N-terminal Transmembrane Domain Research Articles

Ebolavirus Replication and Tetherin/BST-2

Ebolavirus (EBOV) is an enveloped, non-segmented, negative-stranded RNA virus, which consists of five species: Zaire ebolavirus, Sudan ebolavirus, Tai Forest ebolavirus, Bundibugyo ebolavirus, and Reston ebolavirus. EBOV causes a lethal hemorrhagic fever in both humans and non-human primates. The EBOV RNA genome encodes seven viral proteins: NP, VP35, VP40, GP, VP30, VP24, and L. VP40 is a matrix protein and is essential for virus assembly and release from host cells. Expression of VP40 in mammalian cells is sufficient to generate extracellular virus-like particles, which resemble authentic virions. Tetherin/BST-2, which was identified as an effective cellular factor that prevents human immunodeficiency virus-1 release in the absence of viral accessory protein Vpu, has been reported to inhibit ZEBOV VP40-induced VLP release. Tetherin/BST-2 appears to inhibit virus release by physically tethering viral particles to the cell surface via its N-terminal transmembrane domain and C-terminal glycosylphosphatidylinositol anchor. Replication of ZEBOV is not inhibited by tetherin/BST-2 expression, although tetherin/BST-2 was expected to inhibit EBOV release as well as VLP release. Recently, it was reported that viral glycoprotein of EBOV, GP, antagonizes the antiviral effect of tetherin/BST-2. However, the mechanism by which GP antagonizes the antiviral activity of tetherin/BST-2 and whether GP of the other EBOV species function as antagonists of tetherin/BST-2 remain unclear.

Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a plant cell wall pectin biosynthetic homogalacturonan:galacturonosyltransferase complex

Plant cell wall pectic polysaccharides are arguably the most complex carbohydrates in nature. Progress in understanding pectin synthesis has been slow due to its complex structure and difficulties in purifying and expressing the low-abundance, Golgi membrane-bound pectin biosynthetic enzymes. Arabidopsis galacturonosyltransferase (GAUT) 1 is an α-1,4-galacturonosyltransferase (GalAT) that synthesizes homogalacturonan (HG), the most abundant pectic polysaccharide. We now show that GAUT1 functions in a protein complex with the homologous GAUT7. Surprisingly, although both GAUT1 and GAUT7 are type II membrane proteins with single N-terminal transmembrane-spanning domains, the N-terminal region of GAUT1, including the transmembrane domain, is cleaved in vivo. This raises the question of how the processed GAUT1 is retained in the Golgi, the site of HG biosynthesis. We show that the anchoring of GAUT1 in the Golgi requires association with GAUT7 to form the GAUT1:GAUT7 complex. Proteomics analyses also identified 12 additional proteins that immunoprecipitate with the GAUT1:GAUT7 complex. This study provides conclusive evidence that the GAUT1:GAUT7 complex is the catalytic core of an HG:GalAT complex and that cell wall matrix polysaccharide biosynthesis occurs via protein complexes. The processing of GAUT1 to remove its N-terminal transmembrane domain and its anchoring in the Golgi by association with GAUT7 provides an example of how specific catalytic domains of plant cell wall biosynthetic glycosyltransferases could be assembled into protein complexes to enable the synthesis of the complex and developmentally and environmentally plastic plant cell wall.

Overexpression, purification, crystallization and preliminary X-ray crystallographic analysis of the periplasmic domain of outer membrane protein A from Acinetobacter baumannii

Outer membrane protein A from Acinetobacter baumannii (AbOmpA) is a major outer membrane protein and a key player in the bacterial pathogenesis that induces host cell death. AbOmpA is presumed to consist of an N-terminal β-barrel transmembrane domain and a C-terminal periplasmic OmpA-like domain. In this study, the recombinant C-terminal periplasmic domain of AbOmpA was overexpressed in Escherichia coli, purified and crystallized using the vapour-diffusion method. A native diffraction data set was collected to a resolution of 2.0 Å using synchrotron radiation. The space group of the crystal was P2(1), with unit-cell parameters a = 58.24, b = 98.59, c = 97.96 Å, β = 105.92°. The native crystal contained seven or eight molecules per asymmetric unit and had a calculated Matthews coefficient of 2.93 or 2.56 Å(3) Da(-1).

Probing the oligomeric state and interaction surfaces of Fukutin-I in dilauroylphosphatidylcholine bilayers

Fukutin-I is localised to the endoplasmic reticulum or Golgi apparatus within the cell, where it is believed to function as a glycosyltransferase. Its localisation within the cell is thought to to be mediated by the interaction of its N-terminal transmembrane domain with the lipid bilayers surrounding these compartments, each of which possesses a distinctive lipid composition. However, it remains unclear at the molecular level how the interaction between the transmembrane domains of this protein and the surrounding lipid bilayer drives its retention within these compartments. In this work, we employed chemical cross-linking and fluorescence resonance energy transfer measurements in conjunction with multiscale molecular dynamics simulations to determine the oligomeric state of the protein within dilauroylphosphatidylcholine bilayers to identify interactions between the transmembrane domains and to ascertain any role these interactions may play in protein localisation. Our studies reveal that the N-terminal transmembrane domain of Fukutin-I exists as dimer within dilauroylphosphatidylcholine bilayers and that this interaction is driven by interactions between a characteristic TXXSS motif. Furthermore residues close to the N-terminus that have previously been shown to play a key role in the clustering of lipids are shown to also play a major role in anchoring the protein in the membrane.Electronic supplementary materialThe online version of this article (doi:10.1007/s00249-011-0773-5) contains supplementary material, which is available to authorized users.

Misalignment of PLP/DM20 Transmembrane Domains Determines Protein Misfolding in Pelizaeus–Merzbacher Disease

A large number of genetic diseases have been associated with truncated or misfolded membrane proteins trapped in the endoplasmic reticulum (ER). In the ER, they activate the unfolded protein response, which can trigger cell death. Hence, a better understanding of protein misfolding features might help in developing novel therapies. Here, we have studied the molecular basis of Pelizaeus-Merzbacher disease, a leukodystrophy defined by mutations of the PLP1 gene and ER retention of two encoded tetraspan myelin proteins, PLP and DM20. In mouse oligodendroglial cells, mutant isoforms of PLP/DM20 with fewer than all four transmembrane (TM) domains are fully ER retained. Surprisingly, a truncated PLP with only two N-terminal TM domains shows normal cell-surface expression when coexpressed with a second truncated PLP harboring the two C-terminal TM domains. This striking ability to properly self-align the TM domains is disease relevant, as shown for the smaller splice isoform DM20. Here, the increased length of TM domain 3 allows for compensation of the effect of several PLP1 point mutations that impose a conformational constraint onto the adjacent extracellular loop region. We conclude that an important determinant in the quality control of polytopic membrane proteins is the free alignment of their TM domains.

The membrane topological analysis of 3β-hydroxysteroid-Δ24 reductase (DHCR24) on endoplasmic reticulum

DHCR24 encodes 3β-hydroxysteroid-Δ24 reductase, catalyzing the conversion of desmosterol to cholesterol. Our previous study demonstrated that DHCR24 exerts an anti-apoptotic function as a reactive oxygen species (ROS) scavenger, for which it needs its FAD-binding domain. The membrane topology of DHCR24 on endoplasmic reticulum (ER) and the functional significance of its FAD-binding domain are not completely understood. Based on the structure predicted by bioinformatics, we studied the membrane topology of DHCR24 in murine neuroblastoma cells (N2A), using the fluorescent protease protection (FPP) technique. We showed that full-length DHCR24 is localized to the membrane of ER, whereas the predicted transmembrane (TM) domain-deleted DHCR24 mutation is localized to the cytoplasm. The change of DHCR24 localization suggests that the N-terminal TM domain is essential for the ER membrane targeting of DHCR24. The FPP assay demonstrated the membrane topology of DHCR24 with an N-terminal luminal/C-terminal cytoplasmic orientation. Measurement of intracellular ROS using H(2)DCFDA revealed that the ROS levels of cells infected by plasmids driving expression of full-length DHCR24 or the TM domain-deleted DHCR24 mutation after H(2)O(2) exposure were lower than those of control cells, suggesting that the ER membrane targeting of DHCR24 is not required for its enzymatic ROS scavenging activity. Confocal fluorescence microscopy revealed that the DHCR24-overexpressed cells were protected from apoptosis in response to oxidative stress, which was accompanied by a decrease in DHCR24 content on the ER and activation of caspase-3, suggesting that the anti-apoptotic function of DHCR24 is associated with its cleavage by caspase.

Structure of the Newcastle disease virus hemagglutinin-neuraminidase (HN) ectodomain reveals a four-helix bundle stalk

The paramyxovirus hemagglutinin-neuraminidase (HN) protein plays multiple roles in viral entry and egress, including binding to sialic acid receptors, activating the fusion (F) protein to activate membrane fusion and viral entry, and cleaving sialic acid from carbohydrate chains. HN is an oligomeric integral membrane protein consisting of an N-terminal transmembrane domain, a stalk region, and an enzymatically active neuraminidase (NA) domain. Structures of the HN NA domains have been solved previously; however, the structure of the stalk region has remained elusive. The stalk region contains specificity determinants for F interactions and activation, underlying the requirement for homotypic F and HN interactions in viral entry. Mutations of the Newcastle disease virus HN stalk region have been shown to affect both F activation and NA activities, but a structural basis for understanding these dual affects on HN functions has been lacking. Here, we report the structure of the Newcastle disease virus HN ectodomain, revealing dimers of NA domain dimers flanking the N-terminal stalk domain. The stalk forms a parallel tetrameric coiled-coil bundle (4HB) that allows classification of extensive mutational data, providing insight into the functional roles of the stalk region. Mutations that affect both F activation and NA activities map predominantly to the 4HB hydrophobic core, whereas mutations that affect only F-protein activation map primarily to the 4HB surface. Two of four NA domains interact with the 4HB stalk, and residues at this interface in both the stalk and NA domain have been implicated in HN function.

ATP activates ATP-sensitive potassium channels composed of mutant sulfonylurea receptor 1 and Kir6.2 with diminished PIP2 sensitivity

ATP-sensitive potassium (KATP) channels are inhibited by ATP and activated by phosphatidylinositol 4,5-bisphosphate (PIP2). Both channel subunits Kir6.2 and sulfonylurea receptor 1 (SUR1) contribute to gating: while Kir6.2 interacts with ATP and PIP2, SUR1 enhances sensitivity to both ligands. Recently, we showed that a mutation, E128K, in the N-terminal transmembrane domain of SUR1 disrupts functional coupling between SUR1 and Kir6.2, leading to reduced ATP and PIP2 sensitivities resembling channels formed by Kir6.2 alone. We show here that when E128K SUR1 was co-expressed with Kir6.2 mutants known to disrupt PIP2 gating, the resulting channels were surprisingly stimulated rather than inhibited by ATP. To explain this paradoxical gating behavior, we propose a model in which the open state of doubly mutant channels is highly unstable; ATP binding induces a conformational change in ATP-unbound closed channels that is conducive to brief opening when ATP unbinds, giving rise to the appearance of ATP-induced stimulation.

Desensitization of α7 Nicotinic Receptor Is Governed by Coupling Strength Relative to Gate Tightness

Binding of a neurotransmitter to its membrane receptor opens an integral ion conducting pore. However, prolonged exposure to the neurotransmitter drives the receptor to a refractory state termed desensitization, which plays an important role in shaping synaptic transmission. Despite intensive research in the past, the structural mechanism of desensitization is still elusive. Using mutagenesis and voltage clamp in an oocyte expression system, we provide several lines of evidence supporting a novel hypothesis that uncoupling between binding and gating machinery is the underlying mechanism for α7 nicotinic receptor (nAChR) desensitization. First, the decrease in gate tightness was highly correlated to the reduced desensitization. Second, nonfunctional mutants in three important coupling loops (loop 2, loop 7, and the M2-M3 linker) could be rescued by a gating mutant. Furthermore, the decrease in coupling strength in these rescued coupling loop mutants reversed the gating effect on desensitization. Finally, coupling between M1 and hinge region of the M2-M3 linker also influenced the receptor desensitization. Thus, the uncoupling between N-terminal domain and transmembrane domain, governed by the balance of coupling strength and gate tightness, underlies the mechanism of desensitization for the α7 nAChR.

Purification, crystallization and preliminary X-ray crystallographic analysis of the C-terminal cytoplasmic domain of FlhB from Salmonella typhimurium.

FlhB is a key protein in the regulation of protein export by the bacterial flagellar secretion system. It is composed of two domains: an N-terminal transmembrane domain and a C-terminal cytoplasmic domain (FlhBc). FlhBc from Salmonella typhimurium has been successfully crystallized using the vapour-diffusion method. The crystals diffracted to 2.45 Å resolution and belonged to space group P4(2)2(1)2, with unit-cell parameters a=b=49.06, c=142.94 Å. A selenomethionine-containing variant of FlhBc has also been crystallized in the same space group and was used for initial phase calculation by the multiwavelength anomalous dispersion (MAD) method.

The E3 CR1-gamma gene in human adenoviruses associated with epidemic keratoconjunctivitis

Human adenovirus species D type 37 (HAdV-D37) is an important etiologic agent of epidemic keratoconjunctivitis. Annotation of the whole genome revealed an open reading frame (ORF) in the E3 transcription unit predicted to encode a 31.6kDa protein. This ORF, also known as CR1-γ, is predicted to be an integral membrane protein containing N-terminal signal sequence, luminal, transmembrane, and cytoplasmic domains. HAdV-D19 (C), another viral pathogen causing epidemic keratoconjunctivitis, contains an ORF 100% identical to its HAdV-D37 homologue but only 66% identical to other HAdV-D homologues. Kinetics of RNA expression and confirmation of splicing to the adenovirus tripartite leader sequence suggest a role for the protein product of CR1-γ in the late stages of the viral replication cycle. Confocal microscopy is consistent with expression in the cytoplasm. Sequence analysis reveals a hypervariable luminal domain and a conserved cytoplasmic domain. The luminal domain is predicted to contain multiple N-glycosylation sites. The cytoplasmic domain contains a putative protein kinase C phosphorylation site and potential YXXϕ and dileucine (LL) motifs suggesting a potential role in modification of host proteins.

Glycine Dimerization Motif in the N-terminal Transmembrane Domain of the High Density Lipoprotein Receptor SR-BI Required for Normal Receptor Oligomerization and Lipid Transport

Scavenger receptor class B, type I (SR-BI), a CD36 superfamily member, is an oligomeric high density lipoprotein (HDL) receptor that mediates negatively cooperative HDL binding and selective lipid uptake. We identified in the N-terminal transmembrane (N-TM) domain of SR-BI a conserved glycine dimerization motif, G(15)X(2)G(18)X(3)AX(2)G(25), of which the submotif G(18)X(3)AX(2)G(25) significantly contributes to homodimerization and lipid uptake activity. SR-BI variants were generated by mutations (single or multiple Gly → Leu substitutions) or by replacing the N-TM domain with those from other CD36 superfamily members containing (croquemort) or lacking (lysosomal integral membrane protein (LIMP) II) this glycine motif (chimeras). None of the SR-BI variants exhibited altered surface expression (based on antibody binding) or HDL binding. However, the G15L/G18L/G25L triple mutant exhibited reductions in cell surface homo-oligomerization (>10-fold) and the rate of selective lipid uptake (∼ 2-fold). Gly(18) and Gly(25) were necessary for normal lipid uptake activity of SR-BI and the SR-BI/croquemort chimera. The lipid uptake activity of the glycine motif-deficient SR-BI/LIMP II chimera was low but could be increased by introducing glycines at positions 18 and 25. The rate of lipid uptake mediated by SR-BI/LIMP II chimeras was proportional to the extent of receptor oligomerization. Thus, the glycine dimerization motif G(18)X(3)AX(2)G(25) in the N-TM domain of SR-BI contributes substantially to the homo-oligomerization and lipid transport activity of SR-BI but does not influence the negative cooperativity of HDL binding. Oligomerization-independent binding cooperativity suggests that classic allostery is not involved and that the negative cooperativity is probably the consequence of a "lattice effect" (interligand steric interference accompanying binding to adjacent receptors).

β2-Adrenergic Ion-Channel Coupled Receptors as Conformational Motion Detectors

Ion Channel-Coupled Receptors (ICCRs) are artificial proteins comprised of a G protein-coupled receptor and a fused ion channel, engineered to couple channel gating to ligand binding. These novel biological objects have potential use in drug screening and functional characterization, in addition to providing new tools in the synthetic biology repertoire as synthetic K+-selective ligand-gated channels. The ICCR concept was previously validated with fusion proteins between the K+ channel Kir6.2 and muscarinic M2 or dopaminergic D2 receptors. Here, we extend the concept to the distinct, longer β2-adrenergic receptor which, unlike M2 and D2 receptors, displayed barely detectable surface expression in our Xenopus oocyte expression system and did not couple to Kir6.2 when unmodified. Here, we show that a Kir6.2-binding protein, the N-terminal transmembrane domain of the sulfonylurea receptor, can greatly increase plasma membrane expression of β2 constructs. We then demonstrate how engineering of both receptor and channel can produce β2-Kir6.2 ICCRs. Specifically, removal of 62–72 residues from the cytoplasmic C-terminus of the receptor was required to enable coupling, suggesting that ligand-dependent conformational changes do not efficiently propagate to the distal C-terminus. Characterization of the β2 ICCRs demonstrated that full and partial agonists had the same coupling efficacy, that an inverse agonist had no effect and that the stabilizing mutation E122 W reduced agonist-induced coupling efficacy without affecting affinity. Because the ICCRs are expected to report motions of the receptor C-terminus, these results provide novel insights into the conformational dynamics of the β2 receptor.

AtFH8 Is Involved in Root Development under Effect of Low-Dose Latrunculin B in Dividing Cells

Formins have been paid much attention for their potent nucleating activity. However, the connection between the in vivo functions of AtFHs (Arabidopsis thaliana formin homologs) and their effects on actin organization is poorly understood. In this study, we characterized the bundling activity of AtFH8 in vitro and in vivo. Biochemical analysis showed that AtFH8(FH1FH2) could form dimers and bundle preformed actin filaments or induce stellar structures during actin polymerization. Expression of truncated forms of AtFH8 and immunolocalization analysis showed that AtFH8 localized primarily to nuclear envelope in interphase and to the new cell wall after cytokinesis, depending primarily on its N-terminal transmembrane domain. GUS histochemical staining showed AtFH8 was predominantly expressed in Arabidopsis root meristem, vasculature, and outgrowth points of lateral roots. The primary root growth and lateral root initiation of atfh8 could be decreased by latrunculin B (LatB). Analysis of the number of dividing cells in Arabidopsis root tips showed that much fewer dividing cells in Lat B-treated atfh8 plants than wild-type plants, which indicates that AtFH8 was involved in cell division. Actin cytoskeleton in root meristem of atfh8-1 was more sensitive to LatB treatment than that of wild-type. Altogether, our results indicate that AtFH8 is an actin filament nucleator and bundler that functions in cell division and root development.

Solution Structure of the Guanine Nucleotide-binding STAS Domain of SLC26-related SulP Protein Rv1739c from Mycobacterium tuberculosis

The structure and intrinsic activities of conserved STAS domains of the ubiquitous SulP/SLC26 anion transporter superfamily have until recently remained unknown. Here we report the heteronuclear, multidimensional NMR spectroscopy solution structure of the STAS domain from the SulP/SLC26 putative anion transporter Rv1739c of Mycobacterium tuberculosis. The 0.87-Å root mean square deviation structure revealed a four-stranded β-sheet with five interspersed α-helices, resembling the anti-σ factor antagonist fold. Rv1739c STAS was shown to be a guanine nucleotide-binding protein, as revealed by nucleotide-dependent quench of intrinsic STAS fluorescence and photoaffinity labeling. NMR chemical shift perturbation analysis partnered with in silico docking calculations identified solvent-exposed STAS residues involved in nucleotide binding. Rv1739c STAS was not an in vitro substrate of mycobacterial kinases or anti-σ factors. These results demonstrate that Rv1739c STAS binds guanine nucleotides at physiological concentrations and undergoes a ligand-induced conformational change but, unlike anti-σ factor antagonists, may not mediate signals via phosphorylation.

N-terminal transmembrane domain of SUR1 controls gating of Kir6.2 by modulating channel sensitivity to PIP2

Functional integrity of pancreatic adenosine triphosphate (ATP)-sensitive potassium (KATP) channels depends on the interactions between the pore-forming potassium channel subunit Kir6.2 and the regulatory subunit sulfonylurea receptor 1 (SUR1). Previous studies have shown that the N-terminal transmembrane domain of SUR1 (TMD0) interacts with Kir6.2 and is sufficient to confer high intrinsic open probability (Po) and bursting patterns of activity observed in full-length KATP channels. However, the nature of TMD0–Kir6.2 interactions that underlie gating modulation is not well understood. Using two previously described disease-causing mutations in TMD0 (R74W and E128K), we performed amino acid substitutions to study the structural roles of these residues in KATP channel function in the context of full-length SUR1 as well as TMD0. Our results revealed that although R74W and E128K in full-length SUR1 both decrease surface channel expression and reduce channel sensitivity to ATP inhibition, they arrive there via distinct mechanisms. Mutation of R74 uniformly reduced TMD0 protein levels, suggesting that R74 is necessary for stability of TMD0. In contrast, E128 mutations retained TMD0 protein levels but reduced functional coupling between TMD0 and Kir6.2 in mini-KATP channels formed by TMD0 and Kir6.2. Importantly, E128K full-length channels, despite having a greatly reduced Po, exhibit little response to phosphatidylinositol 4,5-bisphosphate (PIP2) stimulation. This is reminiscent of Kir6.2 channel behavior in the absence of SUR1 and suggests that TMD0 controls Kir6.2 gating by modulating Kir6.2 interactions with PIP2. Further supporting this notion, the E128W mutation in full-length channels resulted in channel inactivation that was prevented or reversed by exogenous PIP2. These results identify a critical determinant in TMD0 that controls Kir6.2 gating by controlling channel sensitivity to PIP2. Moreover, they uncover a novel mechanism of KATP channel inactivation involving aberrant functional coupling between SUR1 and Kir6.2.

The Human Transporter Associated with Antigen Processing (TAP): A Computational Study Focused on the Nucleotide Binding Domains

The human Transporter Associated with Antigen Processing (TAP) is the only member of the ATP-Binding Cassette (ABC) transporter family with a crucial role in the adaptive immune response, as it is involved in the antigen presentation pathway. TAP resides in the entoplasmic reticulum (ER) membrane, and shuttles peptides derived from proteasomal degradation into the major histocompatibility complex (MHC) class I molecules, for their further presentation at the cell surface. TAP is a heterodimer formed by two subunits, TAP1 and TAP2, organized in four main domains: two N-terminal transmembrane domains (TMDs), defining the translocation pathway, and two highly conserved C-terminal nucleotide binding domains (NBDs).

Lipids and Topological “rules” of Membrane Protein Assembly: Testing the Generality of Net Charge Balance Rule

Transmembrane domain (TMD) orientation within some membrane proteins is dependent on membrane lipid composition. When the lactose permease (LacY) is assembled in Escherichia coli membranes lacking the major phospholipid phosphatidylethanolamine (PE), the N-terminal TMD bundle is inverted with respect to the C-terminal TMD bundle and the plane of the membrane. This inversion is dependent on the interfacial net positive charge of the protein, the net negative charge of the membrane and a TMD of low hydrophobicity, acting as a molecular hinge between the two halves of the protein by exiting the membrane to the periplasm. Homologous E. coli sucrose permease (CscB) and non-homologous phenylalanine permease (PheP) were investigated to generalize these original observations.CscB function, like that of LacY, is dependent on the presence of PE but topological dependence on membrane lipid composition is less sensitive and only becomes evident after significant changes in the net positive charge of the cytoplasmic surface of the N-terminal bundle. The first cytoplasmic domains of PheP, which are misoriented in PE-lacking cells, have a net negative charge. Decreasing the negative charge density of the extramembrane domains flanking the N-terminal TMD hairpin of PheP favors a reorientation of the N-terminus and adjacent hairpin to their native orientation in the absence of PE as predicted by the above net charge balance rule.Polytopic membrane proteins containing competing opposite charges within their cytoplasmic domains may share a common mechanism for topogenesis dependent on PE. However the degree of sensitivity to phospholipid composition appears to be sequence-specific and might be a result of conformational flexibility, topological preference of individual domains or the availability of mechanical hinge region. Supported by NIH grant R37-GM20478.

Electron Cryomicroscopy Structure of a Membrane-anchored Mitochondrial AAA Protease

FtsH-related AAA proteases are conserved membrane-anchored, ATP-dependent molecular machines, which mediate the processing and turnover of soluble and membrane-embedded proteins in eubacteria, mitochondria, and chloroplasts. Homo- and hetero-oligomeric proteolytic complexes exist, which are composed of homologous subunits harboring an ATPase domain of the AAA family and an H41 metallopeptidase domain. Mutations in subunits of mitochondrial m-AAA proteases have been associated with different neurodegenerative disorders in human, raising questions on the functional differences between homo- and hetero-oligomeric AAA proteases. Here, we have analyzed the hetero-oligomeric yeast m-AAA protease composed of homologous Yta10 and Yta12 subunits. We combined genetic and structural approaches to define the molecular determinants for oligomer assembly and to assess functional similarities between Yta10 and Yta12. We demonstrate that replacement of only two amino acid residues within the metallopeptidase domain of Yta12 allows its assembly into homo-oligomeric complexes. To provide a molecular explanation, we determined the 12 Å resolution structure of the intact yeast m-AAA protease with its transmembrane domains by electron cryomicroscopy (cryo-EM) and atomic structure fitting. The full-length m-AAA protease has a bipartite structure and is a hexamer in solution. We found that residues in Yta12, which facilitate homo-oligomerization when mutated, are located at the interface between neighboring protomers in the hexamer ring. Notably, the transmembrane and intermembrane space domains are separated from the main body, creating a passage on the matrix side, which is wide enough to accommodate unfolded but not folded polypeptides. These results suggest a mechanism regarding how proteins are recognized and degraded by m-AAA proteases.

NMR Structural Studies of a Two-Transmembrane Helix Segment of the Na+/H+ Exchanger Isoform 1

The Na+/H+ exchanger isoform 1 (NHE1) is an integral membrane protein important in the regulation of intracellular pH in the heart by exchanging one intracellular H+ for one extracellular Na+. It contains an N-terminal transmembrane (TM) domain responsible for ion transport and a C-terminal, cytoplasmic, regulatory domain. There is currently no high-resolution structure of NHE1; however, a topology model of the protein by Wakabayashi et al. (J. Biol. Chem.275, 7942) suggests that the membrane domain (amino acids 1-500) contains 12 TM helices. We have previously used NMR spectroscopy to determine the structures of peptides representing several TM helices from NHE1. Structures have been determined for TM IV in organic tri-solvent mixture, and for TM helices VII, IX, XI, and VI in DPC micelles. To provide further insight into the three-dimensional structure of NHE1, we are attempting to express and determine the structures of multiple TM helices. We have recently expressed and purified a two-TM helix segment representing TM VI-VII (amino acids 226-274) of NHE1. These two TM helices have been previously shown to be important in NHE1 function. Furthermore, modelling of NHE1 by Landau et al. (J. Biol. Chem. 282, 37854) suggest that these two helices interact and play key roles in ion transport. We are determining the structure of this region in micelles using NMR. NMR spectra of unlabelled TM VI-VII peptide show good quality spectra for structure determination. Further experiments with 15N labelled peptide will further define the structure. This will provide insight into TM helix interactions in NHE1 and help us to build up the structure of the protein from these TM segments. (Supported by AHFMR, HSF, CIHR, and AIHS).