Higher Order Oligomeric States Research Articles

Unraveling the crosstalk between renin-angiotensin system receptors.

The renin-angiotensin system (RAS) plays a key role in blood pressure regulation. The RAS is a complex interconnected system composed of two axes with opposite effects. The pressor arm, represented by angiotensin (Ang) II and the AT1 receptor (AT1R), mediates the vasoconstrictor, proliferative, hypertensive, oxidative, and pro-inflammatory effects of the RAS, while the depressor/protective arm, represented by Ang-(1-7), its Mas receptor (MasR) and the AT2 receptor (AT2R), opposes the actions elicited by the pressor arm. The AT1R, AT2R, and MasR belong to the G-protein-coupled receptor (GPCR) family. GPCRs operate not only as monomers, but they can also function in dimeric (homo and hetero) or higher-order oligomeric states. Due to the interaction with other receptors, GPCR properties may change: receptor affinity, trafficking, signaling, and its biological function may be altered. Thus, heteromerization provides a newly recognized means of modulation of receptor function, as well as crosstalk between GPCRs. This review is focused on angiotensin receptors, and how their properties are influenced by crosstalk with other receptors, adding more complexity to an already complex system and potentially opening up new therapeutic approaches.

Rapid and Highly Stable Membrane Reconstitution by LAiR Enables the Study of Physiological Integral Membrane Protein Functions.

Functional reintegration into lipid environments represents a major challenge for in vitro investigation of integral membrane proteins (IMPs). Here, we report a new approach, termed LMNG Auto-insertion Reintegration (LAiR), for reintegration of IMPs into lipid bilayers within minutes. The resulting proteoliposomes displayed an unprecedented capability to maintain proton gradients and long-term stability. LAiR allowed for monitoring catalysis of a membrane-bound, physiologically relevant polyisoprenoid quinone substrate by Escherichia coli cytochromes bo 3 (cbo 3) and bd (cbd) under control of the proton motive force. LAiR also facilitated bulk-phase detection and physiological assessment of the "proton leak" in cbo 3, a controversial catalytic state that previously was only approachable at the single-molecule level. LAiR maintained the multisubunit integrity and higher-order oligomeric states of the delicate mammalian F-ATP synthase. Given that LAiR can be applied to both liposomes and planar membrane bilayers and is compatible with IMPs and lipids from prokaryotic and eukaryotic sources, we anticipate LAiR to be applied broadly across basic research, pharmaceutical applications, and biotechnology.

Direct Observation of Na,K‐ATPase Oligomers in The Plasma Membrane of Living Cells by FRET‐FCS

The sodium pump, Na,K‐ATPase, is an integral plasma membrane protein, expressed in all eukaryotic cells. Na,K‐ATPase transforms chemical energy from ATP into a gradient of Na+ and K+ over the plasma membrane by actively exporting three Na+ ions and importing two K+ ions for each hydrolyzed ATP. It is responsible for maintenance of the transmembrane Na+ gradient and is the major determinant of the membrane potential. It provides the driving force for all Na+‐coupled transport processes, thereby controlling essential functions in the cell. Na,K‐ATPase is formed by three subunits alpha/beta/FXYD, where alpha is the catalytic ion‐transporting subunit, beta is a regulatory subunit and FXYD is accessory.Self‐interaction and oligomerization of the Na,K‐ATPase alpha/beta heterodimer in cell membranes has been proposed and discussed for a long time but is still an open question.Here we have used a combination of FRET and Fluorescence Correlation Spectroscopy, FRET‐FCS, in order to detect oligomers of Na,K‐ATPase. Compared to conventional cross‐correlation FCCS, FRET‐FCS is one to two orders of magnitude more sensitive when detecting oligomers. Moreover, FRET‐FCS is inherently insensitive to unbalanced labeling, which is a great advantage during live cell measurements.We hypothesized that Na,K‐ATPase can exist in a higher order oligomeric state and demonstrate the use of FRET‐FCS to test this hypothesisWe have introduced fluorescent labels by using expression of non‐canonical amino acid modified beta subunits. The FRET pair Alexa488 and Alexa647 was directly conjugated to the beta subunits using selective click chemistry. Conventional FCS measurements of labeled cells revealed the absolute density of labeled and unlabeled Na,K‐ATPase. With FRET‐FCS we could observe FRET signals and FCS curves demonstrating the existence of oligomers. Positive controls for the FRET‐FCS measurements were constructed by labeling alpha subunits with Alexa488 and beta subunits with Alexa647.Furthermore, we performed Monte Carlo simulations of Na,K‐ATPase, as monomer and as oligomer of increasing order, together with its ligands in a picket and fence diffusion model of the plasma membrane. The simulations suggest that oligomerization can have an impact on the net efficiency of the Na,K‐ATPase measured as ATP turnover.In conclusion we find that Na,K‐ATPase can be found in the plasma membrane as oligomers. Further we discuss the consequences of oligomerization and propose that it can have a regulatory effect for the Na,K‐ATPase net efficiency.

Oligomerization of THAP9 Transposase via Amino-Terminal Domains.

Active DNA transposases like the Drosophila P element transposase (DmTNP) undergo oligomerization as a prerequisite for transposition. Human THAP9 (hTHAP9) is a catalytically active but functionally uncharacterized homologue of DmTNP. Here we report (using co-immunoprecipitation, pull down, colocalization, and proximity ligation assays) that both full length and truncated hTHAP9 (corresponding to amino-terminal DNA binding and predicted coiled coil domains) undergo homo-oligomerization, predominantly in the nuclei of HEK293T cells. Interestingly, the oligomerization is shown to be partially mediated by DNA. However, mutating the leucines (either individually or together) or deleting the predicted coiled coil region did not significantly affect oligomerization. Thus, we highlight the importance of DNA and the amino-terminal regions of hTHAP9 for their ability to form higher-order oligomeric states. We also report that Hcf-1, THAP1, THAP10, and THAP11 are possible protein interaction partners of hTHAP9. Elucidating the functional relevance of the different putative oligomeric state(s) of hTHAP9 would help answer questions about its interaction partners as well as its unknown physiological roles.

Effect of palmitoylation on the dimer formation of the human dopamine transporter

The human dopamine transporter (hDAT) is one in three members of the monoamine transporter family (MAT). hDAT is essential for regulating the dopamine concentration in the synaptic cleft through dopamine reuptake into the presynaptic neuron; thereby controlling hDAT dopamine signaling. Dysfunction of the transporter is linked to several psychiatric disorders. hDAT and the other MATs have been shown to form oligomers in the plasma membrane, but only limited data exists on which dimeric and higher order oligomeric states are accessible and energetically favorable. In this work, we present several probable dimer conformations using computational coarse-grained self-assembly simulations and assess the relative stability of the different dimer conformations using umbrella sampling replica exchange molecular dynamics. Overall, the dimer conformations primarily involve TM9 and/or TM11 and/or TM12 at the interface. Furthermore, we show that a palmitoyl group (palm) attached to hDAT on TM12 modifies the free energy of separation for interfaces involving TM12, suggesting that S-palmitoylation may change the relative abundance of dimers involving TM12 in a biological context. Finally, a comparison of the identified interfaces of hDAT and palmitoylated hDAT to the human serotonin transporter interfaces and the leucine transporter interface, suggests similar dimer conformations across these protein family.

Nanodisc scaffold peptide (NSPr) replaces detergent by reconstituting acyl-CoA:cholesterol acyltransferase 1 into peptidiscs

To conduct biochemical studies in vitro, membrane proteins (MPs) must be solubilized with detergents. While detergents are great tools, they can also inhibit the biological activity and/or perturb oligomerization of individual MPs. Nanodisc scaffold peptide (NSPr), an amphipathic peptide analog of ApoA1, was recently shown to reconstitute detergent solubilized MPs into peptidiscs in vitro. Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1), also known as sterol O-acyltransferase 1 (SOAT1), plays a key role in cellular cholesterol storage in various cell types and is a drug target to treat multiple human diseases. ACAT1 contains nine transmembrane domains (TMDs) and primarily forms a homotetramer in vitro and in intact cells; deletion of the N-terminal dimerization domain produces a homodimer with full retention in catalytic activity. ACAT1 is prone to inactivation by numerous detergents. Here we pursued the use of NSPr to overcome the detergent-induced inactivation of ACAT1 by generating near detergent-free ACAT1 peptidiscs. Based on native-PAGE analysis, we showed that NSPr reconstitutes ACAT1 into soluble peptidiscs, in which ACAT1 exists predominantly in oligomeric states greater than a homotetramer. The formation of these higher-order oligomeric states was independent of the N-terminal dimerization domain, suggesting that the oligomerization is mediated through hydrophobic interactions of multiple ACAT1 subunits. ACAT1 peptidiscs were still susceptible to heat-mediated inactivation, presumably due to the residual detergent (CHAPS) bound to ACAT1. We then conditioned ACAT1 with phosphatidylcholine (PC) to replace CHAPS prior to the formation of ACAT1 peptidiscs. The results showed, when PC was included, ACAT1 was present mainly in higher-order oligomeric states with greater enzymatic activity. With PC present, the enzymatic activity of ACAT1 peptidiscs was protected from heat-mediated inactivation. These results support the use of NSPr to create a near detergent-free solution of ACAT1 in peptidiscs for various in vitro studies. Our current results also raise the possibility that, under certain conditions, ACAT1 may form higher-order oligomeric states in vivo.

Carbon Dioxide "Trapped" in a β-Carbonic Anhydrase.

Carbonic anhydrases (CAs) are enzymes that catalyze the hydration/dehydration of CO2/HCO3(-) with rates approaching diffusion-controlled limits (kcat/KM ∼ 10(8) M(-1) s(-1)). This family of enzymes has evolved disparate protein folds that all perform the same reaction at near catalytic perfection. Presented here is a structural study of a β-CA (psCA3) expressed in Pseudomonas aeruginosa, in complex with CO2, using pressurized cryo-cooled crystallography. The structure has been refined to 1.6 Å resolution with R(cryst) and R(free) values of 17.3 and 19.9%, respectively, and is compared with the α-CA, human CA isoform II (hCA II), the only other CA to have CO2 captured in its active site. Despite the lack of structural similarity between psCA3 and hCA II, the CO2 binding orientation relative to the zinc-bound solvent is identical. In addition, a second CO2 binding site was located at the dimer interface of psCA3. Interestingly, all β-CAs function as dimers or higher-order oligomeric states, and the CO2 bound at the interface may contribute to the allosteric nature of this family of enzymes or may be a convenient alternative binding site as this pocket has been previously shown to be a promiscuous site for a variety of ligands, including bicarbonate, sulfate, and phosphate ions.

SAMHD1 is a single-stranded nucleic acid binding protein with no active site-associated nuclease activity.

The HIV-1 restriction factor SAMHD1 is a tetrameric enzyme activated by guanine nucleotides with dNTP triphosphate hydrolase activity (dNTPase). In addition to this established activity, there have been a series of conflicting reports as to whether the enzyme also possesses single-stranded DNA and/or RNA 3′-5′ exonuclease activity. SAMHD1 was purified using three chromatography steps, over which the DNase activity was largely separated from the dNTPase activity, but the RNase activity persisted. Surprisingly, we found that catalytic and nucleotide activator site mutants of SAMHD1 with no dNTPase activity retained the exonuclease activities. Thus, the exonuclease activity cannot be associated with any known dNTP binding site. Monomeric SAMHD1 was found to bind preferentially to single-stranded RNA, while the tetrameric form required for dNTPase action bound weakly. ssRNA binding, but not ssDNA, induces higher-order oligomeric states that are distinct from the tetrameric form that binds dNTPs. We conclude that the trace exonuclease activities detected in SAMHD1 preparations arise from persistent contaminants that co-purify with SAMHD1 and not from the HD active site. An in vivo model is suggested where SAMHD1 alternates between the mutually exclusive functions of ssRNA binding and dNTP hydrolysis depending on dNTP pool levels and the presence of viral ssRNA.

High-resolution atomic force microscopy imaging of rhodopsin in rod outer segment disk membranes.

Atomic force microscopy (AFM) is a powerful imaging technique that allows recording topographical information of membrane proteins under near-physiological conditions. Remarkable results have been obtained on membrane proteins that were reconstituted into lipid bilayers. High-resolution AFM imaging of native disk membranes from vertebrate rod outer segments has unveiled the higher-order oligomeric state of the G protein-coupled receptor rhodopsin, which is highly expressed in disk membranes. Based on AFM imaging, it has been demonstrated that rhodopsin assembles in rows of dimers and paracrystals and that the rhodopsin dimer is the fundamental building block of higher-order structures.

Quantitative single-molecule localization microscopy combined with rule-based modeling reveals ligand-induced TNF-R1 reorganization toward higher-order oligomers

We report on the assembly of tumor necrosis factor receptor 1 (TNF-R1) prior to ligand activation and its ligand-induced reorganization at the cell membrane. We apply single-molecule localization microscopy to obtain quantitative information on receptor cluster sizes and copy numbers. Our data suggest a dimeric pre-assembly of TNF-R1, as well as receptor reorganization toward higher oligomeric states with stable populations comprising three to six TNF-R1. Our experimental results directly serve as input parameters for computational modeling of the ligand-receptor interaction. Simulations corroborate the experimental finding of higher-order oligomeric states. This work is a first demonstration how quantitative, super-resolution and advanced microscopy can be used for systems biology approaches at the single-molecule and single-cell level.

Crystal structure of the DdrB/ssDNA complex from Deinococcus radiodurans reveals a DNA binding surface involving higher-order oligomeric states.

The ability of Deinococcus radiodurans to recover from extensive DNA damage is due in part to its ability to efficiently repair its genome, even following severe fragmentation by hundreds of double-strand breaks. The single-strand annealing pathway plays an important role early during the recovery process, making use of a protein, DdrB, shown to greatly stimulate ssDNA annealing. Here, we report the structure of DdrB bound to ssDNA to 2.3 Å. Pentameric DdrB was found to assemble into higher-order structures that coat ssDNA. To gain further mechanistic insight into the protein's function, a number of point mutants were generated altering both DNA binding and higher order oligomerization. This work not only identifies higher-order DdrB associations but also suggests the presence of an extended DNA binding surface running along the ‘top’ surface of a DdrB pentamer and continuing down between two individual subunits of the ring structure. Together this work sheds new insight into possible mechanisms for DdrB function in which higher-order assemblies of DdrB pentamers assist in the pairing of complementary ssDNA using an extended DNA binding surface.

Nucleotide Binding Stabilizes Na,K‐ATPase Quaternary Structure and Facilitates Trafficking to Plasma Membrane

Na,K‐ATPase (NKA) is an integral membrane protein that plays a central role in ionic homeostasis in animals by mediating transport of Na+ and K+ ions against their electrochemical gradients. NKA is a heterodimer of α and β subunits. It is not yet clear if the native structure of NKA consists of αβ‐protomers or higher order oligomeric states. α‐ α associations were earlier demonstrated using in vitro pull down assays with isolated ATP‐binding domain (ABD) and full‐length α (Costa et al., 2002). Our objective is to elucidate the oligomeric state of NKA throughout maturation and trafficking and the role it plays in protein‐protein interactions. Consistent with previous observations, results from our co‐immunoprecipitation experiments using lysates from HEK 293 cells stably expressing α‐subunit with v5‐ or myc‐tagged NKA α‐subunits demonstrate α‐α interaction. Moreover, this self‐association is stabilized by including 2mM Mg‐ATP in the assay. Our preliminary results from confocal imaging of HEK 293 cells co‐expressing GFP‐tagged ABD and mcherry‐tagged full length α‐subunit revealed GFP‐ABD in the plasma membrane (PM), consistent with the isolated ABD being “carried” to the PM with full‐length α‐subunits. We also found that the α‐subunit lacking the M4M5 cytoplasmic loop (αΔ45LL), and thus unable to bind ATP, was retained in ER. Studies are underway to see if expressing the isolated ABD will rescue αΔ45LL and facilitate its delivery to the PM. This work was supported by NIH grant GM061583.

Single Molecule Studies of the General Secretory System

In bacteria and archaea many proteins use the protein conducting channel SecYEG either to transport across the membrane bilayer or to integrate into the bilayer. Further, it is known that the ATPase SecA binds SecYEG to perform translocation. Crystal structures of detergent-solubilized SecYEG and SecA bound to SecYEG have been reported [Nature 427, 36 (2004); Nature 455, 936 (2008)]. In recent years atomic force microscopy (AFM) has emerged as an important complementary tool to study membrane proteins at the single molecule level in near native conditions. In this work we study two central components of the bacterial secretory system (SecYEG and SecA) in membrane via AFM. We have obtained images of proteoliposomes containing just SecYEG, and SecYEG proteoliposomes assembled in the presence of SecA (SecY·A). All samples were adsorbed on mica surfaces and imaged in aqueous buffer solution. We collected several hundred images of each sample to provide statistics. Heights of SecYEG and SecY·A protruding above the lipid bilayer are in close agreement with crystal structure data and the topological asymmetry of SecYEG allows orientation determination. From volume calculations we are able to differentiate SecYEG monomers from dimers and higher order oligomeric states. Images of SecA bound to lipid (i.e, in the absence of SecYEG) were also obtained. In this case, the heights of SecA bound to the lipid are significantly different than the heights of SecY·A suggesting distinct binding modes of SecA to lipid compared to SecA to SecYEG. Further experiments and analysis will be required to conclusively determine the oligomeric state of active SecYEG during translocation.

The Tetrameric α-Helical Membrane Protein GlpF Unfolds via a Dimeric Folding Intermediate

Many membrane proteins appear to be present and functional in higher-order oligomeric states. While few studies have analyzed the thermodynamic stability of α-helical transmembrane (TM) proteins under equilibrium conditions in the past, oligomerization of larger polytopic monomers has essentially not yet been studied. However, it is vital to study the folding of oligomeric membrane proteins to improve our understanding of the general mechanisms and pathways of TM protein folding. To investigate the folding and stability of the aquaglyceroporin GlpF from Escherichia coli, unfolding of the protein in mixed micelles was monitored by steady-state fluorescence and circular dichroism spectroscopy as well as by seminative sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses. On the basis of our results, it appears most likely that GlpF unfolds in a two-step process, involving the equilibrium of tetrameric, dimeric, and monomeric GlpF species. A kinetic analysis also indicates an intermediate along the kinetic GlpF unfolding pathway, and thus, two phases are involved in GlpF unfolding. While three-state unfolding pathways and a dimeric folding intermediate are not uncommon for water-soluble proteins, a stable (un)folding intermediate with a decreased oligomeric structure has not been detected or reported for any α-helical membrane protein.

In Situ Measurement of Oligomerization State of NBCe1‐A in Kidney Tissues using Spatial Fluorescence Intensity Fluctuation Analysis

NBCe1‐A plays an important role in absorbing sodium bicarbonate across the basolateral membrane of the proximal tubule. We have previously showed that minimal functional unit of NBCe1‐A is a monomer, and based on in‐vitro biochemical studies in HEK293 cells, the oligomeric state of the cotransporter was shown to be predominantly dimeric with monomeric and higher oligomeric forms also present. In the present study, we developed an in situ measurement methodology to determine the oligomeric state of NBCe1‐A without requiring tissue disruption. We used fluorescent moment image analysis and spatial intensity distribution analysis (SpIDA) to study the oligomeric state of NBCe1‐A. Both methods allow for unbiased quantitative measurement of fluorescent particle densities and oligomerization states within individual images acquired with laser‐scanning microscopy. Initially we examined basal membranes of highly adherent CHO K1 cells expressing eGFP‐tagged NBCe1‐A, and showed that NBCe1‐A existed primarily in the monomeric state and negligibly in the dimeric state on the cell membrane. We immunolabeled NBCe1‐A in kidney tissues demonstrating the NBCe1‐A is present in dimeric and rarely in higher order oligomeric states. As a control, we compared the results obtained from LSM images of tissues of NBCe1‐A knockout mouse. These experiments demonstrate for the first time the in situ oligomeric state(s) of NBCe1‐A.

In Situ Measurements of Oligomerization State of NBCe1-A in Rat Kidneys Via Spatial Fluorescence Intensity Fluctuation Analysis

NBCe1-A plays an important role in absorbing sodium bicarbonate across the basolateral membrane of the proximal tubule. We have previously showed that minimal functional unit of NBCe1-A is a monomer, and based on in-vitro biochemical studies in HEK293 cells, the oligomeric state of the cotransporter was shown to be predominantly dimeric with monomeric and higher oligomeric forms also present. We developed an in situ measurement methodology to determine the oligomeric state of NBCe1-A without requiring tissue disruption using biochemical methods. We used fluorescent moment image analysis and spatial intensity distribution analysis (SpIDA) to study the oligomeric state of NBCe1-A in cultured cells expressing the cotransporter and in rat kidney tissue. Both methods allow for quantitative measurement of fluorescent particle densities and oligomerization states within individual images acquired with laser-scanning microscopy. Initially we examined basal membranes of highly adherent CHO K1 cells expressing eGFP-tagged NBCe1-A because of their large surface area. As an independent control of monomeric brightness, we used cells expressing monomeric eGFP anchored to the membrane. Taking into account the recovered values of the monomeric eGFP quantal brightness, we show that NBCe1-A exists in monomeric and dimeric states on the cell membrane. We also used an Alexa488-alpha-bungarotoxin conjugate to label cells expressing an NBCe1A-bungarotoxin binding mutant. As a monomeric control, we immobilized Alexa488 dye on cover slips. The spatial fluorescence intensity fluctuation analysis revealed a similar distribution of aggregates as shown for eGFP data. Moreover, we immunolabeled NBCe1-A in rat kidney tissues as well as in cultured HEK293 cells expressing the cotransporter demonstrating the NBCe1-A is present in monomeric, dimeric and rarely in higher order oligomeric states. These experiments demonstrate for the first time the in situ oligomeric state(s) of NBCe1-A.

Crystal structures of autoinhibitory PDZ domain of Tamalin: implications for metabotropic glutamate receptor trafficking regulation

Metabotropic glutamate receptors (mGluRs) function as neuronal G-protein-coupled receptors and this requires efficient membrane targeting through associations with cytoplasmic proteins. However, the molecular mechanism regulating mGluR cell-surface trafficking remains unknown. We report here that mGluR trafficking is controlled by the autoregulatory assembly of a scaffold protein Tamalin. In the absence of mGluR, Tamalin self-assembles into autoinhibited conformations, through its PDZ domain and C-terminal intrinsic ligand motif. X-ray crystallographic analyses visualized integral parts of the oligomeric self-assemblies of Tamalin, which require not only the novel hydrophobic dimerization interface but also canonical and noncanonical PDZ/ligand autoinhibitory interactions. The mGluR cytoplasmic region can competitively bind to Tamalin at a higher concentration, disrupting weak inhibitory interactions. The atomic view of mGluR association suggests that this rearrangement is dominated by electrostatic attraction and repulsion. We also observed in mammalian cells that the association liberates the intrinsic ligand toward a motor protein receptor, thereby facilitating mGluR cell-surface trafficking. Our study suggests a novel regulatory mechanism of the PDZ domain, by which Tamalin switches between the trafficking-inhibited and -active forms depending on mGluR association.

ATRIP Oligomerization Is Required for ATR-dependent Checkpoint Signaling

The ATM and ATR kinases signal cell cycle checkpoint responses to DNA damage. Inactive ATM is an oligomer that is disrupted to form active monomers in response to ionizing radiation. We examined whether ATR is activated by a similar mechanism. We found that the ATRIP subunit of the ATR kinase and ATR itself exist as homooligomers in cells. We did not detect regulation of ATR or ATRIP oligomerization after DNA damage. The predicted coiled-coil domain of ATRIP is essential for ATRIP oligomerization, stable ATR binding, and accumulation of ATRIP at DNA lesions. Additionally, the ATRIP coiled-coil is also required for ATRIP to support ATR-dependent checkpoint signaling to Chk1. Replacing the ATRIP coiled-coil domain with a heterologous dimerization domain restored stable binding to ATR and localization to damage-induced intranuclear foci. Thus, the ATR-ATRIP complex exists in higher order oligomeric states within cells and ATRIP oligomerization is essential for its function.