TY - JOUR AB - G protein-coupled receptors (GPCRs) are involved in a multitude of cellular signaling cascades and consequently are a prominent target for pharmaceutical drugs. In the past decades, a growing number of high-resolution structures of GPCRs has been solved, providing unprecedented insights into their mode of action. However, knowledge on the dynamical nature of GPCRs is equally important for a better functional understanding, which can be obtained by NMR spectroscopy. Here, we employed a combination of size exclusion chromatography, thermal stability measurements and 2D-NMR experiments for the NMR sample optimization of the stabilized neurotensin receptor type 1 (NTR1) variant HTGH4 bound to the agonist neurotensin. We identified the short-chain lipid di-heptanoyl-glycero-phosphocholine (DH7PC) as a promising membrane mimetic for high resolution NMR experiments and obtained a partial NMR backbone resonance assignment. However, internal membrane-incorporated parts of the protein were not visible due to lacking amide proton back-exchange. Nevertheless, NMR and hydrogen deuterium exchange (HDX) mass spectrometry experiments could be used to probe structural changes at the orthosteric ligand binding site in the agonist and antagonist bound states. To enhance amide proton exchange we partially unfolded HTGH4 and observed additional NMR signals in the transmembrane region. However, this procedure led to a higher sample heterogeneity, suggesting that other strategies need to be applied to obtain high-quality NMR spectra of the entire protein. In summary, the herein reported NMR characterization is an essential step toward a more complete resonance assignment of NTR1 and for probing its structural and dynamical features in different functional states. AU - Mohamad, M.* AU - Goricanec, D.* AU - Wagner, G.* AU - Hagn, F. C1 - 67776 C2 - 54254 CY - 525 B St, Ste 1900, San Diego, Ca 92101-4495 Usa TI - NMR sample optimization and backbone assignment of a stabilized neurotensin receptor. JO - J. Struct. Biol. VL - 215 IS - 2 PB - Academic Press Inc Elsevier Science PY - 2023 SN - 1047-8477 ER - TY - JOUR AB - AA amyloidosis is one of the most prevalent forms of systemic amyloidosis and affects both humans and other vertebrates. In this study, we compare MAS solid-state NMR data with a recent cryo-EM study of fibrils involving full-length murine SAA1.1. We address the question whether the specific requirements for the reconstitution of an amyloid fibril structure by cryo-EM can potentially yield a bias towards a particular fibril polymorph. We employ fibril seeds extracted from in to vivo material to imprint the fibril structure onto the biochemically produced protein. Sequential assignments yield the secondary structure elements in the fibril state. Long-range DARR and PAR experiments confirm largely the topology observed in the ex-vivo cryo-EM study. We find that the β-sheets identified in the NMR experiments are similar to the β-sheets found in the cryo-EM study, with the exception of amino acids 33–42. These residues cannot be assigned by solid-state NMR, while they adopt a stable β-sheet in the cryo-EM structure. We suggest that the differences between MAS solid-state NMR and cryo-EM data are a consequence of a second conformer involving residues 33–42. Moreover, we were able to characterize the dynamic C-terminal tail of SAA in the fibril state. The C-terminus is flexible, remains detached from the fibrils, and does not affect the SAA fibril structure as confirmed further by molecular dynamics simulations. As the C-terminus can potentially interact with other cellular components, binding to cellular targets can affect its accessibility for protease digestion. AU - Sundaria, A. AU - Liberta, F.* AU - Savran, D.* AU - Sarkar, R. AU - Rodina, N. AU - Peters, C.* AU - Schwierz, N.* AU - Haupt, C.* AU - Schmidt, M.* AU - Reif, B. C1 - 65818 C2 - 52918 TI - SAA fibrils involved in AA amyloidosis are similar in bulk and by single particle reconstitution: A MAS solid-state NMR study. JO - J. Struct. Biol. VL - 6 PY - 2022 SN - 1047-8477 ER - TY - JOUR AB - Small, ultra-red fluorescence protein (smURFP) introduces the non-native biliverdin (BV) chromophore to phycobiliproteins (PBPs), allowing them to be used as transgenic labels for in vivo mammalian imaging. Presently, no structural information exists for PBPs bound to the non-native BV chromophore, which limits the further development of smURFP and related proteins as imaging labels or indicators. Here we describe the first crystal structure of a PBP bound to BV. The structures of smURFP-Y56R with BV and smURFP-Y56F without BV reveal unique oligomerization interfaces different from those in wild-type PBPs bound to native chromophores. Our structures suggest that the oligomerization interface affects the BV binding site, creating a link between oligomerization and chromophorylation that we confirmed through site-directed mutagenesis and that may help guide efforts to improve the notorious chromophorylation of smURFP and other PBPs engineered to bind BV. AU - Fuenzalida Werner, J.P. AU - Janowski, R. AU - Mishra, K. AU - Weidenfeld, I. AU - Niessing, D. AU - Ntziachristos, V. AU - Stiel, A.-C. C1 - 54472 C2 - 45613 CY - 525 B St, Ste 1900, San Diego, Ca 92101-4495 Usa SP - 519-522 TI - Crystal structure of a biliverdin-bound phycobiliprotein: Interdependence of oligomerization and chromophorylation. JO - J. Struct. Biol. VL - 204 IS - 3 PB - Academic Press Inc Elsevier Science PY - 2018 SN - 1047-8477 ER - TY - JOUR AB - Baculovirus-insect cell expression system has become one of the most widely used eukaryotic expression systems for heterologous protein production in many laboratories. The availability of robust insect cell lines, serum-free media, a range of vectors and commercially-packaged kits have supported the demand for maximizing the exploitation of the baculovirus-insect cell expression system. Naturally, this resulted in varied strategies adopted by different laboratories to optimize protein production. Most laboratories have preference in using either the E. coli transposition-based recombination bacmid technology (e.g. Bac-to-Bac((R)))or homologous recombination transfection within insect cells (e.g. flashBAC (TM)). Limited data is presented in the literature to benchmark the protocols used for these baculovirus vectors to facilitate the selection of a system for optimal production of target proteins. Taking advantage of the Protein Production and Purification Partnership in Europe (P4EU) scientific network, a benchmarking initiative was designed to compare the diverse protocols established in thirteen individual laboratories. This benchmarking initiative compared the expression of four selected intracellular proteins (mouse Dicer-2, 204kDa; human ABL1 wildtype, 126kDa; human FMRP, 68 kDa; viral vNSl-Hl, 76kDa). Here, we present the expression and purification results on these proteins and highlight the significant differences in expression yields obtained using different commercially-packaged baculovirus vectors. The highest expression level for difficult-to-express intracellular protein candidates were observed with the EmBacY baculovirus vector system. AU - Stolt-Bergner, P.* AU - Benda, C.* AU - Bergbrede, T.* AU - Besir, H.* AU - Celie, P.H.N.* AU - Chang, C.J.* AU - Drechsel, D.* AU - Fischer, A.* AU - Geerlof, A. AU - Giabbai, B.* AU - van den Heuvel, J.* AU - Huber, G. AU - Knecht, W.* AU - Lehner, A.* AU - Lemaitre, R.* AU - Nordén, K.* AU - Pardee, G.* AU - Racke, I.* AU - Remans, K.* AU - Sander, A.* AU - Scholz, J.* AU - Stadnik, M.* AU - Storici, P.* AU - Weinbruch, D.* AU - Zaror, I.* AU - Lua, L.H.L.* AU - Suppmann, S.* C1 - 53267 C2 - 44515 CY - 525 B St, Ste 1900, San Diego, Ca 92101-4495 Usa SP - 71-80 TI - Baculovirus-driven protein expression in insect cells: A benchmarking study. JO - J. Struct. Biol. VL - 203 IS - 2 PB - Academic Press Inc Elsevier Science PY - 2018 SN - 1047-8477 ER - TY - JOUR AB - Accurate prediction of intra-molecular interactions from amino acid sequence is an important pre-requisite for obtaining high-quality protein models. Over the recent years, remarkable progress in this area has been achieved through the application of novel co-variation algorithms, which eliminate transitive evolutionary connections between residues. In this work we present a new contact prediction method for α-helical transmembrane proteins, MemConP, in which evolutionary couplings are combined with a machine learning approach. MemConP achieves a substantially improved accuracy (precision: 56.0%, recall: 17.5%, MCC: 0.288) compared to the use of either machine learning or co-evolution methods alone. The method also achieves 91.4% precision, 42.1% recall and a MCC of 0.490 in predicting helix-helix interactions based on predicted contacts. The approach was trained and rigorously benchmarked by cross-validation and independent testing on up-to-date non-redundant datasets of 90 and 30 experimental three dimensional structures, respectively. MemConP is a standalone tool that can be downloaded together with the associated training data from http://webclu.bio.wzw.tum.de/MemConP. AU - Honigschmid, P.* AU - Frishman, D. C1 - 47841 C2 - 39512 CY - San Diego SP - 112-123 TI - Accurate prediction of helix interactions and residue contacts in membrane proteins. JO - J. Struct. Biol. VL - 194 IS - 1 PB - Academic Press Inc Elsevier Science PY - 2016 SN - 1047-8477 ER - TY - JOUR AB - Lysine acetylation is a widespread reversible post-translational modification that regulates a broad spectrum of biological activities across various cellular compartments, cell types, tissues, and disease states. While compartment-specific trends in lysine acetylation have recently been investigated, its tissue-specific preferences remain unexplored. Here we present a comprehensive tissue-based analysis of sequence and structural features of lysine acetylation sites (LASs) based on the recent experimental data of Lundby et al. (2012). We show that acetylated substrates are characterized by tissue-specific motifs both in linear amino acid sequence and in spatial environments. We further demonstrate that the general tendency of LASs to reside in ordered regions and, specifically, in α-helices, is also subject to tissue specific variation. In line with previous findings we show that LASs are generally more evolutionarily conserved than non-LASs, especially in proteins with known function and in structurally regular regions. On the other hand, as revealed by metabolic pathway analysis, LASs have diverse cellular functions in different tissues and are frequently associated with tissue-specific protein domains. These findings may imply the existence of tissue-specific lysine acetyltranferases (KATs) and lysine deacetylases (KDACs). AU - Karabulut, N.P.* AU - Frishman, D. C1 - 45118 C2 - 37209 CY - San Diego SP - 39-48 TI - Tissue-specific sequence and structural environments of lysine acetylation sites. JO - J. Struct. Biol. VL - 191 IS - 1 PB - Academic Press Inc Elsevier Science PY - 2015 SN - 1047-8477 ER - TY - JOUR AB - Structural studies of multi-protein complexes, whether by X-ray diffraction, scattering, NMR spectroscopy or electron microscopy, require stringent quality control of the component samples. The inability to produce 'keystone' subunits in a soluble and correctly folded form is a serious impediment to the reconstitution of the complexes. Co-expression of the components offers a valuable alternative to the expression of single proteins as a route to obtain sufficient amounts of the sample of interest. Even in cases where milligram-scale quantities of purified complex of interest become available, there is still no guarantee that good quality crystals can be obtained. At this step, protein engineering of one or more components of the complex is frequently required to improve solubility, yield or the ability to crystallize the sample. Subsequent characterization of these constructs may be performed by solution techniques such as Small Angle X-ray Scattering and Nuclear Magnetic Resonance to identify 'well behaved' complexes. Herein, we recount our experiences gained at protein production and complex assembly during the European 3D Repertoire project (3DR). The goal of this consortium was to obtain structural information on multi-protein complexes from yeast by combining crystallography, electron microscopy, NMR and in silico modeling methods. We present here representative set case studies of complexes that were produced and analyzed within the 3DR project. Our experience provides useful insight into strategies that are more generally applicable for structural analysis of protein complexes. AU - Collinet, B.* AU - Friberg, A. AU - Brooks, M.A.* AU - van den Elzen, T.* AU - Henriot, V.* AU - Dziembowski, A.* AU - Graille, M.* AU - Durand, D.* AU - Leulliot, N.* AU - Saint André, C.* AU - Lazar, N.* AU - Sattler, M. AU - Séraphin, B.* AU - van Tilbeurgh, H.* C1 - 6639 C2 - 29019 CY - San Diego, CA SP - 147-158 TI - Strategies for the structural analysis of multi-protein complexes: Lessons from the 3D-Repertoire project. JO - J. Struct. Biol. VL - 175 IS - 2 PB - Academic Press PY - 2011 SN - 1047-8477 ER - TY - JOUR AB - Structural analysis of multi-domain protein complexes is a key challenge in current biology and a prerequisite for understanding the molecular basis of essential cellular processes. The use of solution techniques is important for characterizing the quaternary arrangements and dynamics of domains and subunits of these complexes. In this respect solution NMR is the only technique that allows atomic- or residue-resolution structure determination and investigation of dynamic properties of multi-domain proteins and their complexes. As experimental NMR data for large protein complexes are sparse, it is advantageous to combine these data with additional information from other solution techniques. Here, the utility and computational approaches of combining solution state NMR with small-angle X-ray and Neutron scattering (SAXS/SANS) experiments for structural analysis of large protein complexes is reviewed. Recent progress in experimental and computational approaches of combining NMR and SAS are discussed and illustrated with recent examples from the literature. The complementary aspects of combining NMR and SAS data for studying multi-domain proteins, i.e. where weakly interacting domains are connected by flexible linkers, are illustrated with the structural analysis of the tandem RNA recognition motif (RRM) domains (RRM1-RRM2) of the human splicing factor U2AF65 bound to a nine-uridine (U9) RNA oligonucleotide. AU - Madl, T. AU - Gabel, F.* AU - Sattler, M. C1 - 4671 C2 - 28106 SP - 472-482 TI - NMR and small-angle scattering-based structural analysis of protein complexes in solution. JO - J. Struct. Biol. VL - 173 IS - 3 PB - Elsevier PY - 2011 SN - 1047-8477 ER - TY - JOUR AB - The bacterial type II protein secretion (T2S) and type IV piliation (T4P) systems share several common features. In particular, it is well established that the T2S system requires the function of a pilus-like structure, called pseudopilus, which is built upon assembly of pilin-like subunits, called pseudopilins. Pilins and pseudopilins have a hydrophobic N-terminal region, which precedes an extended hydrophilic C-terminal region. In the case of pilins, it was shown that oligomerisation and formation of helical fibers, takes place through interaction between the hydrophobic domains. XcpT, is the most abundant protein of the Pseudomonas aeruginosa T2S, and was proposed to be the main component in the pseudopilus. In this study we present the high-resolution NMR structure of the hydrophilic domain of XcpT (XcpTp). XcpTp is lacking the C-terminal disulfide bridged "D" domain found in type IV pilins and likely involved in receptor binding. This is in agreement with the idea that the XcpT-containing pseudopilus is required for protein secretion and not for bacterial attachment. Interestingly, by solving the 3D structure of XcpTp we revealed that the previously called alphabeta-loop pilin region is in fact highly conserved among major type II pseudopilins and constitutes a specific consensus motif for identifying major pseudopilins, which belong to this family. AU - Alphonse, S.* AU - Durand, E.* AU - Douzi, B.* AU - Wägele, B. AU - Darbon, H.* AU - Filloux, A.* AU - Voulhoux, R.* AU - Bernard, C.* C1 - 2713 C2 - 27057 CY - United States SP - 75-80 TI - Structure of the Pseudomonas aeruginosa XcpT pseudopilin, a major component of the type II secretion system. JO - J. Struct. Biol. VL - 169 IS - 1 PB - Academic Press PY - 2010 SN - 1047-8477 ER -