Heteromeric Protein Complexes Research Articles

Human CRB1 and CRB2 form homo- and heteromeric protein complexes in the retina.

Crumbs homolog 1 (CRB1) is one of the key genes linked to retinitis pigmentosa and Leber congenital amaurosis, which are characterized by a high clinical heterogeneity. The Crumbs family member CRB2 has a similar protein structure to CRB1, and in zebrafish, Crb2 has been shown to interact through the extracellular domain. Here, we show that CRB1 and CRB2 co-localize in the human retina and human iPSC-derived retinal organoids. In retina-specific pull-downs, CRB1 was enriched in CRB2 samples, supporting a CRB1-CRB2 interaction. Furthermore, novel interactors of the crumbs complex were identified, representing a retina-derived protein interaction network. Using co-immunoprecipitation, we further demonstrate that human canonical CRB1 interacts with CRB1 and CRB2, but not with CRB3, which lacks an extracellular domain. Next, we explored how missense mutations in the extracellular domain affect CRB1-CRB2 interactions. We observed no or a mild loss of CRB1-CRB2 interaction, when interrogating various CRB1 or CRB2 missense mutants in vitro. Taken together, our results show a stable interaction of human canonical CRB2 and CRB1 in the retina.

Evaluation of AlphaFold-Multimer prediction on multi-chain protein complexes.

Despite near-experimental accuracy on single-chain predictions, there is still scope for improvement among multimeric predictions. Methods like AlphaFold-Multimer and FoldDock can accurately model dimers. However, how well these methods fare on larger complexes is still unclear. Further, evaluation methods of the quality of multimeric complexes are not well established. We analysed the performance of AlphaFold-Multimer on a homology-reduced dataset of homo- and heteromeric protein complexes. We highlight the differences between the pairwise and multi-interface evaluation of chains within a multimer. We describe why certain complexes perform well on one metric (e.g. TM-score) but poorly on another (e.g. DockQ). We propose a new score, Predicted DockQ version 2 (pDockQ2), to estimate the quality of each interface in a multimer. Finally, we modelled protein complexes (from CORUM) and identified two highly confident structures that do not have sequence homology to any existing structures. All scripts, models, and data used to perform the analysis in this study are freely available at https://gitlab.com/ElofssonLab/afm-benchmark.

Hierarchical TAF1-dependent co-translational assembly of the basal transcription factor TFIID.

Large heteromeric multiprotein complexes play pivotal roles at every step of gene expression in eukaryotic cells. Among them, the 20-subunit basal transcription factor TFIID nucleates the RNA polymerase II preinitiation complex at gene promoters. Here, by combining systematic RNA-immunoprecipitation (RIP) experiments, single-molecule imaging, proteomics and structure-function analyses, we show that human TFIID biogenesis occurs co-translationally. We discovered that all protein heterodimerization steps happen during protein synthesis. We identify TAF1-the largest protein in the complex-as a critical factor for TFIID assembly. TAF1 acts as a flexible scaffold that drives the co-translational recruitment of TFIID submodules preassembled in the cytoplasm. Altogether, our data suggest a multistep hierarchical model for TFIID biogenesis that culminates with the co-translational assembly of the complex onto the nascent TAF1 polypeptide. We envision that this assembly strategy could be shared with other large heteromeric protein complexes.

Structural characterization of a full-length heteromeric glycine receptor in multiple functional states.

Glycine receptors (GlyR) are chloride-conducting ligand-gated ion channels that provide inhibitory neuronal input, particularly in the spinal cord. Synaptic GlyR is a heteromeric protein complex composed of five homologous subunits arranged pseudo-symmetrically about the channel pore. Individual subunits come in two varieties, α and β. Here, we present the structures of full-length zebrafish α1/β GlyR solved without any fiducial marker that may alter the functional state of the protein. This was done in the presence of an antagonist (strychnine), agonist (glycine) and agonist with an allosteric modulator (glycine and ivermectin). These structures confirm other recent findings regarding the stoichiometry of heteromeric GlyR (4α:1β) and also reveal previously uncharacterized subunit-specific features in the extracellular and intracellular domain. The functional state of each structure was assessed by molecular dynamics simulations. The strychnine-bound pore is in a closed state that is mostly symmetric about the five-fold axis. The glycine structures with and without ivermectin both adopted desensitized conformations, but the ivermectin structure has significant asymmetry at the intracellular entrance to the channel pore. Consistent with this observation, ligand density near the ivermectin binding pocket was only observed at the sole α/β interface, and not at α/α interfaces as observed in past homomeric GlyR structures. Simulations show a small number of permeation events for the ivermectin-bound state, suggestive of an asymmetric mechanism for channel opening. Together, these results highlight distinct structural features of heteromeric GlyR and demonstrate the functional range of this physiologically important protein channel.

A plant-unique protein BLISTER coordinates with core retromer to modulate endosomal sorting of plasma membrane and vacuolar proteins

The retromer is a heteromeric protein complex that localizes to endosomal membranes and drives the formation of endosomal tubules that recycle membrane protein cargoes. In plants, the retromer plays essential and canonical functions in regulating the transport of vacuolar storage proteins and the recycle of endocytosed plasma membrane proteins (PM); however, the mechanisms underlying the regulation of assembly, protein stability, and membrane recruitment of the plant retromer complex remain to be elucidated. Inthis study, we identify a plant-unique endosomal regulator termed BLISTER (BLI), which colocalizes and associates with the retromer complex by interacting with the retromer core subunits VPS35 and VPS29. Depletion of BLI perturbs the assembly and membrane recruitment of the retromer core VPS26-VPS35-VPS29 trimer. Consequently, depletion of BLI disrupts retromer-regulated endosomal trafficking function, including transport of soluble vacuolar proteins and recycling of endocytosed PIN-FORMED (PIN) proteins from the endosomes back to the PM. Moreover, genetic analysis in Arabidopsis thaliana mutants reveals BLI and core retromer interact genetically in the regulation of endosomal trafficking. Taken together, we identified BLI as a plant-specific endosomal regulator, which functions in retromer pathway to modulate the recycling of endocytosed PM proteins and the trafficking of soluble vacuolar cargoes.

Molecular characterization and function of the lipid raft protein Lvflotillin-1A from Litopenaeus vannamei

White spot syndrome virus (WSSV) can cause a contagious, high virulent and pandemic disease for crustaceans, especially shrimps. However, the molecular mechanism of WSSV pathogenesis remains unclear. Flotillins are lipid raft-associated proteins, which mainly include flotillin-1 and flotillin-2. They are involved in the formation of large heteromeric protein complexes engaged in diverse signalling pathways at the membrane-cytosol interface. They defined a clathrin-independent endocytic pathway in mammalian cells. Our previous studies suggested that shrimp flotillin-2 might mediate endocytosis involved in WSSV infection. To further explore the function of shrimp flotillin, a flotillin-1 homologous, Lvflotillin-1A was identified and characterized in Litopenaeus vanamei. The transcription of Lvflotillin-1A showed a significant decline at 12h post-infection, followed by complete recovery and a slight up-regulation after the WSSV challenge. Gene silencing revealed that inhibition of Lvflotillin-1A raised the virus infection, suggesting Lvflotillin-1A might play an important role in shrimp immunity. Furthermore, co-immunoprecipitation and immunofluorescence illustrated that Lvflotillin-1A and Lvflotillin-2 could form hetero-oligomers, and co-expression promoted the accumulation of intracellular vesicles. The study revealed that WSSV might up-regulate Lvflotillin-2 expression and alter the subcellular location of Lvflotillin-1 protein to facilitate virus infection. These results will provide information for understanding the interaction between WSSV and shrimp.

DeepTracer-ID: De novo protein identification from cryo-EM maps

The recent revolution in cryo-electron microscopy (cryo-EM) has made it possible to determine macromolecular structures directly from cell extracts. However, identifying the correct protein from the cryo-EM map is still challenging and often needs additional sequence information from other techniques, such as tandem mass spectrometry and/or bioinformatics. Here, we present DeepTracer-ID, a server-based approach to identify the candidate protein in a user-provided organism de novo from a cryo-EM map, without the need for additional information. Our method first uses DeepTracer to generate a protein backbone model that best represents the cryo-EM map, and this model is then searched against the library of AlphaFold2 predictions for all proteins in the given organism. This method is highly accurate and robust for high-resolution cryo-EM maps: in all 13 experimental maps tested blindly, DeepTracer-ID identified the correct proteins as the top candidates. Eight of the maps were of known structures, while the other five unpublished maps were validated by prior protein annotation and careful inspection of the model refined into the map. The program also showed promising results for both homomeric and heteromeric protein complexes. This platform is possible because of the recent breakthroughs in large-scale three-dimensional protein structure prediction.

Identification and functional evaluation of GRIA1 missense and truncation variants in individuals with ID: An emerging neurodevelopmental syndrome

SummaryGRIA1 encodes the GluA1 subunit of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors, which are ligand-gated ion channels that act as excitatory receptors for the neurotransmitter L-glutamate (Glu). AMPA receptors (AMPARs) are homo- or heteromeric protein complexes with four subunits, each encoded by different genes, GRIA1 to GRIA4. Although GluA1-containing AMPARs have a crucial role in brain function, the human phenotype associated with deleterious GRIA1 sequence variants has not been established. Subjects with de novo missense and nonsense GRIA1 variants were identified through international collaboration. Detailed phenotypic and genetic assessments of the subjects were carried out and the pathogenicity of the variants was evaluated in vitro to characterize changes in AMPAR function and expression. In addition, two Xenopus gria1 CRISPR-Cas9 F0 models were established to characterize the in vivo consequences. Seven unrelated individuals with rare GRIA1 variants were identified. One individual carried a homozygous nonsense variant (p.Arg377Ter), and six had heterozygous missense variations (p.Arg345Gln, p.Ala636Thr, p.Ile627Thr, and p.Gly745Asp), of which the p.Ala636Thr variant was recurrent in three individuals. The cohort revealed subjects to have a recurrent neurodevelopmental disorder mostly affecting cognition and speech. Functional evaluation of major GluA1-containing AMPAR subtypes carrying the GRIA1 variant mutations showed that three of the four missense variants profoundly perturb receptor function. The homozygous stop-gain variant completely destroys the expression of GluA1-containing AMPARs. The Xenopus gria1 models show transient motor deficits, an intermittent seizure phenotype, and a significant impairment to working memory in mutants. These data support a developmental disorder caused by both heterozygous and homozygous variants in GRIA1 affecting AMPAR function.

Starch synthase II plays a crucial role in starch biosynthesis and the formation of multienzyme complexes in cassava storage roots

Starch is a glucose polymer synthesized by green plants for energy storage and is crucial for plant growth and reproduction. The biosynthesis of starch polysaccharides is mediated by members of the large starch synthase (SS) protein superfamily. Here, we showed that in cassava storage roots, soluble starch synthase II (MeSSII) plays an important role in starch biosynthesis and the formation of protein complexes with other starch biosynthetic enzymes by directly interacting with MeSSI, MeSBEII, and MeISAII. MeSSII-RNAi cassava lines showed increased amylose content and reduced biosynthesis of the intermediate chain of amylopectin (B1 type) in their storage roots, leading to altered starch physicochemical properties. Furthermore, gel permeation chromatography analysis of starch biosynthetic enzymes between wild type and MeSSII-RNAi lines confirmed the key role of MeSSII in the organization of heteromeric starch synthetic protein complexes. The lack of MeSSII in cassava also reduced the capacity of MeSSI, MeSBEII, MeISAI, and MeISAII to bind to starch granules. These findings shed light on the key components of the starch biosynthesis machinery in root crops.

Control of membrane protein homeostasis by a chaperone-like glial cell adhesion molecule at multiple subcellular locations

The significance of crosstalks among constituents of plasma membrane protein clusters/complexes in cellular proteostasis and protein quality control (PQC) remains incompletely understood. Examining the glial (enriched) cell adhesion molecule (CAM), we demonstrate its chaperone-like role in the biosynthetic processing of the megalencephalic leukoencephalopathy with subcortical cyst 1 (MLC1)-heteromeric regulatory membrane protein complex, as well as the function of the GlialCAM/MLC1 signalling complex. We show that in the absence of GlialCAM, newly synthesized MLC1 molecules remain unfolded and are susceptible to polyubiquitination-dependent proteasomal degradation at the endoplasmic reticulum. At the plasma membrane, GlialCAM regulates the diffusional partitioning and endocytic dynamics of cluster members, including the ClC-2 chloride channel and MLC1. Impaired folding and/or expression of GlialCAM or MLC1 in the presence of diseases causing mutations, as well as plasma membrane tethering compromise the functional expression of the cluster, leading to compromised endo-lysosomal organellar identity. In addition, the enlarged endo-lysosomal compartments display accelerated acidification, ubiquitinated cargo-sorting and impaired endosomal recycling. Jointly, these observations indicate an essential and previously unrecognized role for CAM, where GliaCAM functions as a PQC factor for the MLC1 signalling complex biogenesis and possess a permissive role in the membrane dynamic and cargo sorting functions with implications in modulations of receptor signalling.

Linking transport and translation of mRNAs with endosomes and mitochondria.

In eukaryotic cells, proteins are targeted to their final subcellular locations with precise timing. A key underlying mechanism is the active transport of cognate mRNAs, which in many systems can be linked intimately to membrane trafficking. A prominent example is the long‐distance endosomal transport of mRNAs and their local translation. Here, we describe current highlights of fundamental mechanisms of the underlying transport process as well as of biological functions ranging from endosperm development in plants to fungal pathogenicity and neuronal processes. Translation of endosome‐associated mRNAs often occurs at the cytoplasmic surface of endosomes, a process that is needed for membrane‐assisted formation of heteromeric protein complexes and for accurate subcellular targeting of proteins. Importantly, endosome‐coupled translation of mRNAs encoding mitochondrial proteins, for example, seems to be particularly important for efficient organelle import and for regulating subcellular mitochondrial activity. In essence, these findings reveal a new mechanism of loading newly synthesised proteins onto endocytic membranes enabling intimate crosstalk between organelles. The novel link between endosomes and mitochondria adds an inspiring new level of complexity to trafficking and organelle biology.

A co-fractionation mass spectrometry-based prediction of protein complex assemblies in the developing rice aleurone-subaleurone.

Multiprotein complexes execute and coordinate diverse cellular processes such as organelle biogenesis, vesicle trafficking, cell signaling, and metabolism. Knowledge about their composition and localization provides useful clues about the mechanisms of cellular homeostasis and system-level control. This is of great biological importance and practical significance in heterotrophic rice (Oryza sativa) endosperm and aleurone-subaleurone tissues, which are a primary source of seed vitamins and stored energy. Dozens of protein complexes have been implicated in the synthesis, transport, and storage of seed proteins, lipids, vitamins, and minerals. Mutations in protein complexes that control RNA transport result in aberrant endosperm with shrunken and floury phenotypes, significantly reducing seed yield and quality. The purpose of this study was to broadly predict protein complex composition in the aleurone-subaleurone layers of developing rice seeds using co-fractionation mass spectrometry. Following orthogonal chromatographic separations of biological replicates, thousands of protein elution profiles were subjected to distance-based clustering to enable large-scale multimerization state measurements and protein complex predictions. The predicted complexes had predicted functions across diverse functional categories, including novel heteromeric RNA binding protein complexes that may influence seed quality. This effective and open-ended proteomics pipeline provides useful clues about system-level posttranslational control during the early stages of rice seed development.

A Fusion Protein Complex that Combines IL-12, IL-15, and IL-18 Signaling to Induce Memory-Like NK Cells for Cancer Immunotherapy.

Natural killer (NK) cells are a promising cellular therapy for cancer, with challenges in the field including persistence, functional activity, and tumor recognition. Briefly, priming blood NK cells with recombinant human (rh)IL-12, rhIL-15, and rhIL-18 (12/15/18) results in memory-like NK cell differentiation and enhanced responses against cancer. However, the lack of available, scalable Good Manufacturing Process (GMP)-grade reagents required to advance this approach beyond early-phase clinical trials is limiting. To address this challenge, we developed a novel platform centered upon an inert tissue factor scaffold for production of heteromeric fusion protein complexes (HFPC). The first use of this platform combined IL-12, IL-15, and IL-18 receptor engagement (HCW9201), and the second adds CD16 engagement (HCW9207). This unique HFPC expression platform was scalable with equivalent protein quality characteristics in small- and GMP-scale production. HCW9201 and HCW9207 stimulated activation and proliferation signals in NK cells, but HCW9207 had decreased IL-18 receptor signaling. RNA sequencing and multidimensional mass cytometry revealed parallels between HCW9201 and 12/15/18. HCW9201 stimulation improved NK cell metabolic fitness and resulted in the DNA methylation remodeling characteristic of memory-like differentiation. HCW9201 and 12/15/18 primed similar increases in short-term and memory-like NK cell cytotoxicity and IFNγ production against leukemia targets, as well as equivalent control of leukemia in NSG mice. Thus, HFPCs represent a protein engineering approach that solves many problems associated with multisignal receptor engagement on immune cells, and HCW9201-primed NK cells can be advanced as an ideal approach for clinical GMP-grade memory-like NK cell production for cancer therapy.

Protein interactions studies of the small TbTims chaperons in the mitochondrion of Trypanosoma brucei

Trypanosoma brucei causes a neglected tropical disease known as trypanosomiasis that causes death in population of Sub‐Sahara Africa. T. brucei possesses a single mitochondrion and like other eukaryotes it imports most of its proteins into the mitochondria, however mitochondrial protein import machinery is divergent in this parasite, which makes it a useful target to control this disease. Nucleus‐encoded proteins are imported into T. brucei mitochondrion via two major heteromeric protein complexes in the outer and inner membrane, ATOM and TbTIM, respectively. Besides the membrane integral protein components of these complexes, TbTIM is also peripherally associated with 6 small TbTims (Tim9, 10, 8/13, 11, 12, and 13). At least three of these small TbTims (TbTim8/13, 11, & 12) are unique for T. brucei. Knock‐down of any of these 6 small TbTims destabilized the TbTIM17 complex therefore these proteins are essential in T. brucei. In fungi and human, the small Tims are soluble proteins that form hetero‐hexameric complexes. Each small Tims possesses two pairs of CX3C motifs that are required for proper folding of the monomers by formation of the two intramolecular disulfide bonds. T. brucei small Tims also possess these conserved motifs, except for TbTim12, which has only one pair. However, it is not clear how small TbTims interact with each other and what type of complex structure they form. Thus, our goal for these studies is to find the direct interaction pattern for the small TbTims. To complete this goal, we use yeast two hybrid (Y2H) analysis. Results showed that although all small TbTims can interact with one another, some of these interactions are stronger than others. Overall, we found that TbTim8/13 interacts strongly with TbTim9, TbTim10, and TbTim8/13 while TbTim13 interacts strongly with TbTim13. Based on these results we proposed 3 possible model for small TbTim heterohexameric complexes; 1) random order, and 2) defined order, and 3) modular with different sub‐complexes. We are now testing these models by structure‐modeling and site‐directed mutagenesis studies.Support or Funding InformationThis work is supported by the following NIH grants: 5RO1AI125666, 2SC1GM081146, and R25 GM05994

Circulating Ficolin-2 and Ficolin-3 Form Heterocomplexes.

The complement system constitutes an important part of the innate immune system. The collectins and the ficolins are soluble pattern recognition molecules that contribute to complement activation via the lectin pathway. During previous experiments with ficolin-2 and ficolin-3, we have observed that the molecules may interact. We therefore hypothesized the existence of stable ficolin-2/-3 heterocomplexes. We could demonstrate ficolin-2/-3 heterocomplexes in normal human serum and plasma by ELISA using Abs specific for ficolin-2 and ficolin-3. The formation of heteromeric protein complexes were validated by coimmunoprecipitation and Western blot analysis. When recombinant ficolin-2 and recombinant ficolin-3 were mixed, no complexes were formed. However, when coexpressing ficolin-2 and ficolin-3 in Chinese hamster ovary cells, we could detect ficolin-2/-3 heterocomplexes in the supernatant. Furthermore, we measured concentration of the ficolin-2/-3 heterocomplexes in arbitrary units in 94 healthy individuals. We also established the relationship between the concentrations of ficolin-2, ficolin-3, and the ficolin-2/-3 heterocomplexes. We observed that the concentration of the ficolin-2/-3 heterocomplex correlated significantly with ficolin-2 (ρ: 0.24, p < 0.018) and ficolin-3 concentrations (ρ: 0.46, p < 0.0001). In conclusion, we describe a novel protein complex between ficolin-2 and ficolin-3 present in serum and plasma, which might be of additional biological relevance apart from the native ficolin-2 and ficolin-3 molecules.

Type 2β Corticotrophin Releasing Factor Receptor Forms a Heteromeric Complex With Dopamine D1 Receptor in Living Cells.

Corticotrophin releasing factor (CRF) and its related peptides differentially bind to CRF receptors to modulate stress-related behaviors. CRF receptors comprise two G-protein coupled receptors (GPCR), type-1 CRF receptors (CRF1), and type-2 CRF receptors (CRF2). CRF2 encompasses three spliced variants in humans, alpha (CRF2α), beta (CRF2β), and gamma (CRF2γ), which differ in their N-terminal extracellular domains and expression patterns. Previously, we showed that CRF2α form a heteromeric protein complex with dopamine D1 receptors (D1R), leading to changes in the signaling of D1R. Based on the high sequence identity between CRF2α and CRF2β, we hypothesized that CRF2β also heteromerize with D1R. To test the hypothesis, we compared the expression and localization of both CRF2 isoforms and whether CRF2β form stable protein complexes with D1R in HEK293 and ATR75 cell lines. We observed that the immunoreactivity for CRF2β was similar to that of CRF2α in the endoplasmic compartment but significantly higher in the Golgi compartment. Immunoprecipitation analysis showed that CRF2β forms a heteromeric protein complex with D1R. Furthermore, the protein complex formed by CRF2β and D1R was stable enough to change the sub-cellular localization of CRF2β when it was co-expressed with a construct of D1R bearing a nuclear localization signal. Immunofluorescence in A7R5 cells, which endogenously express CRF2β and D1R, shows significant colocalization of CRF2β with D1R. In conclusion, our results show that CRF2β forms a stable heteromeric protein complex with D1R, a potential new therapeutic target in tissues where both receptors are co-expressed, such as the septum in the brain, and heart, kidney, and skeletal muscle in the periphery.

Super-resolution Microscopy-based Bimolecular Fluorescence Complementation to Study Protein Complex Assembly and Co-localization.

Numerous experimental approaches exist to study interactions between two subunits of a large macromolecular complex. However, most methods do not provide spatial and temporal information about binding, which are critical for dissecting the mechanism of assembly of nanosized complexes in vivo. While recent advances in super-resolution microscopy techniques have provided insights into biological structures beyond the diffraction limit, most require extensive expertise and/or special sample preparation, and it is a challenge to extend beyond binary, two color experiments. Using HyVolution, a super-resolution technique that combines confocal microscopy at sub-airy unit pinhole sizes with computational deconvolution, we achieved 140 nm resolution in both live and fixed samples with three colors, including two fluorescent proteins (mTurquoise2 and GFP) with significant spectral overlap that were distinguished by means of shifting the excitation wavelength away from common wavelengths. By combining HyVolution super-resolution fluorescence microscopy with bimolecular fluorescence complementation (SRM-BiFC), we describe a new assay capable of visualizing protein-protein interactions in vivo at sub-diffraction resolution. This method was used to improve our understanding of the ordered assembly of the Saccharomyces cerevisiae spindle pole body (SPB), a ~1 giga-Dalton heteromeric protein complex formed from 18 structural components present in multiple copies. We propose that SRM-BiFC is a powerful tool for examination of direct interactions between protein complex subunits at sub-diffraction resolution in live cells.

A heteromeric cis-prenyltransferase is responsible for the biosynthesis of glycosyl carrier lipids in Methanosarcina mazei

Cis-prenyltransferases are enzymes responsible for the biosynthesis of glycosyl carrier lipids, natural rubber, and some secondary metabolites. Certain organisms, including some archaeal species, possess multiple genes encoding cis-prenyltransferase homologs, and the physiological roles of these seemingly-redundant genes are often obscure. Cis-prenyltransferases usually form homomeric complexes, but recent reports have demonstrated that certain eukaryotic enzymes are heteromeric protein complexes consisting of two homologous subunits. In this study, three cis-prenyltransferase homolog proteins, MM_0014, MM_0618, and MM_1083, from the methanogenic archaeon Methanosarcina mazei are overexpressed in Escherichia coli and partially purified for functional characterization. Coexistence of MM_0618 and MM_1083 exhibits prenyltransferase activity, while each of them alone has almost no activity. The chain-lengths of the products of this heteromeric enzyme are in good agreement with those of glycosyl carrier lipids extracted from M. mazei, which are likely di- and tetra-hydrogenated decaprenyl phosphates, suggesting that the MM_0618/MM_1083 heteromer is involved in glycosyl carrier lipid biosynthesis. MM_0014 acts as a typical homomeric cis-prenyltransferase and produces shorter products.

The structure of lipopolysaccharide transport protein B (LptB) from Burkholderia pseudomallei.

The thick outer membrane (OM) of Gram-negative bacteria performs an important protective role against hostile environments, supports cell integrity, and contributes to surface adhesion and in some cases also to virulence. A major component of the OM is lipopolysaccharide (LPS), a complex glycolipid attached to a core containing fatty-acyl chains. The assembly and transport of lipid A, the membrane anchor for LPS, to the OM begins when a heteromeric LptB2FG protein complex extracts lipid A from the outer leaflet of the inner membrane. This process requires energy, and upon hydrolysis of ATP one component of the heteromeric assembly, LptB, triggers a conformational change in LptFG in support of lipid A transport. A structure of LptB from the intracellular pathogen Burkholderia pseudomallei is reported here. LptB forms a dimer that displays a relatively fixed structure irrespective of whether it is in complex with LptFG or in isolation. Highly conserved sequence and structural features are discussed that allow LptB to fuel the transport of lipid A.

Discovery of Pathologic GPCR Aggregation.

The family of G-protein-coupled receptors (GPCRs) is one of the most important drug targets. Mechanisms underlying GPCR activation and signaling are therefore of great pharmacologic interest. It was long thought that GPCRs exist and function as monomers. This feature was considered to distinguish GPCRs from other membrane receptors such as receptor tyrosine kinases or cytokine receptors, which signal from dimeric receptor complexes. But during the last two decades it was increasingly recognized that GPCRs can undergo aggregation to form dimers and higher order oligomers, resulting in homomeric and/or heteromeric protein complexes with different stoichiometries. Moreover, this protein complex formation could modify GPCR signaling and function. We contributed to this paradigm shift in GPCR pharmacology by the discovery of the first pathologic GPCR aggregation, which is the protein complex formation between the angiotensin II AT1 receptor and the bradykinin B2 receptor. Increased AT1-B2 heteromerization accounts for the angiotensin II hypersensitivity of pregnant women with preeclampsia hypertension. Since the discovery of AT1-B2, other pathologic GPCR aggregates were found, which contribute to atherosclerosis, neurodegeneration and Alzheimer's disease. As a result of our findings, pathologic GPCR aggregation appears as an independent and disease-specific process, which is increasingly considered as a novel target for pharmacologic intervention.