TY  - JOUR
AB  - Interleukin 12 (IL-12) family cytokines connect the innate and adaptive branches of the immune system and regulate immune responses. A unique characteristic of this family is that each member is anα:βheterodimer. For human αsubunits it has been shown that they depend on theirβsubunit for structure formation and secretion from cells. Since subunits are shared within the family and IL-12 as well as IL-23 use the same βsubunit, subunit competition may influence cytokine secretion and thus downstream immunological functions. Here, we rationally design a folding-competent human IL-23α subunit that does not depend on itsβsubunit for structure formation. This engineered variant still forms a functional heterodimeric cytokine but shows less chaperone dependency and stronger affinity in assembly with its βsubunit. It forms IL-23 more efficiently than its natural counterpart, skewing the balance of IL-12 and IL-23 towards more IL-23 formation. Together, our study shows that folding-competent human IL-12 familyαsubunits are obtainable by only few mutations and compatible with assembly and function of the cytokine. These findings might suggest that human α subunits have evolved for assembly-dependent folding to maintain and regulate correct IL-12 family member ratios in the light of subunit competition.
AU  - Aschenbrenner, I.*
AU  - Siebenmorgen, T.
AU  - Lopez, A.
AU  - Parr, M.*
AU  - Ruckgaber, P.*
AU  - Kerle, A.*
AU  - Rührnößl, F.*
AU  - Catici, D.*
AU  - Haslbeck, M.*
AU  - Frishman, D.*
AU  - Sattler, M.
AU  - Zacharias, M.*
AU  - Feige, M.J.*
C1  - 68684
C2  - 54892
CY  - 24-28 Oval Rd, London Nw1 7dx, England
TI  - Assembly-dependent structure formation shapes human interleukin-23 versus interleukin-12 secretion.
JO  - J. Mol. Biol.
VL  - 435
IS  - 23
PB  - Academic Press Ltd- Elsevier Science Ltd
PY  - 2023
SN  - 0022-2836
ER  - 

TY  - JOUR
AB  - N-degron E3 ubiquitin ligases recognize specific residues at the N-termini of substrates. Although molecular details of N-degron recognition are known for several E3 ligases, the range of N-terminal motifs that can bind a given E3 substrate binding domain remains unclear. Here, we discovered capacity of Gid4 and Gid10 substrate receptor subunits of yeast “GID”/human “CTLH” multiprotein E3 ligases to tightly bind a wide range of N-terminal residues whose recognition is determined in part by the downstream sequence context. Screening of phage displaying peptide libraries with exposed N-termini identified novel consensus motifs with non-Pro N-terminal residues binding Gid4 or Gid10 with high affinity. Structural data reveal that conformations of flexible loops in Gid4 and Gid10 complement sequences and folds of interacting peptides. Together with analysis of endogenous substrate degrons, the data show that degron identity, substrate domains harboring targeted lysines, and varying E3 ligase higher-order assemblies combinatorially determine efficiency of ubiquitylation and degradation.
AU  - Chrustowicz, J.*
AU  - Sherpa, D.*
AU  - Teyra, J.*
AU  - Loke, M.S.*
AU  - Popowicz, G.M.
AU  - Basquin, J.*
AU  - Sattler, M.
AU  - Prabu, J.R.*
AU  - Sidhu, S.S.*
AU  - Schulman, B.A.*
C1  - 63611
C2  - 51602
CY  - 24-28 Oval Rd, London Nw1 7dx, England
TI  - Multifaceted N-degron recognition and ubiquitylation by GID/CTLH E3 ligases.
JO  - J. Mol. Biol.
VL  - 434
IS  - 2
PB  - Academic Press Ltd- Elsevier Science Ltd
PY  - 2022
SN  - 0022-2836
ER  - 

TY  - JOUR
AB  - Membrane mimetics are essential for structural and functional studies of membrane proteins. A promising lipid-based system are phospholipid nanodiscs, where two copies of a so-called membrane scaffold protein (MSP) wrap around a patch of lipid bilayer. Consequently, the size of a nanodisc is determined by the length of the MSP. Furthermore, covalent MSP circularization was reported to improve nanodisc stability. However, a more detailed comparative analysis of the biophysical properties of circularized and linear MSP nanodiscs for their use in high-resolution NMR has not been conducted so far. Here, we analyze the membrane fluidity and temperature-dependent size variability of circularized and linear nanodiscs using a large set of analytical methods. We show that MSP circularization does not alter the membrane fluidity in nanodiscs. Further, we show that the phase transition temperature increases for circularized versions, while the cooperativity decreases. We demonstrate that circularized nanodiscs keep a constant size over a large temperature range, in contrast to their linear MSP counterparts. Due to this size stability, circularized nanodiscs are beneficial for high-resolution NMR studies of membrane proteins at elevated temperatures. Despite their slightly larger size as compared to linear nanodiscs, 3D NMR experiments of the voltage-dependent anion channel 1 (VDAC1) in circularized nanodiscs have a markedly improved spectral quality in comparison to VDAC1 incorporated into linear nanodiscs of a similar size. This study provides evidence that circularized MSP nanodiscs are a promising tool to facilitate high-resolution NMR studies of larger and challenging membrane proteins in a native lipid environment.
AU  - Daniilidis, M.*
AU  - Brandl, M.J.*
AU  - Hagn, F.
C1  - 66592
C2  - 53222
CY  - 24-28 Oval Rd, London Nw1 7dx, England
TI  - The advanced properties of circularized MSP nanodiscs facilitate high-resolution NMR studies of membrane proteins: Advanced NMR properties of circular MSP nanodiscs.
JO  - J. Mol. Biol.
VL  - 434
IS  - 24
PB  - Academic Press Ltd- Elsevier Science Ltd
PY  - 2022
SN  - 0022-2836
ER  - 

TY  - JOUR
AB  - MicroRNAs (miRNAs) are small, noncoding RNAs that mediate post-transcriptional downregulation of specific target genes. These transcripts are the products of a two-step processing pathway; primary miRNAs (pri-miRNAs) are processed by Drosha into individual precursor miRNA (pre-miRNA) hairpins, which are subsequently processed by Dicer into mature miRNAs. Single nucleotide polymorphisms (SNPs) that occur in pri-miRNAs, pre-miRNAs and mature miRNAs have been shown to affect the processing of specific target genes by modulating Drosha and Dicer processing or interactions with RNA binding proteins (RBPs). Using NMR and single-molecule optical tweezer experiments, we have investigated the conformational effects of a cancer-linked G/A mutation in the terminal loop of pri-miR-30c RNA, and how this influences binding by the SRSF3 and hnRNP A1 RBPs, which are implicated in its processing. Our results reveal that the wildtype and G/A variant pri-miR-30c RNAs adopt very similar elongated stem-loop structures, both of which are bound by SRSF3. However, while both wildtype and G/A pri-miR-30c RNAs can form dimeric kissing hairpin structures, the G to A mutation results in partial destabilization of the dimer in the variant transcript. This promotes recognition and binding by hnRNP A1, an RBP that enhances pri-miR-30c processing. Our data provide structural insight into the conformational effects of a G/A mutation in pri-miR-30c RNA and how this could affect processing and promote cancer.
AU  - Jones, A.
AU  - Walbrun, A.*
AU  - Falleroni, F.*
AU  - Rief, M.*
AU  - Sattler, M.
C1  - 65515
C2  - 52709
TI  - Conformational effects of a cancer-linked mutation in pri-miR-30c RNA.
JO  - J. Mol. Biol.
VL  - 434
IS  - 18
PY  - 2022
SN  - 0022-2836
ER  - 

TY  - JOUR
AB  - Human PEX14 plays a dual role as docking protein in peroxisomal protein import and as peroxisomal anchor for microtubules (MT), which relates to peroxisome motility. For docking, the conserved N-terminal domain of PEX14 (PEX14-NTD) binds amphipathic alpha-helical ligands, typically comprising one or two aromatic residues, of which human PEX5 possesses eight. Here, we show that the PEX14-NTD also binds to microtubular filaments in vitro with a dissociation constant in nanomolar range. PEX14 interacts with two motifs in the C-terminal region of human ß-tubulin. At least one of the binding motifs is in spatial proximity to the binding site of microtubules (MT) for kinesin. Both PEX14 and kinesin can bind to MT simultaneously. Notably, binding of PEX14 to tubulin can be prevented by its association with PEX5. The data suggest that PEX5 competes peroxisome anchoring to MT by occupying the ß-tubulin-binding site of PEX14. The competitive correlation of matrix protein import and motility may facilitate the homogeneous dispersion of peroxisomes in mammalian cells.
AU  - Reuter, M.*
AU  - Kooshapur, H.
AU  - Suda, J.G.*
AU  - Gaussmann, S.
AU  - Neuhaus, A.*
AU  - Brühl, L.*
AU  - Bharti, P.*
AU  - Jung, M.*
AU  - Schliebs, W.*
AU  - Sattler, M.
AU  - Erdmann, R.*
C1  - 61061
C2  - 50025
CY  - 24-28 Oval Rd, London Nw1 7dx, England
TI  - Competitive microtubule binding of PEX14 coordinates peroxisomal protein import and motility.
JO  - J. Mol. Biol.
VL  - 433
IS  - 5
PB  - Academic Press Ltd- Elsevier Science Ltd
PY  - 2021
SN  - 0022-2836
ER  - 

TY  - JOUR
AB  - MPV17 is an integral inner mitochondrial membrane protein, whose loss-of-function is linked to the hepatocerebral form of the mitochondrial-DNA-depletion syndrome, leading to a tissue-specific reduction of mitochondrial DNA and organ failure in infants. Several disease-causing mutations in MPV17 have been identified and earlier studies with reconstituted protein suggest that MPV17 forms a high conductivity channel in the membrane. However, the molecular and structural basis of the MPV17 functionality remain only poorly understood. In order to make MPV17 accessible to high-resolution structural studies, we here present an efficient protocol for its high-level production in E. coli and refolding into detergent micelles. Using biophysical and NMR methods, we show that refolded MPV17 in detergent micelles adopts a compact structure consisting of six membrane-embedded α-helices. Furthermore, we demonstrate that MPV17 forms oligomers in a lipid bilayer that are further stabilized by disulfide-bridges. In line with these findings, MPV17 could only be inserted into lipid nanodiscs of 8-12 nm in diameter if intrinsic cysteines were either removed by mutagenesis or blocked by chemical modification. Using this nanodisc reconstitution approach, we could show that disease-linked mutations in MPV17 abolish its oligomerization properties in the membrane. These data suggest that, induced by oxidative stress, MPV17 can alter its oligomeric state from a properly folded monomer to a disulfide-stabilized oligomeric pore which might be required for the transport of metabolic DNA precursors into the mitochondrial matrix to compensate for the damage caused by reactive oxygen species.
AU  - Sperl, L.E.*
AU  - Hagn, F.
C1  - 62346
C2  - 50799
CY  - 24-28 Oval Rd, London Nw1 7dx, England
TI  - NMR structural and biophysical analysis of the disease-linked inner mitochondrial membrane protein MPV17.
JO  - J. Mol. Biol.
VL  - 433
IS  - 15
PB  - Academic Press Ltd- Elsevier Science Ltd
PY  - 2021
SN  - 0022-2836
ER  - 

TY  - JOUR
AB  - The study of complex and dynamic biomolecular assemblies is a key challenge in structural biology and requires the use of multiple methodologies providing complementary spatial and temporal information. NMR spectroscopy is a powerful technique that allows high-resolution structure determination of biomolecules as well as investigating their dynamic properties in solution. However, for high-molecular-weight systems, such as biomolecular complexes or multi-domain proteins, it is often only possible to obtain sparse NMR data, posing significant challenges to structure determination. Combining NMR data with information obtained from other solution techniques is therefore an attractive approach. The combination of NMR with small-angle X-ray and/or neutron scattering has been shown to be particularly fruitful. These scattering approaches provide low-resolution information of biomolecules in solution and reflect ensemble-averaged contributions of dynamic conformations for scattering molecules up to megadalton molecular weight. Here, we review recent developments in the combination of nuclear magnetic resonance spectroscopy (NMR) and small-angle scattering (SAS) experiments. We briefly outline the different types of information that are provided by these techniques. We then discuss computational methods that have been developed to integrate NMR and SAS data, particularly considering the presence of dynamic structural ensembles and flexibility of the investigated biomolecules. Finally, recent examples of the successful combination of NMR and SAS are presented to illustrate the utility of their combination. (C) 2020 Elsevier Ltd. All rights reserved.
AU  - Delhommel, F.
AU  - Gabel, F.*
AU  - Sattler, M.
C1  - 58829
C2  - 48582
CY  - 24-28 Oval Rd, London Nw1 7dx, England
SP  - 2890-2912
TI  - Current approaches for integrating solution NMR spectroscopy and small-angle scattering to study the structure and dynamics of biomolecular complexes.
JO  - J. Mol. Biol.
VL  - 432
IS  - 9
PB  - Academic Press Ltd- Elsevier Science Ltd
PY  - 2020
SN  - 0022-2836
ER  - 

TY  - JOUR
AB  - The multi-domain RNA binding protein RBM5 is a molecular signature of metastasis. RBM5 regulates alternative splicing of apoptotic genes including the cell death receptor Fas and the initiator Caspase-2. The RBM5 RanBP2-type zinc finger (Zf1) is known to specifically recognize single-stranded RNAs with high affinity. Here, we study the structure and conformational dynamics of the Zf1 zinc finger of human RBM5 using NMR. We show that the presence of a non-canonical cysteine in Zf1 kinetically destabilizes the protein. Metal-exchange kinetics show that mutation of the cysteine establishes high-affinity coordination of the zinc. Our data indicate that selection of such a structurally destabilizing mutation during the course of evolution could present an opportunity for functional adaptation of the protein.
AU  - Soni, K.
AU  - Martinez-Lumbreras, S.
AU  - Sattler, M.
C1  - 59299
C2  - 48775
CY  - 24-28 Oval Rd, London Nw1 7dx, England
SP  - 4127-4138
TI  - Conformational dynamics from ambiguous zinc coordination in the RanBP2-type zinc finger of RBM5.
JO  - J. Mol. Biol.
VL  - 432
IS  - 14
PB  - Academic Press Ltd- Elsevier Science Ltd
PY  - 2020
SN  - 0022-2836
ER  - 

TY  - JOUR
AB  - 6A10 is a CA XII inhibitory monoclonal antibody, which was demonstrated to reduce the growth of cancer cells in vitro and in a xenograft model of lung cancer. It was also shown to enhance chemosensitivity of multiresistant cancer cell lines and to significantly reduce the number of lung metastases in combination with doxorubicin in mice carrying human triple-negative breast cancer xenografts. Starting from these data, we report here on the development of the 6A10 antigen-binding fragment (Fab), termed Fab6A10, and its functional, biochemical, and structural characterization. In vitro binding and inhibition assays demonstrated that Fab6A10 selectively binds and inhibits CA XII, whereas immunohistochemistry experiments highlighted its capability to stain malignant glioma cells in contrast to the surrounding brain tissue. Finally, the crystallographic structure of CA XII/Fab6A10 complex provided insights into the inhibition mechanism of Fab6A10, showing that upon binding, it obstructs the substrate access to the enzyme active site and interacts with CA XII His64 freezing it in its out conformation. Altogether, these data indicate Fab6A10 as a new promising therapeutic tool against cancer.
AU  - Alterio, V.*
AU  - Kellner, M.
AU  - Esposito, D.*
AU  - Liesche-Starnecker, F.*
AU  - Bua, S.*
AU  - Supuran, C.T.*
AU  - Monti, S.M.*
AU  - Zeidler, R.
AU  - De Simone, G.*
C1  - 57606
C2  - 47843
CY  - 24-28 Oval Rd, London Nw1 7dx, England
SP  - 4910-4921
TI  - Biochemical and structural insights into Carbonic Anhydrase XII/Fab6A10 complex.
JO  - J. Mol. Biol.
VL  - 431
IS  - 24
PB  - Academic Press Ltd- Elsevier Science Ltd
PY  - 2019
SN  - 0022-2836
ER  - 

TY  - JOUR
AB  - The recent discovery of biologically active fully disordered, so called random fuzzy protein protein interactions leads to the question of how the high flexibility of these protein complexes correlates to aggregation and pathologic misfolding.We identify the structural mechanism by which a random fuzzy protein complex composed of the intrinsically disordered proteins alpha-Synuclein and SERF1a is able to potentiate cytotoxic aggregation. A structural model derived from an integrated NMR/SAXS analysis of the reconstituted aSyn:SERF1a complex enabled us to observe the partial deprotection of one precise aSyn amyloid nucleation element in the fully unstructured ensemble. This minimal exposure was sufficient to increase the amyloidogenic tendency of SERF1a-bound aSyn.Our findings provide a structural explanation of the previously observed pro-amyloid activity of SERF1a. They further demonstrate that random fuzziness can trigger a structurally organized disease-associated reaction such as amyloid polymerization.
AU  - Merle, D.A.*
AU  - Witternigg, A.*
AU  - Tam-Amersdorfer, C.*
AU  - Hartlmüller, C.
AU  - Spreitzer, E.*
AU  - Schrank, E.*
AU  - Wagner-Lichtenegger, S.*
AU  - Werzer, O.*
AU  - Zangger, K.*
AU  - Kungl, A.J.*
AU  - Madl, T.*
AU  - Meyer, N.H.*
AU  - Falsone, S.F.*
C1  - 55949
C2  - 46727
CY  - 24-28 Oval Rd, London Nw1 7dx, England
SP  - 2581-2598
TI  - Increased aggregation tendency of alpha-synuclein in a fully disordered protein complex.
JO  - J. Mol. Biol.
VL  - 431
IS  - 14
PB  - Academic Press Ltd- Elsevier Science Ltd
PY  - 2019
SN  - 0022-2836
ER  - 

TY  - JOUR
AB  - Following ingestion, gastrointestinal pathogens compete against the gastrointestinal microbiota and overcome host immune defenses in order to cause infections. Besides employing direct killing mechanisms, the commensal microbiota occupies metabolic niches to outcompete invading pathogens. Salmonella enterica serovar Typhimurium (S. Typhimurium) uses several strategies to successfully colonize the gut and establish infection, of which an increasing number is based on phenotypic heterogeneity within the S. Typhimurium population. The utilization of myo-inositol (MI) and the production of colicin confer a selective advantage over the microbiota in terms of exploitative and interference competition, respectively. In this review, we summarize the genetic basis underlying bistability of MI catabolism and colicin production. As demonstrated by single-cell analyses, a stochastic switch in the expression of the genes responsible for colicin production and MI degradation constitutes the heterogeneity of the two phenotypes. Both genetic systems are tightly regulated to avoid their expression under non-appropriate conditions and possible detrimental effects on bacterial fitness. Moreover, evolutionary mechanisms underlying formation and stability of these phenotypes in S. Typhimurium are discussed. We propose that both MI catabolism and colicin production create a bet-hedging strategy, which provides an adaptive benefit for S. Typhimurium in the fluctuating environment of the mammalian gut.
AU  - Müller, J.
AU  - Spriewald, S.*
AU  - Stecher, B.*
AU  - Stadler, E.*
AU  - Fuchs, T.M.*
C1  - 56565
C2  - 47131
CY  - 24-28 Oval Rd, London Nw1 7dx, England
SP  - 4732-4748
TI  - Evolutionary stability of salmonella competition with the gut microbiota: How the environment fosters heterogeneity in exploitative and interference competition.
JO  - J. Mol. Biol.
VL  - 431
IS  - 23
PB  - Academic Press Ltd- Elsevier Science Ltd
PY  - 2019
SN  - 0022-2836
ER  - 

TY  - JOUR
AB  - BET proteins such as BRD3 are oncogenic transcriptional coactivators. SPOP binding triggers their proteasomal degradation. In both endometrial and prostate cancers, SPOP mutations occur in the MATH domain, but with opposed influence on drug susceptibility. In prostate cancer, SPOP mutations presumably cause increased BET levels, decreasing BET inhibitor drug susceptibility. As opposed, in endometrial cancer, decreased BET levels concomitant with higher BET inhibitor drug susceptibility were observed. Here, we present the to our knowledge first co-crystal structure of SPOP and a bromodomain containing protein (BRD3). Our structural and biophysical data confirm the suggested loss-of-function in prostate cancer-associated SPOP mutants and provide mechanistic explanation. As opposed to previous literature, our data on endometrial cancer-associated SPOP mutants do not show altered binding behavior compared to wild-type SPOP, indicating a more complex regulatory mechanism. SPOP mutation screening may thus be considered a valuable personalized medicine tool for effective antitumor therapy. (C) 2019 Elsevier Ltd. All rights reserved.
AU  - Ostertag, M.S.
AU  - Hutwelker, W.
AU  - Plettenburg, O.
AU  - Sattler, M.
AU  - Popowicz, G.M.
C1  - 55944
C2  - 46688
CY  - 24-28 Oval Rd, London Nw1 7dx, England
SP  - 2213-2221
TI  - Structural insights into BET client recognition of endometrial and prostate cancer-associated SPOP mutants.
JO  - J. Mol. Biol.
VL  - 431
IS  - 11
PB  - Academic Press Ltd- Elsevier Science Ltd
PY  - 2019
SN  - 0022-2836
ER  - 

TY  - JOUR
AB  - The voltage-dependent anion channel (VDAC) resides in the outer mitochondrial membrane and can adopt a closed or open configuration, most likely depending on whether the N-terminal segment (NTS) occupies the pore or protrudes into the cytoplasm. In this study, we calculate the free energy of releasing the NTS from the pore using molecular dynamics simulation. This is complicated by the flexible nature of the NTS, in particular its disordered structure in aqueous solution compared to the pore lumen. We carried out potential of mean force calculations using enhanced sampling or conformational restraints to address the conformational sampling problem. For the binding to the VDAC pore, two systems were considered, featuring either the native VDAC system or a modified system where the NTS is detached from the pore, that is, noncovalently bound in the pore lumen. The calculated free energies required to translocate the NTS from the pore into the solvent moiety are 83.8 or 74.3 kJ mol−1, respectively. The dissociation pathway in VDAC presents two in-pore minima, separated by a low free energy barrier and a membrane-bound intermediate state. Since we observe small changes in pore shape along the NTS dissociation pathway, we suggest that rigidification of the VDAC pore might impair NTS dissociation. The stability of the membrane-bound state of the VDAC NTS is confirmed by independent molecular dynamics simulations showing spontaneous membrane binding of a NTS-derived peptide as well as nuclear magnetic resonance experiments where chemical shift perturbations of the NTS-derived peptide evidence binding to phospholipid nanodiscs.
AU  - Reif, M.M.*
AU  - Fischer, M.*
AU  - Fredriksson, K.*
AU  - Hagn, F.
AU  - Zacharias, M.*
C1  - 54549
C2  - 45657
SP  - 223-243
TI  - The N-Terminal segment of the voltage-dependent anion channel: A possible membrane-bound intermediate in pore unbinding.
JO  - J. Mol. Biol.
VL  - 431
IS  - 2
PY  - 2019
SN  - 0022-2836
ER  - 

TY  - JOUR
AB  - The antibody light chain (LC) consists of two domains and is essential for antigen binding in mature immunoglobulins. The two domains are connected by a highly conserved linker that comprises the structurally important Arg108 residue. In antibody light chain (AL) amyloidosis, a severe protein amyloid disease, the LC and its N-terminal variable domain (V-L) convert to fibrils deposited in the tissues causing organ failure. Understanding the factors shaping the architecture of the LC is important for basic science, biotechnology and for deciphering the principles that lead to fibril formation. In this study, we examined the structure and properties of LC variants with a mutated or extended linker. We show that under destabilizing conditions, the linker modulates the amyloidogenicity of the LC. The fibril formation propensity of LC linker variants and their susceptibility to proteolysis directly correlate implying an interplay between the two LC domains. Using NMR and residual dipolar coupling-based simulations, we found that the linker residue Arg108 is a key factor regulating the relative orientation of the VL and CL domains, keeping them in a bent and dense, but still flexible conformation. Thus, inter-domain contacts and the relative orientation of VL and CL to each other are of major importance for maintaining the structural integrity of the full-length LC. (C) 2018 Elsevier Ltd. All rights reserved.
AU  - Weber, B.*
AU  - Hora, M.*
AU  - Kazman, P.*
AU  - Göbl, C.
AU  - Camilloni, C.*
AU  - Reif, B.*
AU  - Buchner, J.*
C1  - 54713
C2  - 45777
CY  - 24-28 Oval Rd, London Nw1 7dx, England
SP  - 4925-4940
TI  - The antibody light-chain linker regulates domain orientation and amyloidogenicity.
JO  - J. Mol. Biol.
VL  - 430
IS  - 24
PB  - Academic Press Ltd- Elsevier Science Ltd
PY  - 2018
SN  - 0022-2836
ER  - 

TY  - JOUR
AB  - The aggregation of mostly antibody light chain variable (VL) domains into amyloid fibrils in various tissues is the main cause of death in systemic amyloid light chain amyloidosis. Point mutations within the domain are important to shift the VL into the fibrillar pathway. But why and how only some site-specific mutations achieve this still remains elusive. We show here that both destabilizing and surprisingly stable mutants readily predispose an amyloid resistant VL domain to amyloid formation. The decreased thermodynamic stability of the destabilizing mutant results in the accumulation of non-native intermediates that readily populate the amyloid state. Interestingly, the stable mutants establish site-specific non-native interactions with especially nearby serine/threonine residues that unexpectedly do not affect the folding behavior of the VL domain but rather readily induce and stabilize the fibril structure, a previously unrecognized mechanism. These findings provide a new concept for the molecular mechanism of amyloid fibril formation.
AU  - Nokwe, C.N.*
AU  - Hora, M.
AU  - Zacharias, M.*
AU  - Yagi, H.*
AU  - Peschek, J.*
AU  - Reif, B.
AU  - Goto, Y.*
AU  - Buchner, J.*
C1  - 47798
C2  - 39505
CY  - London
SP  - 1315-1332
TI  - A stable mutant predisposes antibody domains to amyloid formation through specific non-native interactions.
JO  - J. Mol. Biol.
VL  - 428
IS  - 6
PB  - Academic Press Ltd- Elsevier Science Ltd
PY  - 2016
SN  - 0022-2836
ER  - 

TY  - JOUR
AU  - Simons, K.*
AU  - Coskun, Ü.
C1  - 50272
C2  - 42244
CY  - London
SP  - 4737-4738
TI  - Membrane lipid galore.
JO  - J. Mol. Biol.
VL  - 428
IS  - 24
PB  - Academic Press Ltd- Elsevier Science Ltd
PY  - 2016
SN  - 0022-2836
ER  - 

TY  - JOUR
AB  - The pH-responsive one component signaling system CadC in Escherichia coli belongs to the family of ToxR-like proteins, whose members share a conserved modular structure, with an N-terminal cytoplasmic winged helix-turn-helix DNA-binding domain being followed by a single transmembrane helix and a C-terminal periplasmic pH-sensing domain. In E. coli CadC a cytoplasmic linker comprising approximately 50 amino acids is essential for transmission of the signal from the sensor to the DNA-binding domain. However, the mechanism of transduction is poorly understood. Using NMR spectroscopy, we demonstrate here that the linker region is intrinsically disordered in solution. Furthermore, mutational analyses showed that it tolerates a range of amino acid substitutions (altering polarity, rigidity, α-helix-forming propensity), is robust to extension, but is sensitive to truncation. Indeed, truncations either reversed the expression profile of the target operon cadBA or decoupled expression from external pH altogether. CadC dimerizes via its periplasmic domain, but light scattering analysis provided no evidence for dimerization of the isolated DNA-binding domain, with or without the linker region. However, bacterial two-hybrid analysis revealed that CadC forms stable dimers in a stimulus- and linker-dependent manner, interacting only at pH<6.8. Strikingly, a variant with reversed cadBA expression profile, which lacks most of the linker, dimerizes preferentially at higher pH. Thus, we propose that the disordered CadC linker is required for transducing the pH-dependent response of the periplasmic sensor into a structural rearrangement which facilitates dimerization of the cytoplasmic CadC DNA-binding domain.
AU  - Buchner, S.*
AU  - Schlundt, A.
AU  - Lassak, J.*
AU  - Sattler, M.
AU  - Jung, K.*
C1  - 44902
C2  - 37141
CY  - London
SP  - 2548-2561
TI  - Structural and functional analysis of the signal transducing linker in the pH-responsive one component system CadC of Escherichia coli.
JO  - J. Mol. Biol.
VL  - 427
IS  - 15
PB  - Academic Press Ltd- Elsevier Science Ltd
PY  - 2015
SN  - 0022-2836
ER  - 

TY  - JOUR
AB  - Glycosphingolipid metabolism relies on selective recruitment of the pleckstrin homology (PH) domains of FAPP proteins to the trans-Golgi network. The mechanism involved is unclear but requires recognition of phosphatidylinositol-4-phosphate (PI4P) within the Golgi membrane. We investigated the molecular basis of FAPP1-PH domain interactions with PI4P bilayers in liposome sedimentation and membrane partitioning assays. Our data reveals a mechanism in which FAPP-PH proteins preferentially target PI4P-containing liquid disordered membranes, while liquid ordered membranes were disfavored. Additionally, NMR spectroscopy was used to identify the binding determinants responsible for recognizing trans-Golgi network-like bicelles including phosphoinositide and neighboring lipid molecules. Membrane penetration by the FAPP1-PH domain was mediated by an exposed, conserved hydrophobic wedge next to the PI4P recognition site and ringed by a network of complementary polar residues and basic charges. Our data illuminates how insertion of a structured loop provides selectivity for sensing membrane fluidity and targeting to defined membrane zones and organelles. The determinants of this membrane sensing process are conserved across the CERT, OSBP and FAPP family. Hence, lipid gradients not only result in differential membrane ordering along the secretory pathway but also specifically localize diverse proteins through recognition of ensembles of lipid ligands in dynamic and deformable bilayers in order to promote anterograde trafficking.
AU  - Lenoir, M.*
AU  - Grzybek, M.
AU  - Majkowski, M.*
AU  - Rajesh, S.*
AU  - Kaur, J.*
AU  - Whittaker, S.B.*
AU  - Coskun, Ü.
AU  - Overduin, M.*
C1  - 43508
C2  - 36532
CY  - London
SP  - 966-981
TI  - Structural basis of dynamic membrane recognition by trans-Golgi network specific FAPP proteins.
JO  - J. Mol. Biol.
VL  - 427
IS  - 4
PB  - Academic Press Ltd- Elsevier Science Ltd
PY  - 2015
SN  - 0022-2836
ER  - 

TY  - JOUR
AB  - The green tea compound epigallocatechin-3-gallate (EGCG) inhibits Alzheimer's disease beta-amyloid peptide (A beta) neurotoxicity. Solution-state NMR allows probing initial EGCG-A beta interactions. We show that EGCG-induced A beta oligomers adopt a well-defined structure and are amenable for magic angle spinning solid-state NMR investigations. We find that EGCG interferes with the aromatic hydrophobic core of A beta. The C-terminal part of the A beta peptide (residues 22-39) adopts a beta-sheet conformation, whereas the N-terminus (residues 1-20) is unstructured. The characteristic salt bridge involving residues D23 and K28 is present in the structure of these oligomeric A beta aggregates as well. The structural analysis of small-molecule-induced amyloid aggregates will open new perspectives for Alzheimer's disease drug development.
AU  - Lopez del Amo, J.M.
AU  - Fink, U.*
AU  - Dasari, M.
AU  - Grelle, G.*
AU  - Wanker, E.E.*
AU  - Bieschke, J.*
AU  - Reif, B.
C1  - 8628
C2  - 30433
SP  - 517-524
TI  - Structural properties of EGCG-induced, nontoxic Alzheimer's disease Aβ oligomers.
JO  - J. Mol. Biol.
VL  - 421
IS  - 4-5
PB  - Elsevier 
PY  - 2012
SN  - 0022-2836
ER  - 

TY  - JOUR
AB  - Steroids and retinoids are signaling molecules that control a variety of physiological processes. 17beta-Hydroxysteroid dehydrogenase type 1 (17beta-HSD1) catalyzes the reduction of estrone to estradiol, supplying biologically active estrogen-regulating sex-specific differentiation. Photoreceptor-associated retinol dehydrogenase (prRDH) is evolutionarily closely related to 17beta-HSD1 but reduces all-trans retinal to all-trans retinol, contributing to rhodopsin regeneration in the visual cycle. Sequence alignment revealed a new enzyme-specific conserved amino acid close to the active site: methionine (position 144 in human enzyme) in prRDH and glycine (position 145) in 17beta-HSD1. We investigated the role of this residue in substrate discrimination in human and zebrafish enzymes. Both recombinant enzymes were expressed in HEK 293 cells followed by normalization of expression by semiquantitative Western blots. Changing of the prRDH-specific methionine to glycine resulted in a gain of function: the mutants now catalyzed the reduction of estrone and all-trans retinal. Human and zebrafish wild-type 17beta-HSD1s efficiently catalyzed the reduction of all-trans retinal to its alcohol. Exchange of glycine for methionine increased the catalytic activity of 17beta-HSD1 toward all-trans retinal in zebrafish but not in the human enzyme, in which the opposite effect was observed. Molecular modeling showed that the zebrafish 17beta-HSD1 substrate-binding pocket is similar to that of prRDH and methionine insertion benefits all-trans retinal reduction. In contrast, in human 17beta-HSD1, the insertion of the bulky methionine causes a disruption of substrate-binding site. We demonstrate for the first time the role of a single amino acid in the evolution of these functionally diverse enzymes and suggest new physiological functions for 17beta-HSD1 in retinoid metabolism. This has implications for the validation of inhibitors of 17beta-HSD1 developed for cancer treatment.
AU  - Haller, F.
AU  - Moman, E.*
AU  - Hartmann, R.W.*
AU  - Adamski, J.
AU  - Mindnich, R.*
C1  - 1528
C2  - 27269
SP  - 255-267
TI  - Molecular framework of steroid/retinoid discrimination in 17β-hydroxysteroid dehydrogenase type 1 and photoreceptor-associated retinol dehydrogenase.
JO  - J. Mol. Biol.
VL  - 399
IS  - 2
PB  - Elsevier
PY  - 2010
SN  - 0022-2836
ER  - 

TY  - JOUR
AB  - The Tudor-SN protein (p100, SND1) has been implicated in a variety of cellular processes, Such as transcription, processing of edited double-stranded RNA, and splicing regulation. Molecular details of these functions are not vet understood. Tudor domains have previously been shown to bind methylated ligands, such as methylated lysines and arginines. It has been suggested that the role of TUdor-SN in splicing may involve binding to such methylated ligands or to the methylated 5' cap of spliceosomal snRNAs. Here, we report the crystal structure of the extended Tudor domain of Tudor-SN from Drosophila melanogaster to a resolution of 2.1 angstrom. NMR secondary chemical shifts, relaxation data, and residual dipolar Couplings indicate that the solution and Crystal Structures are similar. Binding of various ligands was investigated by NMR. Binding sites and affinities were characterized by chemical shift perturbations. We show that the aromatic cage of the Tudor domain specifically binds a peptide containing symmetrically dimethylated arginines (sbMA) with micromolar affinity, while the same peptide comprising nonmethylated arginines does not show significant chemical shift perturbations. Tudor-SN preferentially recognizes sDMA over asymmetrically dimethylated arginine (aDMA). In contrast, two 5' cap analogues with different methylation patterns, as well as mono-, di-, and trimethyllysines, show no binding. Our data demonstrate that the Tudor domain of Tudor-SN specifically recognizes sDMA-containing ligands. The aromatic cage of TUdor-SN is very similar to the one in the Tudor domain of the survival of motor neuron protein, which also recognizes sDMA peptides, indicating a conserved binding motif for this methylation mark. Recognition of sDMA in the C-terminal tails of spliceosomal Sm proteins suggests how Tudor-SN may interact with small nuclear ribonucleoprotein particles during the regulation of splicing.
AU  - Friberg, A.R.
AU  - Corsini, L.*
AU  - Mourao, A.
AU  - Sattler, M.
C1  - 1843
C2  - 26887
SP  - 921-934
TI  - Structure and ligand binding of the extended Tudor domain of D. melanogaster Tudor-SN.
JO  - J. Mol. Biol.
VL  - 387
IS  - 4
PB  - Elsevier
PY  - 2009
SN  - 0022-2836
ER  - 

TY  - JOUR
AU  - Antonov, A.V.
AU  - Mewes, H.-W.
C1  - 4447
C2  - 24035
SP  - 289-296
TI  - Complex functionality of gene groups identified from high-throughput data.
JO  - J. Mol. Biol.
VL  - 363
PY  - 2006
SN  - 0022-2836
ER  - 

TY  - JOUR
AU  - Greenwood, A.D.*
AU  - Horsch, M.
AU  - Stengel, A.
AU  - Vorberg, I.*
AU  - Lutzny, G.*
AU  - Maas, E.*
AU  - Schädler, S.
AU  - Erfle, V.
AU  - Beckers, J.
AU  - Schätzl, H.*
AU  - Leib-Mösch, C.
C1  - 5355
C2  - 22778
SP  - 487-500
TI  - Cell line dependent RNA expression profiles of prion-infected mouse neuronal cells.
JO  - J. Mol. Biol.
VL  - 349
PY  - 2005
SN  - 0022-2836
ER  - 

TY  - JOUR
AU  - Pagel, P.
AU  - Wong, P.
AU  - Frishman, D.*
C1  - 4103
C2  - 22395
SP  - 1331-1346
TI  - A domain interaction map based on phylogenetic profiling.
JO  - J. Mol. Biol.
VL  - 344
PY  - 2004
SN  - 0022-2836
ER  - 

TY  - JOUR
AB  - The evolutionarily conserved Notch signal transduction pathway regulates cell fate and cellular differentiation in various tissues and has essential functions in embryonic patterning and tumorigenesis. Cell-cell signaling by the Notch pathway is mediated by the interaction of the transmembrane receptor Notch with its ligands Delta or Jagged presented on adjacent cells. Whereas signal transduction to Notch expressing cells has been described, it is unclear whether Delta-dependent signaling may exist within the Delta-expressing cell. Here, we report on the identification of Acvrinp1, a MAGUK family member, interacting with the intracellular domain of Delta1 (Dll1). We confirmed the interaction between Dll1 and Acvrinp1 by pull-down experiments in vitro and in a mammalian two-hybrid system in vivo. We delimited the fourth PDZ domain of Acvrinp1 and the PDZ-binding domain of Dll1 as major interacting domains. In situ expression analyses in mouse embryos revealed that Dll1 and Acvrinp1 show partly overlapping but distinct expression patterns, for example, in the central nervous system and the vibrissae buds. Further, we found that expression of Acvrinp1 is altered in Dll1 loss-of-function mouse embryos. © 2003 Elsevier Ltd. All rights reserved.
AU  - Pfister, S.
AU  - Przemeck, G.K.H.
AU  - Gerber, J.-K.
AU  - Beckers, J.
AU  - Adamski, J.
AU  - Hrabě de Angelis, M.
C1  - 10056
C2  - 21168
SP  - 229-235
TI  - Interaction of the MAGUK family member Acvrinp1 and the cytoplasmic domain of the notch ligand Delta1.
JO  - J. Mol. Biol.
VL  - 333
IS  - 2
PY  - 2003
SN  - 0022-2836
ER  - 

TY  - JOUR
AU  - Roulet, E.*
AU  - Bucher, P.*
AU  - Schneider, R.
AU  - Wingender, E.*
AU  - Dusserre, Y.*
AU  - Werner, T.
AU  - Mermod, N.*
C1  - 21611
C2  - 19742
SP  - 833-848
TI  - Experimental analysis and computer prediction of CTF/NFI transcription factor DNA binding sites.
JO  - J. Mol. Biol.
VL  - 297
PY  - 2000
SN  - 0022-2836
ER  - 

TY  - JOUR
AU  - Scherf, M.
AU  - Klingenhoff, A.
AU  - Werner, T.
C1  - 1544
C2  - 22799
SP  - 599-606
TI  - Highly specific localization of promoter regions in large genomic sequences by promoterInspector: A novel context analysis approach.
JO  - J. Mol. Biol.
VL  - 297
PY  - 2000
SN  - 0022-2836
ER  - 

TY  - JOUR
AB  - Mutator strains of the fission yeast Schizosaccharomyces pombe produce mitochondrial respiratory deficient mutants at a high rate, and roughly 20% of these mutants carry deletions in the range of 50 to 1500 base-pairs. To elucidate the mechanism of deletion we have sequenced ten deletion mutants in the mosaic gene encoding apocytochrome b (cob) and three in the split gene coding for the first subunit of cytochrome c oxidase (cox1). Of 13 deletions, ten are correlated with the presence of direct repeats, which could promote deletions by slipped mispairing during DNA replication. In some of these mutants, the termini are located in possible DNA secondary structures. In three independently isolated mutants with identical deletions in the cob gene, the 5′ deletion endpoint coincides with the 3′ splice point of the intron, whereas the 3′ endpoint of the deletion exhibits pronounced homology with the 5′ splice point of the intron. This result suggests that these deletions might be initiated by erroneous RNA splicing.
AU  - Ahne, A.
AU  - Muller-Derlich, J.*
AU  - Merlos-Lange, A.M.*
AU  - Kanbay, F.*
AU  - Wolf, K.*
AU  - Lang, B.F.*
C1  - 41713
C2  - 38214
SP  - 725-734
TI  - Two distinct mechanisms for deletion in mitochondrial DNA of Schizosaccharomyces pombe mutator strains. Slipped mispairing mediated by direct repeats and erroneous intron splicing.
JO  - J. Mol. Biol.
VL  - 202
IS  - 4
PY  - 1988
SN  - 0022-2836
ER  -