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001-es BibID:BIBFORM062460
035-os BibID:(Cikkazonosító)18397 (WOS)000367035500001 (Scopus)84951782397
Első szerző:Bartók Ádám (biotechnológus)
Cím:An engineered scorpion toxin analogue with improved Kv1.3 selectivity displays reduced conformational flexibility / Adam Bartok, Krisztina Fehér, Andrea Bodor, Kinga Rákosi, Gábor K. Tóth, Katalin E. Kövér, Gyorgy Panyi, Zoltan Varga
Dátum:2015
ISSN:2045-2322
Megjegyzések:The voltage-gated Kv1.3 K(+) channel plays a key role in the activation of T lymphocytes. Kv1.3 blockers selectively suppress immune responses mediated by effector memory T cells, which indicates the great potential of selective Kv1.3 inhibitors in the therapy of certain autoimmune diseases. Anuroctoxin (AnTx), a 35-amino-acid scorpion toxin is a high affinity blocker of Kv1.3, but also blocks Kv1.2 with similar potency. We designed and produced three AnTx variants: ([F32T]-AnTx, [N17A]-AnTx, [N17A/F32T]-AnTx) using solid-phase synthesis with the goal of improving the selectivity of the toxin for Kv1.3 over Kv1.2 while keeping the high affinity for Kv1.3. We used the patch-clamp technique to determine the blocking potency of the synthetic toxins on hKv1.3, mKv1.1, hKv1.2 and hKCa3.1 channels. Of the three variants [N17A/F32T]-AnTx maintained the high affinity of the natural peptide for Kv1.3 but became more than 16000-fold selective over Kv1.2. NMR data and molecular dynamics simulations suggest that the more rigid structure with restricted conformational space of the double substituted toxin compared to the flexible wild-type one is an important determinant of toxin selectivity. Our results provide the foundation for the possibility of the production and future therapeutic application of additional, even more selective toxins targeting various ion channels.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
folyóiratcikk
T lymphocytes
scorpion toxin
patch-clamp technique
Megjelenés:Scientific Reports. - 5 : 18397 (2015), p. 1-13. -
További szerzők:Fehér Krisztina (1974-) (vegyész) Bodor Andrea Rákosi Kinga Tóth Gábor K. Kövér Katalin, E. (1956-2023) (vegyész) Panyi György (1966-) (biofizikus) Varga Zoltán (1969-) (biofizikus, szakfordító)
Pályázati támogatás:TÁMOP-4.2.2/A-11/1/KONV-2012-0025
TÁMOP
TÁMOP-4.2.2/A-11/1/ KONV-2012-0035
TÁMOP
TÁMOP-4.2.1./B-09/KMR-2010-0003
TÁMOP
K 75904
OTKA
NK 101337
OTKA
K 105459
OTKA
4.2.4.A/2-11/1-2012-0001
TÁMOP
KTIA_ NAP_13-2-2015-0009
Egyéb
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DOI
Intézményi repozitóriumban (DEA) tárolt változat
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2.

001-es BibID:BIBFORM084647
Első szerző:Bodor Andrea
Cím:DYNLL2 Dynein Light Chain Binds to an Extended Linear Motif of Myosin 5a Tail That Has Structural Plasticity / Bodor Andrea, Radnai László, Hetényi Csaba, Rapali Péter, Láng András, E. Kövér Katalin, Perczel András, Weixiao Y. Wahlgren, Katona Gergely, Nyitray László
Dátum:2014
ISSN:0006-2960
Megjegyzések:LC8 dynein light chains (DYNLL) are conserved homodimeric eukaryotic hub proteins that participate in diverse cellular processes. Among the binding partners of DYNLL2, myosin 5a (myo5a) is a motor protein involved in cargo transport. Here we provide a profound characterization of the DYNLL2 binding motif of myo5a in free and DYNLL2 bound form by using NMR spectroscopy, Xray crystallography and molecular dynamics simulations. In the free form the DYNLL2 binding region, located in an intrinsically disordered domain of the myo5a tail, has a nascent helical character. The motif becomes structured and folds into a ?-strand upon binding to DYNLL2. Despite all differences of the myo5a sequence from the consensus binding motif, it accommodates into the same DYNLL2 binding groove as all other partners do. Interestingly, while the core motif shows similar interaction pattern in the binding groove as seen inother complexes, the flanking residues make several additional contacts, thereby lengthening the binding motif. The N-terminal extension folds back and partially blocks the free edge of the ?-sheet formed by the binding motif itself. The C-terminal extension contacts the dimer interface and interacts with symmetry related residues of the second myo5a peptide. The involvement of flanking residues of the core binding site of myo5a could modify the quaternary structure of the full-length myo5a and affect its biological functions. The presented structural data widen our understanding of the diverse partner recognition of DYNLL proteins and provide an example of a Janus-faced linear motif. The LC8 dynein light chains were originally described as the smallest subunit of the microtubuleassociated cytoplasmic dynein motor complex.They show a highly conserved amino acid sequence throughout evolution and they were later shown to bind to more than 70 other proteins involved in various biological processes, therefore they are now considered hub proteins (1, 2).Vertebrates contain two closely related LC8 paralogs, DYNLL1 and DYNLL2 with partially overlapping functions(1). Under physiological conditions LC8proteins arestable homodimers (3).Atomic resolution structureswere determined both for the apo and ligand-bound form using X-ray crystallographyand NMR spectroscopy (4-6). Five ?-strands make up two core ?-sheets; one sideof each sheet is flanked by two ?-helices, and the otherside forms the dimer interface. The ?3-strand of one subunit pairs to the?2?-strandof the other subunitin an antiparallel mode; and the dimer is stabilized by interface contacts through hydrophobic interactions and side chain H-bonds. Two equivalent grooves are formed and they represent the target binding pockets of the dimer. LC8 binding motifs are usually localized near coiled-coil or other dimerization domains in intrinsically disordered segmentsof theirinteracting proteins. Unstructured polypeptide regions are often involved in interactions that are mediated by sequential binding sitesknown as linear motifs (7, 8). The LC8 binding motif presents a relatively weak consensus sequence [DS]-4K-3X-2[TVI]-1Q0[TV]1[DE]2 with a conserved glutamine in the arbitrary chosen coreposition 0 (9). A few binding motifs differ considerably from the above consensus sequence; among them is myosin 5a in which position 0 is substituted by a methionine. Myosin 5a (myo5a) is an intracellular motor protein involved in short-range vesicle transport along actin filaments playing various roles in different cell types (10, 11). Its structureis divided into three main parts: (i)the N-terminal motor domain responsible for force generation and ATP hydrolysis; (ii)the long, helical neck domain stabilized by the binding of six light chains; (iii) the coiled-coil tail ending in two globular cargo binding domains(12) (Figure 1).Within the long tail domain three coiled-coil segments, interrupted by flexible non-coiled-coil loops, are responsible for dimerization of the two heavy chains. The primary transcript of the human MYO5A geneis processed by alternative splicing (13). Three exons within the tail domain (B, D, and F) are expressed in a cell type-specific manner; the predominant melanocyte- and brain-specific isoforms contains exons D, F, and exon B, respectively (13). The exon pattern of myo5a tail likely determines which cargo binds to the motor, because these sequences could serve as binding sites of adaptor proteins: exon D and F are necessary for the binding of Rab10 and melanophilin, respectively(14), (15). In a previous study, we identified the DYNLL2 binding motif of myo5a in a short sequence (Ile1280-Ile1294) situated between the medial and distal coiled-coils of the tail (16). The three-residue-long alternatively expressed exon B within the binding motif was shown to be necessary for the interaction with DYNLL2. Binding of DYNLL2 stabilizes the flanking coiled-coil sequences (16, 17). Detailed kinetic and thermodynamic characterization of this interaction using monoand dimeric myo5a fragments revealed that the dissociation constant is somewhat weaker than that of the canonical motifs(~1-5 ?M for a monomeric peptide) andbivalency of the ligand leads to pronounced avidity (18). LC8 proteinsare subunits not only of the dynein (19), but also of the myo5a motor complex (20), and they are known binding partners of several proteins transported by these motors (e.g. nNOS (21, 22), Pak1 (23-25), Bmf (26), Bim (27), Bassoon (28), GKAP (29), and viral proteins (30, 31)), it was hypothesized that DYNLL may provide direct physical linkage between the motor and the cargos (26, 28, 29). However, as the majority of the binding partners (including the motors) are dimeric, and therefore they are bivalent ligands of LC8 and the formation of highly stable dimer-to-dimer complexes is preferred (18). Therefore, the validity of the original cargo adaptor hypothesis is questioned. Here we extend our previous studies (16) and extensively characterize the DYNLL2 binding motif of myo5a and its complex with DYNLL2. NMR experiments show that the 23-residue-long DYNLL2-binding region of myo5a is unstructured with a nascent ?-helical element in theapo form whichundergoes a disorder-to-order transitionand folds into a ?-sheet upon complex formation. Our results suggest that the interacting surface in the DYNLL2-myo5a peptide complex is more extended compared to other LC8-partner complexes. NMR results were supported by molecular dynamics simulations performed on the free peptide and on the complex. Working with a shorter (14residues) myo5a peptide, we solved the crystal structure of the DYNLL2 complex at 1.85?A resolution. The structure explains the role of the M0 instead of the canonical Q0residue in the consensus binding sequence.
Tárgyszavak:Természettudományok Kémiai tudományok idegen nyelvű folyóiratközlemény külföldi lapban
folyóiratcikk
Megjelenés:Biochemistry. - 53 : 45 (2014), p. 7107-7122. -
További szerzők:Radnai László Hetényi Csaba Rapali Péter Láng András Kövér Katalin, E. (1956-2023) (vegyész) Perczel András Wahlgren, Weixiao Y. Katona Gergely Nyitray László
Pályázati támogatás:OTKA-NK81950
OTKA
OTKA-K108437
OTKA
OTKA-NK101072
OTKA
OTKA-K105459
OTKA
TÁMOP 4.2.1./B-09/KMR-2010-0003
TÁMOP
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Intézményi repozitóriumban (DEA) tárolt változat
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3.

001-es BibID:BIBFORM081445
Első szerző:Bodor Andrea
Cím:Membrane interactions in small fast-tumbling bicelles as studied by 31P NMR / Andrea Bodor, Katalin E. Kövér, Lena Mäler
Dátum:2015
ISSN:0005-2736
Megjegyzések:Small fast-tumbling bicelles are ideal for studies of membrane interactions at molecular level; they allow analysis of lipid properties using solution-state NMR. In the present study we used 31P NMR relaxation to obtain detailed information on lipid head-group dynamics. We explored the effect of two topologically different membrane-interacting peptides on bicelles containing either dimyristoylphosphocholine (DMPC), or a mixture of DMPC and dimyristoylphosphoglycerol (DMPG), and dihexanoylphosphocholine (DHPC). KALP21 is a model transmembrane peptide, designed to span a DMPC bilayer and dynorphin B is a membrane surface active neuropeptide. KALP21 causes significant increase in bicelle size, as evidenced by both dynamic light scattering and 31P T2 relaxation measurements. The effect of dynorphin B on bicelle size is more modest, although significant effects on T2 relaxation are observed at higher temperatures. A comparison of 31P T1 values for the lipids with and without the peptides showed that dynorphin B has a greater effect on lipid head-group dynamics than KALP21, especially at elevated temperatures. From the field-dependence of T1 relaxation data, a correlation time describing the overall lipid motion was derived. Results indicate that the positively charged dynorphin B decreases the mobility of the lipid molecules - in particular for the negatively charged DMPG - while KALP21 has a more modest influence. Our results demonstrate that while a transmembrane peptide has severe effects on overall bilayer properties, the surface bound peptide has a more dramatic effect in reducing lipid head-group mobility. These observations may be of general importance for understanding peptide?membrane interactions.
Tárgyszavak:Természettudományok Kémiai tudományok idegen nyelvű folyóiratközlemény külföldi lapban
folyóiratcikk
bicelle
Solution31P NMR spectroscopy
Relaxation study
Membrane protein
Dynamic light scattering
Megjelenés:Biochimica et Biophysica Acta (BBA). Biomembranes. - 1848 : 3 (2015), p. 760-766. -
További szerzők:Kövér Katalin, E. (1956-2023) (vegyész) Mäler, Lena
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Intézményi repozitóriumban (DEA) tárolt változat
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