Összesen 1 találat.


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
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
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
TÁMOP 4.2.1./B-09/KMR-2010-0003
Internet cím:Szerző által megadott URL
Intézményi repozitóriumban (DEA) tárolt változat
Rekordok letöltése1