TY - JOUR AB - ICA512/PTPRN is a receptor tyrosine-like phosphatase implicated in the biogenesis and turnover of the insulin secretory granules (SGs) in pancreatic islet beta cells. Previously we found biophysical evidence that its luminal RESP18 homology domain (RESP18HD) forms a biomolecular condensate and interacts with insulin in vitro at close-to-neutral pH, that is, in conditions resembling those present in the early secretory pathway. Here we provide further evidence for the relevance of these findings by showing that at pH 6.8 RESP18HD interacts also with proinsulin-the physiological insulin precursor found in the early secretory pathway and the major luminal cargo of β-cell nascent SGs. Our light scattering analyses indicate that RESP18HD and proinsulin, but also insulin, populate nanocondensates ranging in size from 15 to 300 nm and 10e2 to 10e6 molecules. Co-condensation of RESP18HD with proinsulin/insulin transforms the initial nanocondensates into microcondensates (size >1 μm). The intrinsic tendency of proinsulin to self-condensate implies that, in the ER, a chaperoning mechanism must arrest its spontaneous intermolecular condensation to allow for proper intramolecular folding. These data further suggest that proinsulin is an early driver of insulin SG biogenesis, in a process in which its co-condensation with RESP18HD participates in their phase separation from other secretory proteins in transit through the same compartments but destined to other routes. Through the cytosolic tail of ICA512, proinsulin co-condensation with RESP18HD may further orchestrate the recruitment of cytosolic factors involved in membrane budding and fission of transport vesicles and nascent SGs. AU - Toledo, P.L.* AU - Vazquez, D.S.* AU - Gianotti, A.R.* AU - Abate, M.B.* AU - Wegbrod, C. AU - Torkko, J.M. AU - Solimena, M. AU - Ermácora, M.R.* C1 - 67817 C2 - 54295 CY - 111 River St, Hoboken 07030-5774, Nj Usa TI - Condensation of the β-cell secretory granule luminal cargoes pro/insulin and ICA512 RESP18 homology domain. JO - Protein Sci. VL - 32 IS - 6 PB - Wiley PY - 2023 SN - 0961-8368 ER - TY - JOUR AU - Gestaut, D.* AU - Roh, S.H.* AU - Ma, B.* AU - Pintile, G.* AU - Joachimiak, L.* AU - Leitner, A.* AU - Walzthoeni, T. AU - Aebersold, R.* AU - Chiu, W.* AU - Frydman, J.* C1 - 57138 C2 - 47559 CY - 111 River St, Hoboken 07030-5774, Nj Usa SP - 44-44 TI - The chaperonin TRIC/CCT associates with prefoldin through a conserved electrostatic interface essential for cellular proteostasis. JO - Protein Sci. VL - 28 PB - Wiley PY - 2019 SN - 0961-8368 ER - TY - JOUR AB - Increased efforts have been undertaken to better understand the formation of signaling complexes at cellular membranes. Since the preparation of proteins containing a transmembrane domain or a prenylation motif is generally challenging an alternative membrane anchoring unit that is easy to attach, water-soluble and binds to different membrane mimetics would find broad application. The 33-residue long FATC domain of yeast TOR1 (y1fatc) fulfills these criteria and binds to neutral and negatively charged micelles, bicelles, and liposomes. As a case study, we fused it to the FKBP506-binding region of the protein FKBP38 (FKBP38-BD) and used H-1-N-15 NMR spectroscopy to characterize localization of the chimeric protein to micelles, bicelles, and liposomes. Based on these and published data for y1fatc, its use as a C-terminally attachable membrane anchor for other proteins is compatible with a wide range of buffer conditions (pH circa 6-8.5, NaCl 0 to >150 mM, presence of reducing agents, different salts such as MgCl2 and CaCl2). The high water-solubility of y1fatc enables its use for titration experiments against a membrane-localized interaction partner of the fused target protein. Results from studies with peptides corresponding to the C-terminal 17-11 residues of the 33-residue long domain by 1D H-1 NMR and CD spectroscopy indicate that they still can interact with membrane mimetics. Thus, they may be used as membrane anchors if the full y1fatc sequence is disturbing or if a chemically synthesized y1fatc peptide shall be attached by native chemical ligation, for example, unlabeled peptide to N-15-labeled target protein for NMR studies. AU - de Cicco, M.* AU - Milroy, L.G.* AU - Dames, S.A. C1 - 52809 C2 - 44201 CY - Hoboken SP - 546-560 TI - Target of rapamycin FATC domain as a general membrane anchor: The FKBP-12 like domain of FKBP38 as a case study. JO - Protein Sci. VL - 27 IS - 2 PB - Wiley PY - 2018 SN - 0961-8368 ER - TY - JOUR AB - Structural biology provides essential information for elucidating molecular mechanisms that underlie biological function. Advances in hardware, sample preparation, experimental methods and computational approaches now enable structural analysis of protein complexes with increasing complexity that more closely represent biologically entities in the cellular environment. Integrated multi-disciplinary approaches are required to overcome limitations of individual methods and take advantage of complementary aspects provided by different structural biology techniques. Although X-ray crystallography remains the method of choice for structural analysis of large complexes, crystallization of flexible systems is often difficult and does typically not provide insights into conformational dynamics present in solution. Nuclear magnetic resonance spectroscopy (NMR) is on well-suited to study dynamics at picosecond to second time scales, and to map binding interfaces even of large systems at residue-resolution, but suffers from poor sensitivity with increasing molecular weight. Small angle scattering (SAS) methods provide low resolution information in solution and can characterize dynamics and conformational equilibria complementary to crystallography and NMR. The combination of NMR, crystallography and small angle scattering is thus very useful for analysis of the structure and conformational dynamics of (large) protein complexes in solution. In high molecular weight systems, where NMR data are often sparse, small angle scattering provides additional structural information and can differentiate between NMR-derived models. Scattering data can also validate the solution conformation of a crystal structure and indicate the presence of conformational equilibria. Here, we review current state-of-the-art approaches for combining NMR, crystallography and small angle scattering data to characterize protein complexes in solution. AU - Hennig, J. AU - Sattler, M. C1 - 30968 C2 - 34070 CY - Hoboken SP - 669-682 TI - The dynamic duo: Combining NMR and small angle scattering in structural biology. JO - Protein Sci. VL - 23 IS - 6 PB - Wiley-blackwell PY - 2014 SN - 0961-8368 ER - TY - JOUR AU - Xiao, Y.* AU - Lee, T.* AU - Latham, M.P.* AU - Warner, L. AU - Tanimoto, A.* AU - Pardi, A.* AU - Ahn, N.G.* C1 - 31967 C2 - 34911 CY - Hoboken SP - 263-264 TI - Phosphorylation releases constraints to domain motion in ERK2. JO - Protein Sci. VL - 23 PB - Wiley-Blackwell PY - 2014 SN - 0961-8368 ER - TY - JOUR AB - Post-translational modifications of histone tails are among the most prominent epigenetic marks and play a critical role in transcriptional control at the level of chromatin. The Polycomblike (Pcl) protein is part of a histone methyltransferase complex (Pcl-PRC2) responsible for high levels of histone H3 K27 trimethylation. Studies in Drosophila larvae suggest that Pcl is required for anchoring Pcl-PRC2 at target genes, but how this is achieved is unknown. Pcl comprises a Tudor domain and two PHD fingers. These domains are known to recognize methylated lysine or arginine residues and could contribute to targeting of Pcl-PRC2. Here, we report an NMR structure of the Tudor domain from Drosophila Pcl (Pcl-Tudor) and binding studies with putative ligands. Pcl-Tudor contains an atypical, incomplete aromatic cage that does not interact with known Tudor domain ligands, such as methylated lysines or arginines. Interestingly, human Pcl orthologs exhibit a complete aromatic cage, suggesting that they may recognize methylated lysines. Structural comparison with other Tudor domains suggests that Pcl-Tudor may engage in intra- or intermolecular interactions through an exposed hydrophobic surface patch. AU - Friberg, A. AU - Oddone, A.* AU - Klymenko, T.* AU - Müller, J.* AU - Sattler, M. C1 - 5735 C2 - 27562 SP - 1906-1916 TI - Structure of an atypical Tudor domain in the Drosophila Polycomblike protein. JO - Protein Sci. VL - 19 IS - 10 PB - Wiley-Blackwell PY - 2010 SN - 0961-8368 ER - TY - JOUR AB - Structural investigations are frequently hindered by difficulties in obtaining diffracting crystals of the target protein. Here, we report the crystallization and structure solution of the U2AF homology motif (UHM) domain of splicing factor Puf60 fused to Escherichia coli thioredoxin A. Both modules make extensive crystallographic contacts, contributing to a well-defined crystal lattice with clear electron density for both the thioredoxin and the Puf60-UHM module. We compare two short linker sequences between the two fusion domains, GSAM and GSPPM, for which only the GSAM-linked fusion protein yielded diffracting crystals. While specific interdomain contacts are not observed for both fusion proteins, NMR relaxation data in solution indicate reduced interdomain mobility between the Trx and Puf60-UHM modules. The GSPPM-linked fusion protein is significantly more flexible, albeit both linker sequences have the same number of degrees of torsional freedom. Our analysis provides a rationale for the crystallization of the GSAM-linked fusion protein and indicates that in this case, a four-residue linker between thioredoxin A and the fused target may represent the maximal length for crystallization purposes. Our data provide an experimental basis for the rational design of linker sequences in carrier-driven crystallization and identify thioredoxin A as a powerful fusion partner that can aid crystallization of difficult targets. AU - Corsini, L.* AU - Hothorn, M.* AU - Scheffzek, K.* AU - Sattler, M. AU - Stier, G.* C1 - 4893 C2 - 25937 SP - 2070-2079 TI - Thioredoxin as a fusion tag for carrier-driven crystallization. JO - Protein Sci. VL - 17 IS - 12 PB - Wiley-Blackwell PY - 2008 SN - 0961-8368 ER -