Center for Membrane Proteomics (CMP)
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Antigenic and 3D structural characterization of soluble X4 and hybrid X4-R5 HIV-1 Env trimers
(2014)
Background: HIV-1 is decorated with trimeric glycoprotein spikes that enable infection by engaging CD4 and a chemokine coreceptor, either CCR5 or CXCR4. The variable loop 3 (V3) of the HIV-1 envelope protein (Env) is the main determinant for coreceptor usage. The predominant CCR5 using (R5) HIV-1 Env has been intensively studied in function and structure, whereas the trimeric architecture of the less frequent, but more cytopathic CXCR4 using (X4) HIV-1 Env is largely unknown, as are the consequences of sequence changes in and near V3 on antigenicity and trimeric Env structure.
Results: Soluble trimeric gp140 Env constructs were used as immunogenic mimics of the native spikes to analyze their antigenic properties in the context of their overall 3D structure. We generated soluble, uncleaved, gp140 trimers from a prototypic T-cell line-adapted (TCLA) X4 HIV-1 strain (NL4-3) and a hybrid (NL4-3/ADA), in which the V3 spanning region was substituted with that from the primary R5 isolate ADA. Compared to an ADA (R5) gp140, the NL4-3 (X4) construct revealed an overall higher antibody accessibility, which was most pronounced for the CD4 binding site (CD4bs), but also observed for mAbs against CD4 induced (CD4i) epitopes and gp41 mAbs. V3 mAbs showed significant binding differences to the three constructs, which were refined by SPR analysis. Of interest, the NL4-3/ADA construct with the hybrid NL4-3/ADA CD4bs showed impaired CD4 and CD4bs mAb reactivity despite the presence of the essential elements of the CD4bs epitope. We obtained 3D reconstructions of the NL4-3 and the NL4-3/ADA gp140 trimers via electron microscopy and single particle analysis, which indicates that both constructs inherit a propeller-like architecture. The first 3D reconstruction of an Env construct from an X4 TCLA HIV-1 strain reveals an open conformation, in contrast to recently published more closed structures from R5 Env. Exchanging the X4 V3 spanning region for that of R5 ADA did not alter the open Env architecture as deduced from its very similar 3D reconstruction.
Conclusions: 3D EM analysis showed an apparent open trimer configuration of X4 NL4-3 gp140 that is not modified by exchanging the V3 spanning region for R5 ADA.
The blue light-dependent interaction between the proteins iLID and Nano allows recruiting and patterning proteins on GUV membranes, which thereby capture key features of patterns observed in nature. This photoswitchable protein interaction provides non-invasive, reversible and dynamic control over protein patterns of different sizes with high specificity and spatiotemporal resolution.
MTO1-deficient mouse model mirrors the human phenotype showing complex I defect and cardiomyopathy
(2014)
Recently, mutations in the mitochondrial translation optimization factor 1 gene (MTO1) were identified as causative in children with hypertrophic cardiomyopathy, lactic acidosis and respiratory chain defect. Here, we describe an MTO1-deficient mouse model generated by gene trap mutagenesis that mirrors the human phenotype remarkably well. As in patients, the most prominent signs and symptoms were cardiovascular and included bradycardia and cardiomyopathy. In addition, the mutant mice showed a marked worsening of arrhythmias during induction and reversal of anaesthesia. The detailed morphological and biochemical workup of murine hearts indicated that the myocardial damage was due to complex I deficiency and mitochondrial dysfunction. In contrast, neurological examination was largely normal in Mto1-deficient mice. A translational consequence of this mouse model may be to caution against anaesthesia-related cardiac arrhythmias which may be fatal in patients.
The development of single-photon-counting detectors, such as the PILATUS, has been a major recent breakthrough in macromolecular crystallography, enabling noise-free detection and novel data-acquisition modes. The new EIGER detector features a pixel size of 75 × 75 µm, frame rates of up to 3000 Hz and a dead time as low as 3.8 µs. An EIGER 1M and EIGER 16M were tested on Swiss Light Source beamlines X10SA and X06SA for their application in macromolecular crystallography. The combination of fast frame rates and a very short dead time allows high-quality data acquisition in a shorter time. The ultrafine φ-slicing data-collection method is introduced and validated and its application in finding the optimal rotation angle, a suitable rotation speed and a sufficient X-ray dose are presented. An improvement of the data quality up to slicing at one tenth of the mosaicity has been observed, which is much finer than expected based on previous findings. The influence of key data-collection parameters on data quality is discussed.
Relative orientation of POTRA domains from cyanobacterial Omp85 studied by pulsed EPR spectroscopy
(2016)
Many proteins of the outer membrane of Gram-negative bacteria and of the outer envelope of the endosymbiotically derived organelles mitochondria and plastids have a β-barrel fold. Their insertion is assisted by membrane proteins of the Omp85-TpsB superfamily. These proteins are composed of a C-terminal β-barrel and a different number of N-terminal POTRA domains, three in the case of cyanobacterial Omp85. Based on structural studies of Omp85 proteins, including the five POTRA-domain-containing BamA protein of Escherichia coli, it is predicted that anaP2 and anaP3 bear a fixed orientation, whereas anaP1 and anaP2 are connected via a flexible hinge. We challenged this proposal by investigating the conformational space of the N-terminal POTRA domains of Omp85 from the cyanobacterium Anabaena sp. PCC 7120 using pulsed electron-electron double resonance (PELDOR, or DEER) spectroscopy. The pronounced dipolar oscillations observed for most of the double spin-labeled positions indicate a rather rigid orientation of the POTRA domains in frozen liquid solution. Based on the PELDOR distance data, structure refinement of the POTRA domains was performed taking two different approaches: 1) treating the individual POTRA domains as rigid bodies; and 2) using an all-atom refinement of the structure. Both refinement approaches yielded ensembles of model structures that are more restricted compared to the conformational ensemble obtained by molecular dynamics simulations, with only a slightly different orientation of N-terminal POTRA domains anaP1 and anaP2 compared with the x-ray structure. The results are discussed in the context of the native environment of the POTRA domains in the periplasm.
As a centerpiece of antigen processing, the ATP-binding cassette transporter associated with antigen processing (TAP) became a main target for viral immune evasion. The herpesviral ICP47 inhibits TAP function, thereby suppressing an adaptive immune response. Here, we report on a thermostable ICP47-TAP complex, generated by fusion of different ICP47 fragments. These fusion complexes allowed us to determine the direction and positioning in the central cavity of TAP. ICP47-TAP fusion complexes are arrested in a stable conformation, as demonstrated by MHC I surface expression, melting temperature, and the mutual exclusion of herpesviral TAP inhibitors. We unveiled a conserved region next to the active domain of ICP47 as essential for the complete stabilization of the TAP complex. Binding of the active domain of ICP47 arrests TAP in an open inward facing conformation rendering the complex inaccessible for other viral factors. Based on our findings, we propose a dual interaction mechanism for ICP47. A per se destabilizing active domain inhibits the function of TAP, whereas a conserved C-terminal region additionally stabilizes the transporter. These new insights into the ICP47 inhibition mechanism can be applied for future structural analyses of the TAP complex.
ATP-binding cassette (ABC) transporters, a superfamily of integral membrane proteins, catalyse the translocation of substrates across the cellular membrane by ATP hydrolysis. Here we demonstrate by nucleotide turnover and binding studies based on 31P solid-state NMR spectroscopy that the ABC exporter and lipid A flippase MsbA can couple ATP hydrolysis to an adenylate kinase activity, where ADP is converted into AMP and ATP. Single-point mutations reveal that both ATPase and adenylate kinase mechanisms are associated with the same conserved motifs of the nucleotide-binding domain. Based on these results, we propose a model for the coupled ATPase-adenylate kinase mechanism, involving the canonical and an additional nucleotide-binding site. We extend these findings to other prokaryotic ABC exporters, namely LmrA and TmrAB, suggesting that the coupled activities are a general feature of ABC exporters.
Motivation: Arabidopsis thaliana is a well-established model system for the analysis of the basic physiological and metabolic pathways of plants. Nevertheless, the system is not yet fully understood, although many mechanisms are described, and information for many processes exists. However, the combination and interpretation of the large amount of biological data remain a big challenge, not only because data sets for metabolic paths are still incomplete. Moreover, they are often inconsistent, because they are coming from different experiments of various scales, regarding, for example, accuracy and/or significance. Here, theoretical modeling is powerful to formulate hypotheses for pathways and the dynamics of the metabolism, even if the biological data are incomplete. To develop reliable mathematical models they have to be proven for consistency. This is still a challenging task because many verification techniques fail already for middle-sized models. Consequently, new methods, like decomposition methods or reduction approaches, are developed to circumvent this problem.
Methods: We present a new semi-quantitative mathematical model of the metabolism of Arabidopsis thaliana. We used the Petri net formalism to express the complex reaction system in a mathematically unique manner. To verify the model for correctness and consistency we applied concepts of network decomposition and network reduction such as transition invariants, common transition pairs, and invariant transition pairs.
Results: We formulated the core metabolism of Arabidopsis thaliana based on recent knowledge from literature, including the Calvin cycle, glycolysis and citric acid cycle, glyoxylate cycle, urea cycle, sucrose synthesis, and the starch metabolism. By applying network decomposition and reduction techniques at steady-state conditions, we suggest a straightforward mathematical modeling process. We demonstrate that potential steady-state pathways exist, which provide the fixed carbon to nearly all parts of the network, especially to the citric acid cycle. There is a close cooperation of important metabolic pathways, e.g., the de novo synthesis of uridine-5-monophosphate, the γ-aminobutyric acid shunt, and the urea cycle. The presented approach extends the established methods for a feasible interpretation of biological network models, in particular of large and complex models.
The transporter associated with antigen processing (TAP) selectively translocates antigenic peptides into the endoplasmic reticulum. Loading onto major histocompatibility complex class I molecules and proofreading of these bound epitopes are orchestrated within the macromolecular peptide-loading complex, which assembles on TAP. This heterodimeric ABC-binding cassette (ABC) transport complex is therefore a major component in the adaptive immune response against virally or malignantly transformed cells. Its pivotal role predestines TAP as a target for infectious diseases and malignant disorders. The development of therapies or drugs therefore requires a detailed comprehension of structure and function of this ABC transporter, but our knowledge about various aspects is still insufficient. This review highlights recent achievements on the structure and dynamics of antigenic peptides in complex with TAP. Understanding the binding mode of antigenic peptides in the TAP complex will crucially impact rational design of inhibitors, drug development, or vaccination strategies.
Neuronal ceroid lipofuscinosis (NCL) is a group of neurodegenerative lysosomal storage disorders characterized by vision loss, mental and motor deficits, and spontaneous seizures. Neuropathological analyses of autopsy material from NCL patients and animal models revealed brain atrophy closely associated with glial activity. Earlier reports also noticed loss of retinal cells and reactive gliosis in some forms of NCL. To study this phenomenon in detail, we analyzed the ocular phenotype of CLN6nclf mice, an established mouse model for variant-late infantile NCL. Retinal morphometry, immunohistochemistry, optokinetic tracking, electroretinography, and mRNA expression were used to characterize retinal morphology and function as well as the responses of Müller cells and microglia. Our histological data showed a severe and progressive degeneration in the CLN6nclf retina co-inciding with reactive Müller glia. Furthermore, a prominent phenotypic transformation of ramified microglia to phagocytic, bloated, and mislocalized microglial cells was identified in CLN6nclf retinas. These events overlapped with a rapid loss of visual perception and retinal function. Based on the strong microglia reactivity we hypothesized that dietary supplementation with immuno-regulatory compounds, curcumin and docosahexaenoic acid (DHA), could ameliorate microgliosis and reduce retinal degeneration. Our analyses showed that treatment of three-week-old CLN6nclf mice with either 5% DHA or 0.6% curcumin for 30 weeks resulted in a reduced number of amoeboid reactive microglia and partially improved retinal function. DHA-treatment also improved the morphology of CLN6nclf retinas with a preserved thickness of the photoreceptor layer in most regions of the retina. Our results suggest that microglial reactivity closely accompanies disease progression in the CLN6nclf retina and both processes can be attenuated with dietary supplemented immuno-modulating compounds.