interface or on the surface of the dimer. In particular, a cluster of aromatic amino acids, which includes F508 and straddles the two NBDs, might be directly involved in the interaction of the NBD1/NBD2 heterodimer with the channel-forming membrane-spanning domains.Received 24 May 2005; received after revision 13 June 2005; accepted 18 June 2005     

Abtransport von Schwefelverbindungen aus Bohnenprimärblättern (Phaseolus vulgaris) nach Begasung mit H2 35S

Summary Some unnatural sulphur amino acids can be detected in the phloem of the vascular bundles from Phaseolus vulgaris after the application of H₂ ³⁵S to the primary leaves.     

Preferential hydrophobic interactions are responsible for a preference of D-amino acids in the aminoacylation of 5′-AMP with hydrophobic amino acids

We have studied the chemistry of aminoacyl AMP to model reactions at the 3 terminus of aminoacyl tRNA for the purpose of understanding the origin of protein synthesis. The present studies relate to the D, L preference in the esterification of 5-AMP. All N-acetyl amino acids we studied showed faster reaction of the D-isomer, with a generally decreasing preference for D-isomer as the hydrophobicity of the amino acid decreased. The -branched amino acids, Ile and Val, showed an extreme preference for D-isomer. Ac-Leu, the -branched amino acid, showed a slightly low D/L ratio relative to its hydrophobicity. The molecular basis for these preferences for D-isomer is understandable in the light of our previous studies and seems to be due to preferential hydrophobic interaction of the D-isomer with ademine. The preference for hydrophobic D-amino acids can be decreased by addition of an organic solvent to the reaction medium. Conversely, peptidylation with Ac-PhePhe shows a preference for the LL isomer over the DD isomer.