176 research outputs found
Chapter Refinement of Protein Tertiary Structure by Using Spin-Spin Coupling Constants from Nuclear Magnetic Resonance Measurements
Communications engineering / telecommunication
The parallel G-quadruplex structure of vertebrate telomeric repeat sequences is not the preferred folding topology under physiological conditions
G-quadruplex topologies of telomeric repeat sequences from vertebrates were investigated in the presence of molecular crowding (MC) mimetics, namely polyethylene glycol 200 (PEG), Ficoll 70 as well as Xenopus laevis egg extract by CD and NMR spectroscopy and native PAGE. Here, we show that the conformational behavior of the telomeric repeats in X. laevis egg extract or in Ficoll is notably different from that observed in the presence of PEG. While the behavior of the telomeric repeat in X. laevis egg extract or in Ficoll resembles results obtained under dilute conditions, PEG promotes the formation of high-order parallel topologies. Our data suggest that PEG should not be used as a MC mimetic
Dynamics of Bacteriorhodopsin in the DarkâAdapted State from Solution NMR
To achieve efficient proton pumping in the light-driven proton pump bacteriorhodopsin, the protein must be tightly coupled to the retinal to rapidly convert retinal isomerization into protein structural rearrangements. Methyl group dynamics of bR embedded in lipid nanodiscs were determined in the dark-adapted state, and were found to be mostly well-ordered at the cytosolic side. Methyl groups in the M145A mutant of bR, which displays only 10% residual proton pumping activity, are less well ordered suggesting a link between side chain dynamics on the cytosolic side of the bR cavity and proton pumping activity. In addition, slow conformational exchange, attributed to low frequency motions of aromatic rings, was indirectly observed for residues on the extracellular side of the bR cavity. This may be related to reorganization of the water network. These observations provide a detailed picture of previously undescribed equilibrium dynamics on different time scales for ground-state bR
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Regulation of the Activity in the p53 Family Depends on the Organization of the Transactivation Domain.
Despite high sequence homology among the p53 family members, the regulation of their transactivation potential is based on strikingly different mechanisms. Previous studies revealed that the activity of TAp63α is regulated via an autoinhibitory mechanism that keeps inactive TAp63α in a dimeric conformation. While all p73 isoforms are constitutive tetramers, their basal activity is much lower compared with tetrameric TAp63. We show that the dimeric state of TAp63α not only reduces DNA binding affinity, but also suppresses interaction with the acetyltransferase p300. Exchange of the transactivation domains is sufficient to transfer the regulatory characteristics between p63 and p73. Structure determination of the transactivation domains of p63 and p73 in complex with the p300 Taz2 domain further revealed that, in contrast to p53 and p73, p63 has a single transactivation domain. Sequences essential for stabilizing the closed dimer of TAp63α have evolved into a second transactivation domain in p73 and p53.The research was funded by the DFG (DO 545/8 and DO 545/13), the Center for Biomolecular Magnetic Resonance (BMRZ), and the Cluster of Excellence Frankfurt (Macromolecular Complexes). M.T. was supported by a fellowship from the Fonds of the Chemical Industry
Crystal Structure of a PCP/Sfp Complex Reveals the Structural Basis for Carrier Protein Posttranslational Modification
SummaryPhosphopantetheine transferases represent a class of enzymes found throughout all forms of life. From a structural point of view, they are subdivided into three groups, with transferases from group II being the most widespread. They are required for the posttranslational modification of carrier proteins involved in diverse metabolic pathways. We determined the crystal structure of the group II phosphopantetheine transferase Sfp from Bacillus in complex with a substrate carrier protein in the presence of coenzyme A and magnesium, and observed two protein-protein interaction sites. Mutational analysis showed that only the hydrophobic contacts between the carrier proteinâs second helix and the C-terminal domain of Sfp are essential for their productive interaction. Comparison with a similar structure of a complex of human proteins suggests that the mode of interaction is highly conserved in all domains of life
The UBA domain of conjugating enzyme Ubc1/Ube2K facilitates assembly of K48/K63âbranched ubiquitin chains
The assembly of a specific polymeric ubiquitin chain on a target protein is a key event in the regulation of numerous cellular processes. Yet, the mechanisms that govern the selective synthesis of particular polyubiquitin signals remain enigmatic. The homologous ubiquitin-conjugating (E2) enzymes Ubc1 (budding yeast) and Ube2K (mammals) exclusively generate polyubiquitin linked through lysine 48 (K48). Uniquely among E2 enzymes, Ubc1 and Ube2K harbor a ubiquitin-binding UBA domain with unknown function. We found that this UBA domain preferentially interacts with ubiquitin chains linked through lysine 63 (K63). Based on structural modeling, in vitro ubiquitination experiments, and NMR studies, we propose that the UBA domain aligns Ubc1 with K63-linked polyubiquitin and facilitates the selective assembly of K48/K63-branched ubiquitin conjugates. Genetic and proteomics experiments link the activity of the UBA domain, and hence the formation of this unusual ubiquitin chain topology, to the maintenance of cellular proteostasis.Deutsche Forschungsgemeinschaft (DFG)
http://dx.doi.org/10.13039/501100001659MaxâPlanckâGesellschaft (MPG)
http://dx.doi.org/10.13039/501100004189Peer Reviewe
Structural and Functional analysis of the GABARAP interaction motif (GIM)
© 2017 The Authors. Published under the terms of the CC BY 4.0 license. Through the canonical LC3 interaction motif (LIR), [W/F/Y]-X 1 -X 2 -[I/L/V], protein complexes are recruited to autophagosomes to perform their functions as either autophagy adaptors or receptors. How these adaptors/receptors selectively interact with either LC3 or GABARAP families remains unclear. Herein, we determine the range of selectivity of 30 known core LIR motifs towards individual LC3s and GABARAPs. From these, we define a GABARAP Interaction Motif (GIM) sequence ([W/F] -[V/I]-X 2 -V) that the adaptor protein PLEKHM1 tightly conforms to. Using biophysical and structural approaches, we show that the PLEKHM1-LIR is indeed 11-fold more specific for GABARAP than LC3B. Selective mutation of the X 1 and X 2 positions either completely abolished the interaction with all LC3 and GABARAPs or increased PLEKHM1-GIM selectivity 20-fold towards LC3B. Finally, we show that conversion of p62/SQSTM1, FUNDC1 and FIP200 LIRs into our newly defined GIM, by introducing two valine residues, enhances their interaction with endogenous GABARAP over LC3B. The identification of a GABARAP-specific interaction motif will aid the identification and characterization of the expanding array of autophagy receptor and adaptor proteins and their in vivo functions
International Consensus Conference for Advanced Breast Cancer, Lisbon 2019: ABC5 Consensus â Assessment by a German Group of Experts
The 5th International Consensus Conference for Advanced
Breast Cancer (ABC5) took place on November 14â16, 2019,
in Lisbon, Portugal. Its aim is to standardize the treatment of
advanced breast cancer based on the available evidence and
to ensure that all breast cancer patients worldwide receive
adequate treatment and access to new therapies. This year,
the conference focused on developments and study results
in the treatment of patients with hormone receptor-positive/HER2-negative breast cancer as well as precision medicine. As in previous years, patient advocates from around the
world were integrated into the ABC conference and had seats on the ABC consensus panel. In the present paper, a
working group of German breast cancer experts comments
on the results of the on-site ABC5 consensus votes by ABC
panelists regarding their applicability for routine treatment
in Germany. These comments take the recommendations of
the Breast Committee of the Gynecological Oncology Working Group (Arbeitsgemeinschaft GynÀkologische Onkologie;
AGO) into account. The report and assessment presented
here pertain to the preliminary results of the ABC5 consensus. The final version of the statements will be published in
Annals of Oncology and The Breast
Neurocalcin Delta Suppression Protects against Spinal Muscular Atrophy in Humans and across Species by Restoring Impaired Endocytosis
This document is the Accepted Manuscript version of the following article: Riessland et al., 'Neurocalcin Delta Suppression Protects against Spinal Muscular Atrophy in Humans and across Species by Restoring Impaired Endocytosis', The American Journal of Human Genetics, Vol. 100 (2): 297-315, first published online 26 January 2017. The final, published version is available online at doi: http://dx.doi.org/10.1016/j.ajhg.2017.01.005 © 2017 American Society of Human Genetics.Homozygous SMN1 loss causes spinal muscular atrophy (SMA), the most common lethal genetic childhood motor neuron disease. SMN1 encodes SMN, a ubiquitous housekeeping protein, which makes the primarily motor neuron-specific phenotype rather unexpected. SMA-affected individuals harbor low SMN expression from one to six SMN2 copies, which is insufficient to functionally compensate for SMN1 loss. However, rarely individuals with homozygous absence of SMN1 and only three to four SMN2 copies are fully asymptomatic, suggesting protection through genetic modifier(s). Previously, we identified plastin 3 (PLS3) overexpression as an SMA protective modifier in humans and showed that SMN deficit impairs endocytosis, which is rescued by elevated PLS3 levels. Here, we identify reduction of the neuronal calcium sensor Neurocalcin delta (NCALD) as a protective SMA modifier in five asymptomatic SMN1-deleted individuals carrying only four SMN2 copies. We demonstrate that NCALD is a Ca(2+)-dependent negative regulator of endocytosis, as NCALD knockdown improves endocytosis in SMA models and ameliorates pharmacologically induced endocytosis defects in zebrafish. Importantly, NCALD knockdown effectively ameliorates SMA-associated pathological defects across species, including worm, zebrafish, and mouse. In conclusion, our study identifies a previously unknown protective SMA modifier in humans, demonstrates modifier impact in three different SMA animal models, and suggests a potential combinatorial therapeutic strategy to efficiently treat SMA. Since both protective modifiers restore endocytosis, our results confirm that endocytosis is a major cellular mechanism perturbed in SMA and emphasize the power of protective modifiers for understanding disease mechanism and developing therapies.Peer reviewedFinal Accepted Versio
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