@article{WolffGastEversetal.2021, author = {Wolff, Martin and Gast, Klaus and Evers, Andreas and Kurz, Michael and Pfeiffer-Marek, Stefania and Sch{\"u}ler, Anja and Seckler, Robert and Thalhammer, Anja}, title = {A Conserved Hydrophobic Moiety and Helix-Helix Interactions Drive the Self-Assembly of the Incretin Analog Exendin-4}, series = {Biomolecules}, volume = {11}, journal = {Biomolecules}, number = {9}, publisher = {MDPI}, address = {Basel}, issn = {2218-273X}, doi = {10.3390/biom11091305}, pages = {20}, year = {2021}, abstract = {Exendin-4 is a pharmaceutical peptide used in the control of insulin secretion. Structural information on exendin-4 and related peptides especially on the level of quaternary structure is scarce. We present the first published association equilibria of exendin-4 directly measured by static and dynamic light scattering. We show that exendin-4 oligomerization is pH dependent and that these oligomers are of low compactness. We relate our experimental results to a structural hypothesis to describe molecular details of exendin-4 oligomers. Discussion of the validity of this hypothesis is based on NMR, circular dichroism and fluorescence spectroscopy, and light scattering data on exendin-4 and a set of exendin-4 derived peptides. The essential forces driving oligomerization of exendin-4 are helix-helix interactions and interactions of a conserved hydrophobic moiety. Our structural hypothesis suggests that key interactions of exendin-4 monomers in the experimentally supported trimer take place between a defined helical segment and a hydrophobic triangle constituted by the Phe22 residues of the three monomeric subunits. Our data rationalize that Val19 might function as an anchor in the N-terminus of the interacting helix-region and that Trp25 is partially shielded in the oligomer by C-terminal amino acids of the same monomer. Our structural hypothesis suggests that the Trp25 residues do not interact with each other, but with C-terminal Pro residues of their own monomers.}, language = {en} } @misc{WolffGastEversetal.2021, author = {Wolff, Martin and Gast, Klaus and Evers, Andreas and Kurz, Michael and Pfeiffer-Marek, Stefania and Sch{\"u}ler, Anja and Seckler, Robert and Thalhammer, Anja}, title = {A Conserved Hydrophobic Moiety and Helix-Helix Interactions Drive the Self-Assembly of the Incretin Analog Exendin-4}, series = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, journal = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, number = {9}, issn = {1866-8372}, doi = {10.25932/publishup-52208}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-522081}, pages = {22}, year = {2021}, abstract = {Exendin-4 is a pharmaceutical peptide used in the control of insulin secretion. Structural information on exendin-4 and related peptides especially on the level of quaternary structure is scarce. We present the first published association equilibria of exendin-4 directly measured by static and dynamic light scattering. We show that exendin-4 oligomerization is pH dependent and that these oligomers are of low compactness. We relate our experimental results to a structural hypothesis to describe molecular details of exendin-4 oligomers. Discussion of the validity of this hypothesis is based on NMR, circular dichroism and fluorescence spectroscopy, and light scattering data on exendin-4 and a set of exendin-4 derived peptides. The essential forces driving oligomerization of exendin-4 are helix-helix interactions and interactions of a conserved hydrophobic moiety. Our structural hypothesis suggests that key interactions of exendin-4 monomers in the experimentally supported trimer take place between a defined helical segment and a hydrophobic triangle constituted by the Phe22 residues of the three monomeric subunits. Our data rationalize that Val19 might function as an anchor in the N-terminus of the interacting helix-region and that Trp25 is partially shielded in the oligomer by C-terminal amino acids of the same monomer. Our structural hypothesis suggests that the Trp25 residues do not interact with each other, but with C-terminal Pro residues of their own monomers.}, language = {en} } @article{BraigKriegsVoigtlaenderetal.2017, author = {Braig, Friederike and Kriegs, Malte and Voigtlaender, Minna and Habel, Beate and Grob, Tobias and Biskup, Karina and Blanchard, Veronique and Sack, Markus and Thalhammer, Anja and Ben Batalla, Isabel and Braren, Ingke and Laban, Simon and Danielczyk, Antje and Goletz, Steffen and Jakubowicz, Elzbieta and Maerkl, Bruno and Trepel, Martin and Knecht, Rainald and Riecken, Kristoffer and Fehse, Boris and Loges, Sonja and Bokemeyer, Carsten and Binder, Mascha}, title = {Cetuximab Resistance in Head and Neck Cancer Is Mediated by EGFR-K-521 Polymorphism}, series = {Cancer research}, volume = {77}, journal = {Cancer research}, number = {5}, publisher = {American Association for Cancer Research}, address = {Philadelphia}, issn = {0008-5472}, doi = {10.1158/0008-5472.CAN-16-0754}, pages = {1188 -- 1199}, year = {2017}, abstract = {Head and neck squamous cell carcinomas (HNSCC) exhibiting resistance to the EGFR-targeting drug cetuximab poses a challenge to their effective clinical management. Here, we report a specific mechanism of resistance in this setting based upon the presence of a single nucleotide polymorphism encoding EGFR-K-521 (K-allele), which is expressed in > 40\% of HNSCC cases. Patients expressing the K-allele showed significantly shorter progressionfree survival upon palliative treatment with cetuximab plus chemotherapy or radiation. In several EGFR-mediated cancer models, cetuximab failed to inhibit downstream signaling or to kill cells harboring a high K-allele frequency. Cetuximab affinity for EGFR-K-521 was reduced slightly, but ligand-mediated EGFR acti-vation was intact. We found a lack of glycan sialyation on EGFR-K-521 that associated with reduced protein stability, suggesting a structural basis for reduced cetuximab efficacy. CetuGEX, an antibody with optimized Fc glycosylation targeting the same epitope as cetuximab, restored HNSCC sensitivity in a manner associated with antibody-dependent cellular cytotoxicity rather than EGFR pathway inhibition. Overall, our results highlight EGFR-K-521 expression as a key mechanism of cetuximab resistance to evaluate prospectively as a predictive biomarker in HNSCC patients. Further, they offer a preclinical rationale for the use of ADCC-optimized antibodies to treat tumors harboring this EGFR isoform.}, language = {en} } @article{ShouBremerRindfleischetal.2019, author = {Shou, Keyun and Bremer, Anne and Rindfleisch, Tobias and Knox-Brown, Patrick and Hirai, Mitsuhiro and Rekas, Agata and Garvey, Christopher J. and Hincha, Dirk K. and Stadler, Andreas M. and Thalhammer, Anja}, title = {Conformational selection of the intrinsically disordered plant stress protein COR15A in response to solution osmolarity - an X-ray and light scattering study}, series = {Physical chemistry, chemical physics : a journal of European Chemical Societies}, volume = {21}, journal = {Physical chemistry, chemical physics : a journal of European Chemical Societies}, number = {34}, publisher = {Royal Society of Chemistry}, address = {Cambridge}, issn = {1463-9076}, doi = {10.1039/c9cp01768b}, pages = {18727 -- 18740}, year = {2019}, abstract = {The plant stress protein COR15A stabilizes chloroplast membranes during freezing. COR15A is an intrinsically disordered protein (IDP) in aqueous solution, but acquires an alpha-helical structure during dehydration or the increase of solution osmolarity. We have used small- and wide-angle X-ray scattering (SAXS/WAXS) combined with static and dynamic light scattering (SLS/DLS) to investigate the structural and hydrodynamic properties of COR15A in response to increasing solution osmolarity. Coarse-grained ensemble modelling allowed a structure-based interpretation of the SAXS data. Our results demonstrate that COR15A behaves as a biomacromolecule with polymer-like properties which strongly depend on solution osmolarity. Biomacromolecular self-assembly occurring at high solvent osmolarity is initiated by the occurrence of two specific structural subpopulations of the COR15A monomer. The osmolarity dependent structural selection mechanism is an elegant way for conformational regulation and assembly of COR15A. It highlights the importance of the polymer-like properties of IDPs for their associated biological function.}, language = {en} } @misc{SowemimoKnoxBrownBorcherdsetal.2019, author = {Sowemimo, Oluwakemi T. and Knox-Brown, Patrick and Borcherds, Wade and Rindfleisch, Tobias and Thalhammer, Anja and Daughdrill, Gary W.}, title = {Conserved glycines control disorder and function in the cold-regulated protein, COR15A}, series = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, journal = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, number = {1089}, issn = {1866-8372}, doi = {10.25932/publishup-47221}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-472217}, pages = {19}, year = {2019}, abstract = {Cold-regulated (COR) 15A is an intrinsically disordered protein (IDP) from Arabidopsis thaliana important for freezing tolerance. During freezing-induced cellular dehydration, COR15A transitions from a disordered to mostly alpha-helical structure. We tested whether mutations that increase the helicity of COR15A also increase its protective function. Conserved glycine residues were identified and mutated to alanine. Nuclear magnetic resonance (NMR) spectroscopy was used to identify residue-specific changes in helicity for wildtype (WT) COR15A and the mutants. Circular dichroism (CD) spectroscopy was used to monitor the coil-helix transition in response to increasing concentrations of trifluoroethanol (TFE) and ethylene glycol. The impact of the COR15A mutants on the stability of model membranes during a freeze-thaw cycle was investigated by fluorescence spectroscopy. The results of these experiments showed the mutants had a higher content of alpha-helical structure and the increased alpha-helicity improved membrane stabilization during freezing. Comparison of the TFE- and ethylene glycol-induced coil-helix transitions support our conclusion that increasing the transient helicity of COR15A in aqueous solution increases its ability to stabilize membranes during freezing. Altogether, our results suggest the conserved glycine residues are important for maintaining the disordered structure of COR15A but are also compatible with the formation of alpha-helical structure during freezing induced dehydration.}, language = {en} } @article{SowemimoKnoxBrownBorcherdsetal.2019, author = {Sowemimo, Oluwakemi T. and Knox-Brown, Patrick and Borcherds, Wade and Rindfleisch, Tobias and Thalhammer, Anja and Daughdrill, Gary W.}, title = {Conserved Glycines Control Disorder and Function in the Cold-Regulated Protein, COR15A}, series = {Biomolecules}, volume = {9}, journal = {Biomolecules}, number = {3}, publisher = {MDPI}, address = {Basel}, issn = {2218-273X}, doi = {10.3390/biom9030084}, pages = {17}, year = {2019}, abstract = {Cold-regulated (COR) 15A is an intrinsically disordered protein (IDP) from Arabidopsis thaliana important for freezing tolerance. During freezing-induced cellular dehydration, COR15A transitions from a disordered to mostly alpha-helical structure. We tested whether mutations that increase the helicity of COR15A also increase its protective function. Conserved glycine residues were identified and mutated to alanine. Nuclear magnetic resonance (NMR) spectroscopy was used to identify residue-specific changes in helicity for wildtype (WT) COR15A and the mutants. Circular dichroism (CD) spectroscopy was used to monitor the coil-helix transition in response to increasing concentrations of trifluoroethanol (TFE) and ethylene glycol. The impact of the COR15A mutants on the stability of model membranes during a freeze-thaw cycle was investigated by fluorescence spectroscopy. The results of these experiments showed the mutants had a higher content of alpha-helical structure and the increased alpha-helicity improved membrane stabilization during freezing. Comparison of the TFE- and ethylene glycol-induced coil-helix transitions support our conclusion that increasing the transient helicity of COR15A in aqueous solution increases its ability to stabilize membranes during freezing. Altogether, our results suggest the conserved glycine residues are important for maintaining the disordered structure of COR15A but are also compatible with the formation of alpha-helical structure during freezing induced dehydration.}, language = {en} } @misc{SowemimoBorcherdsKnoxBrownetal.2019, author = {Sowemimo, Oluwakemi and Borcherds, Wade and Knox-Brown, Patrick and Rindfleisch, Tobias and Thalhammer, Anja and Daughdrill, Gary}, title = {Evolution of Transient Helicity and Disorder in Late Embryogenesis Abundant Protein COR15A}, series = {Biophysical journal}, volume = {116}, journal = {Biophysical journal}, number = {3}, publisher = {Cell Press}, address = {Cambridge}, issn = {0006-3495}, doi = {10.1016/j.bpj.2018.11.2553}, pages = {473A -- 473A}, year = {2019}, abstract = {Cold regulated protein 15A (COR15A) is a nuclear encoded, intrinsically disordered protein that is found in Arabidopsis thaliana. It belongs to the Late Embryogenesis Abundant (LEA) family of proteins and is responsible for increased freezing tolerance in plants. COR15A is intrinsically disordered in dilute solutions and adopts a helical structure upon dehydration or in the presence of co-solutes such as TFE and ethylene glycol. This helical structure is thought to be important for protecting plants from dehydration induced by freezing. Multiple protein sequence alignments revealed the presence of several conserved glycine residues that we hypothesize keeps COR15A from becoming helical in dilute solutions. Using AGADIR, the change in helical content of COR15A when these conserved glycine residues were mutated to alanine residues was predicted. Based on the predictions, glycine to alanine mutants were made at position 68, and 54,68,81, and 84. Labeled samples of wildtype COR15A and mutant proteins were purified and NMR experiments were performed to examine any structural changes induced by the mutations. To test the effects of dehydration on the structure of COR15A, trifluoroethanol, an alcohol based co solvent that is proposed to induce/stabilize helical structure in peptides was added to the NMR samples, and the results of the experiment showed an increase in helical content, compared to the samples without TFE. To test the functional differences between wild type and the mutants, liposome leakage assays were performed. The results from these assays suggest the more helical mutants may augment membrane stability.}, language = {en} } @article{NavarroRetamalBremerIngolfssonetal.2018, author = {Navarro-Retamal, Carlos and Bremer, Anne and Ingolfsson, Helgi I. and Alzate-Morales, Jans and Caballero, Julio and Thalhammer, Anja and Gonzalez, Wendy and Hincha, Dirk K.}, title = {Folding and Lipid Composition Determine Membrane Interaction of the Disordered Protein COR15A}, series = {Biophysical journal}, volume = {115}, journal = {Biophysical journal}, number = {6}, publisher = {Cell Press}, address = {Cambridge}, issn = {0006-3495}, doi = {10.1016/j.bpj.2018.08.014}, pages = {968 -- 980}, year = {2018}, abstract = {Plants from temperate climates, such as the model plant Arabidopsis thaliana, are challenged with seasonal low temperatures that lead to increased freezing tolerance in fall in a process termed cold acclimation. Among other adaptations, this involves the accumulation of cold-regulated (COR) proteins, such as the intrinsically disordered chloroplast-localized protein COR15A. Together with its close homolog COR15B, it stabilizes chloroplast membranes during freezing. COR15A folds into amphipathic alpha-helices in the presence of high concentrations of low-molecular-mass crowders or upon dehydration. Under these conditions, the (partially) folded protein binds peripherally to membranes. In our study, we have used coarse-grained molecular dynamics simulations to elucidate the details of COR15A-membrane binding and its effects on membrane structure and dynamics. Simulation results indicate that at least partial folding of COR15A and the presence of highly unsaturated galactolipids in the membranes are necessary for efficient membrane binding. The bound protein is stabilized on the membrane by interactions of charged and polar amino acids with galactolipid headgroups and by interactions of hydrophobic amino acids with the upper part of the fatty acyl chains. Experimentally, the presence of liposomes made from a mixture of lipids mimicking chloroplast membranes induces additional folding in COR15A under conditions of partial dehydration, in agreement with the simulation results.}, language = {en} } @article{BremerWolffThalhammeretal.2017, author = {Bremer, Anne and Wolff, Martin and Thalhammer, Anja and Hincha, Dirk K.}, title = {Folding of intrinsically disordered plant LEA proteins is driven by glycerol-induced crowding and the presence of membranes}, series = {The FEBS journal}, volume = {284}, journal = {The FEBS journal}, publisher = {Wiley}, address = {Hoboken}, issn = {1742-464X}, doi = {10.1111/febs.14023}, pages = {919 -- 936}, year = {2017}, abstract = {Late embryogenesis abundant (LEA) proteins are related to cellular dehydration tolerance. Most LEA proteins are predicted to have no stable secondary structure in solution, i.e., to be intrinsically disordered proteins (IDPs), but they may acquire alpha-helical structure upon drying. In the model plant Arabidopsis thaliana, the LEA proteins COR15A and COR15B are highly induced upon cold treatment and are necessary for the plants to attain full freezing tolerance. Freezing leads to increased intracellular crowding due to dehydration by extracellular ice crystals. In vitro, crowding by high glycerol concentrations induced partial folding of COR15 proteins. Here, we have extended these investigations to two related proteins, LEA11 and LEA25. LEA25 is much longer than LEA11 and COR15A, but shares a conserved central sequence domain with the other two proteins. We have created two truncated versions of LEA25 (2H and 4H) to elucidate the structural and functional significance of this domain. Light scattering and CD spectroscopy showed that all five proteins were largely unstructured and monomeric in dilute solution. They folded in the presence of increasing concentrations of trifluoroethanol and glycerol. Additional folding was observed in the presence of glycerol and membranes. Fourier transform infra red spectroscopy revealed an interaction of the LEA proteins with membranes in the dry state leading to a depression in the gel to liquid-crystalline phase transition temperature. Liposome stability assays revealed a cryoprotective function of the proteins. The C- and N-terminal extensions of LEA25 were important in cryoprotection, as the central domain itself (2H, 4H) only provided a low level of protection.}, language = {en} } @misc{BroekerKieleCasjensetal.2018, author = {Broeker, Nina K. and Kiele, Franziska and Casjens, Sherwood R. and Gilcrease, Eddie B. and Thalhammer, Anja and Koetz, Joachim}, title = {In Vitro Studies of Lipopolysaccharide-Mediated DNA Release of Podovirus HK620}, series = {Viruses}, journal = {Viruses}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-417493}, pages = {15}, year = {2018}, abstract = {Gram-negative bacteria protect themselves with an outermost layer containing lipopolysaccharide (LPS). O-antigen-specific bacteriophages use tailspike proteins (TSP) to recognize and cleave the O-polysaccharide part of LPS. However, O-antigen composition and structure can be highly variable depending on the environmental conditions. It is important to understand how these changes may influence the early steps of the bacteriophage infection cycle because they can be linked to changes in host range or the occurrence of phage resistance. In this work, we have analyzed how LPS preparations in vitro trigger particle opening and DNA ejection from the E. coli podovirus HK620. Fluorescence-based monitoring of DNA release showed that HK620 phage particles in vitro ejected their genome at velocities comparable to those found for other podoviruses. Moreover, we found that HK620 irreversibly adsorbed to the LPS receptor via its TSP at restrictive low temperatures, without opening the particle but could eject its DNA at permissive temperatures. DNA ejection was solely stimulated by LPS, however, the composition of the O-antigen dictated whether the LPS receptor could start the DNA release from E. coli phage HK620 in vitro. This finding can be significant when optimizing bacteriophage mixtures for therapy, where in natural environments O-antigen structures may rapidly change.}, language = {en} }