FMP Publications

Our publications are recorded in a searchable database since 2010, updates will be added regularly.

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Direct assessment of substrate binding to the Neurotransmitter:Sodium Symporter LeuT by solid state NMR
Erlendsson(*), S., Gotfryd(*), K., Larsen, F. H., Mortensen(*), J. S., Geiger, M. A., van Rossum, B. J., Oschkinat, H., Gether(*), U., Teilum(*), K.; Loland(*), C. J.
Elife, 6

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: The Neurotransmitter:Sodium Symporters (NSSs) represent an important class of proteins mediating sodium-dependent uptake of neurotransmitters from the extracellular space. The substrate binding stoichiometry of the bacterial NSS protein, LeuT, and thus the principal transport mechanism, has been heavily debated. Here we used solid state NMR to specifically characterize the bound leucine ligand and probe the number of binding sites in LeuT. We were able to produce high-quality NMR spectra of substrate bound to microcrystalline LeuT samples and identify one set of sodium-dependent substrate-specific chemical shifts. Furthermore, our data show that the binding site mutants F253A and L400S, which probe the major S1 binding site and the proposed S2 binding site, respectively, retain sodium-dependent substrate binding in the S1 site similar to the wild-type protein. We conclude that under our experimental conditions there is only one detectable leucine molecule bound to LeuT.

Claudins are essential for cell shape changes and convergent extension movements during neural tube closure
Baumholtz(*), A. I., Simard(*), A., Nikolopoulou(*), E., Oosenbrug(*), M., Collins(*), M. M., Piontek, A., Krause, G., Piontek(*), J., Greene(*), N. D. E.; Ryan(*), A. K.
Developmental biology, 428:25-38

Tags: Structural Bioinformatics and Protein Design (Krause, G.)

Abstract: During neural tube closure, regulated changes at the level of individual cells are translated into large-scale morphogenetic movements to facilitate conversion of the flat neural plate into a closed tube. Throughout this process, the integrity of the neural epithelium is maintained via cell interactions through intercellular junctions, including apical tight junctions. Members of the claudin family of tight junction proteins regulate paracellular permeability, apical-basal cell polarity and link the tight junction to the actin cytoskeleton. Here, we show that claudins are essential for neural tube closure: the simultaneous removal of Cldn3, -4 and -8 from tight junctions caused folate-resistant open neural tube defects. Their removal did not affect cell type differentiation, neural ectoderm patterning nor overall apical-basal polarity. However, apical accumulation of Vangl2, RhoA, and pMLC were reduced, and Par3 and Cdc42 were mislocalized at the apical cell surface. Our data showed that claudins act upstream of planar cell polarity and RhoA/ROCK signaling to regulate cell intercalation and actin-myosin contraction, which are required for convergent extension and apical constriction during neural tube closure, respectively.

Design of S-Allylcysteine in Situ Production and Incorporation Based on a Novel Pyrrolysyl-tRNA Synthetase Variant
Exner(*), M. P., Kuenzl(*), T., To(*), T. M., Ouyang(*), Z., Schwagerus, S., Hoesl(*), M. G., Hackenberger, C. P., Lensen(*), M. C., Panke(*), S.; Budisa(*), N.
Chembiochem, 18:85-90

Tags: Chemical Biology II (Hackenberger)

Abstract: The noncanonical amino acid S-allyl cysteine (Sac) is one of the major compounds of garlic extract and exhibits a range of biological activities. It is also a small bioorthogonal alkene tag capable of undergoing controlled chemical modifications, such as photoinduced thiol-ene coupling or Pd-mediated deprotection. Its small size guarantees minimal interference with protein structure and function. Here, we report a simple protocol efficiently to couple in-situ semisynthetic biosynthesis of Sac and its incorporation into proteins in response to amber (UAG) stop codons. We exploited the exceptional malleability of pyrrolysyl-tRNA synthetase (PylRS) and evolved an S-allylcysteinyl-tRNA synthetase (SacRS) capable of specifically accepting the small, polar amino acid instead of its long and bulky aliphatic natural substrate. We succeeded in generating a novel and inexpensive strategy for the incorporation of a functionally versatile amino acid. This will help in the conversion of orthogonal translation from a standard technique in academic research to industrial biotechnology.

Intersectin associates with synapsin and regulates its nanoscale localization and function
Gerth(*), F., Jäpel, M., Pechstein, A., Kochlamazashvili, G., Lehmann, M., Puchkov, D., Onofri(*), F., Benfenati(*), F., Nikonenko(*), A. G., Maritzen, T., Freund(*), C.; Haucke, V.
Proc Natl Acad Sci U S A, 114:12057-12062

Tags: Molecular Pharmacology and Cell Biology (Haucke); Membrane Traffic and Cell Motility (Maritzen)

Abstract: Neurotransmission is mediated by the exocytic release of neurotransmitters from readily releasable synaptic vesicles (SVs) at the active zone. To sustain neurotransmission during periods of elevated activity, release-ready vesicles need to be replenished from the reserve pool of SVs. The SV-associated synapsins are crucial for maintaining this reserve pool and regulate the mobilization of reserve pool SVs. How replenishment of release-ready SVs from the reserve pool is regulated and which other factors cooperate with synapsins in this process is unknown. Here we identify the endocytic multidomain scaffold protein intersectin as an important regulator of SV replenishment at hippocampal synapses. We found that intersectin directly associates with synapsin I through its Src-homology 3 A domain, and this association is regulated by an intramolecular switch within intersectin 1. Deletion of intersectin 1/2 in mice alters the presynaptic nanoscale distribution of synapsin I and causes defects in sustained neurotransmission due to defective SV replenishment. These phenotypes were rescued by wild-type intersectin 1 but not by a locked mutant of intersectin 1. Our data reveal intersectin as an autoinhibited scaffold that serves as a molecular linker between the synapsin-dependent reserve pool and the presynaptic endocytosis machinery.

Structural Basis of the Oncogenic Interaction of Phosphatase PRL-1 with the Magnesium Transporter CNNM2
Gimenez-Mascarell(*), P., Oyenarte(*), I., Hardy(*), S., Breiderhoff(*), T., Stuiver, M., Kostantin(*), E., Diercks(*), T., Pey(*), A. L., Ereno-Orbea(*), J., Martinez-Chantar(*), M. L., Khalaf-Nazzal(*), R., Claverie-Martin(*), F., Müller(*), D., Tremblay(*), M. L.; Martinez-Cruz(*), L. A.
J Biol Chem, 292:786-801

Tags: In-Cell NMR (Selenko)

Abstract: Phosphatases of regenerating liver (PRLs), the most oncogenic of all protein-tyrosine phosphatases (PTPs), play a critical role in metastatic progression of cancers. Recent findings established a new paradigm by uncovering that their association with magnesium transporters of the cyclin M (CNNM) family causes a rise in intracellular magnesium levels that promote oncogenic transformation. Recently, however, essential roles for regulation of the circadian rhythm and reproduction of the CNNM family have been highlighted. Here, we describe the crystal structure of PRL-1 in complex with the Bateman module of CNNM2 (CNNM2BAT), which consists of two cystathionine beta-synthase (CBS) domains (IPR000664) and represents an intracellular regulatory module of the transporter. The structure reveals a heterotetrameric association, consisting of a disc-like homodimer of CNNM2BAT bound to two independent PRL-1 molecules, each one located at opposite tips of the disc. The structure highlights the key role played by Asp-558 at the extended loop of the CBS2 motif of CNNM2 in maintaining the association between the two proteins and proves that the interaction between CNNM2 and PRL-1 occurs via the catalytic domain of the phosphatase. Our data shed new light on the structural basis underlying the interaction between PRL phosphatases and CNNM transporters and provides a hypothesis about the molecular mechanism by which PRL-1, upon binding to CNNM2, might increase the intracellular concentration of Mg2+ thereby contributing to tumor progression and metastasis. The availability of this structure sets the basis for the rational design of compounds modulating PRL-1 and CNNM2 activities.

Small Molecules Targeting Human N-Acetylmannosamine Kinase
Hinderlich(*), S., Neuenschwander, M., Wratil(*), P. R., Oder, A., Lisurek, M., Nguyen(*), L. D., von Kries, J. P.; Hackenberger, C. P. R.

Tags: Chemical Biology II (Hackenberger), Screening Unit ( von Kries)

Abstract: N-Acetylmannosamine kinase (MNK) plays a key role in the biosynthesis of sialic acids and glycosylation of proteins. Sialylated glycoconjugates affect a large number of biological processes, including immune modulation and cancer transformation. In search of effective inhibitors of MNK we applied high-throughput screening of drug-like small molecules. By applying different orthogonal assays for their validation we identified four potential MNK-specific inhibitors with IC50 values in the low-micromolar range. Molecular modelling of the inhibitors into the active site of MNK supports their binding to the sugar or the ATP-binding pocket of the enzyme or both. These compounds are promising for downregulation of the sialic acid content of glycoconjugates and for studying the functional contribution of sialic acids to disease development.

Heparan Sulfates Support Pyramidal Cell Excitability, Synaptic Plasticity, and Context Discrimination
Minge(*), D., Senkov(*), O., Kaushik(*), R., Herde(*), M. K., Tikhobrazova(*), O., Wulff(*), A. B., Mironov(*), A., van Kuppevelt(*), T. H., Oosterhof(*), A., Kochlamazashvili, G., Dityatev(*), A.; Henneberger(*), C.
Cerebral cortex (New York, N.Y. : 1991), 27:903-918

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: Heparan sulfate (HS) proteoglycans represent a major component of the extracellular matrix and are critical for brain development. However, their function in the mature brain remains to be characterized. Here, acute enzymatic digestion of HS side chains was used to uncover how HSs support hippocampal function in vitro and in vivo. We found that long-term potentiation (LTP) of synaptic transmission at CA3-CA1 Schaffer collateral synapses was impaired after removal of highly sulfated HSs with heparinase 1. This reduction was associated with decreased Ca2+ influx during LTP induction, which was the consequence of a reduced excitability of CA1 pyramidal neurons. At the subcellular level, heparinase treatment resulted in reorganization of the distal axon initial segment, as detected by a reduction in ankyrin G expression. In vivo, digestion of HSs impaired context discrimination in a fear conditioning paradigm and oscillatory network activity in the low theta band after fear conditioning. Thus, HSs maintain neuronal excitability and, as a consequence, support synaptic plasticity and learning.

Structure of the competence pilus major pilin ComGC in Streptococcus pneumoniae
Muschiol(*), S., Erlendsson(*), S., Aschtgen(*), M. S., Oliveira(*), V., Schmieder, P., de Lichtenberg(*), C., Teilum(*), K., Boesen(*), T., Akbey(*), Ü.; Henriques-Normark(*), B.
J Biol Chem, 292:14134-14146

Tags: Solution NMR (Schmieder)

Abstract: Type IV pili are important virulence factors on the surface of many pathogenic bacteria and have been implicated in a wide range of diverse functions, including attachment, twitching motility, biofilm formation, and horizontal gene transfer. The respiratory pathogen Streptococcus pneumoniae deploys type IV pili to take up DNA during transformation. These "competence pili" are composed of the major pilin protein ComGC and exclusively assembled during bacterial competence, but their biogenesis remains unclear. Here, we report the high resolution NMR structure of N-terminal truncated ComGC revealing a highly flexible and structurally divergent type IV pilin. It consists of only three alpha-helical segments forming a well-defined electronegative cavity and confined electronegative and hydrophobic patches. The structure is particularly flexible between the first and second alpha-helix with the first helical part exhibiting slightly slower dynamics than the rest of the pilin, suggesting that the first helix is involved in forming the pilus structure core and that parts of helices two and three are primarily surface-exposed. Taken together, our results provide the first structure of a type IV pilin protein involved in the formation of competence-induced pili in Gram-positive bacteria and corroborate the remarkable structural diversity among type IV pilin proteins.

Loss of the Na+/H+ exchanger NHE8 causes male infertility in mice by disrupting acrosome formation
Oberheide, K., Puchkov, D.; Jentsch, T. J.
J Biol Chem,

Tags: Physiology and Pathology of Ion Transport (Jentsch), Cellular Imaging (Wiesner/Puchkov)

Abstract: Mammalian sperm feature a specialized secretory organelle on the anterior part of the sperm nucleus, the acrosome, which is essential for male fertility. It is formed by a fusion of Golgi-derived vesicles. We show here that the predominantly Golgi-resident Na+/H+ exchanger NHE8 localizes to the developing acrosome of spermatids. Similar to wild-type mice, Nhe8-/- mice generated Golgi-derived vesicles positive for acrosomal markers and attached to nuclei, but these vesicles failed to form large acrosomal granules and the acrosomal cap. Spermatozoa from Nhe8-/- mice completely lacked acrosomes, were round-headed, exhibited abnormal mitochondrial distribution and displayed decreased motility, resulting in selective male infertility. Of note, similar features are also found in globozoospermia, one of the causes of male infertility in humans. Germ cell-specific, but not Sertoli cell-specific Nhe8 disruption recapitulated the globozoospermia phenotype, demonstrating that NHE8's role in spermiogenesis is germ cell-intrinsic. Our work has uncovered a crucial role of NHE8 in acrosome biogenesis and suggests that some forms of human globozoospermia might be caused by a loss of function of this Na+/H+ exchanger. It points to NHE8 as a candidate gene for human globozoospermia and a possible drug target for male contraception.

Stable Positioning of Unc13 Restricts Synaptic Vesicle Fusion to Defined Release Sites to Promote Synchronous Neurotransmission
Reddy-Alla(*), S., Böhme, M. A., Reynolds(*), E., Beis(*), C., Grasskamp, A. T., Mampell(*), M. M., Maglione, M., Jusyte, M., Rey(*), U., Babikir(*), H., McCarthy, A. W., Quentin(*), C., Matkovic(*), T., Bergeron(*), D. D., Mushtaq, Z., Goettfert(*), F., Owald(*), D., Mielke(*), T., Hell(*), S. W., Sigrist(*), S. J.; Walter, A. M.

Tags: Molecular and Theoretical Neuroscience (Walter)

Abstract: Neural information processing depends on precisely timed, Ca2+-activated synaptic vesicle exocytosis from release sites within active zones (AZs), but molecular details are unknown. Here, we identify that the (M)Unc13-family member Unc13A generates release sites and show the physiological relevance of their restrictive AZ targeting. Super-resolution and intravital imaging of Drosophila neuromuscular junctions revealed that (unlike the other release factors Unc18 and Syntaxin-1A) Unc13A was stably and precisely positioned at AZs. Local Unc13A levels predicted single AZ activity. Different Unc13A portions selectively affected release site number, position, and functionality. An N-terminal fragment stably localized to AZs, displaced endogenous Unc13A, and reduced the number of release sites, while a C-terminal fragment generated excessive sites at atypical locations, resulting in reduced and delayed evoked transmission that displayed excessive facilitation. Thus, release site generation by the Unc13A C terminus and their specific AZ localization via the N terminus ensure efficient transmission and prevent ectopic, temporally imprecise release.

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Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
Robert-Roessle-Str. 10
13125 Berlin, Germany
+4930 94793 - 100 
+4930 94793 - 109 (Fax)

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