Biomedical subjects

Martin Margittai

Publications and source records attributed to Martin Margittai.

8 recordsLinked to original sources

PubMed2006

Side chain-dependent stacking modulates tau filament structure.

The misfolding of proteins into highly ordered fibrils with similar physical properties is a hallmark of many degenerative diseases. Here, we use the microtubule associated protein tau as a model system to investigate the role of amino acid side chains in the formation of such fibrils. We identify a region (positions 272-289) in the tau protein that, in the fibrillar state, either forms part of a core of parallel, in-register, beta-strands, or remains unfolded. Single point mutations are sufficient to control this conformational switch with disease mutants G272V and DeltaK280 (found in familial forms of dementia) inducing a folded state. Through systematic mutagenesis we derive a propensity scale for individual amino acids to form fibrils with parallel, in-register, beta-strands. This scale should not only apply to tau fibrils but generally to all fibrils with same strand arrangement.

Amino Acid Sequence↗

PubMed2006

Spin labeling analysis of amyloids and other protein aggregates.

Because of the enormous size of amyloid fibrils and their low tendency to form crystal lattices, it has been difficult to obtain high-resolution structural information on these aggregates. Magnetic resonance methods, such as solid-state nuclear magnetic resonance spectroscopy and electron paramagnetic resonance (EPR) spectroscopy, are promising new technologies by which to obtain molecular models. This chapter will focus on the application of EPR spectroscopy to amyloids and other protein aggregates. Site-directed spin labeling (SDSL), in combination with EPR spectroscopy, has been successfully used to study protein structure and the dynamics of soluble as well as membrane proteins. Recent studies indicate that this strategy is also well suited for studying amyloid fibrils. For example, an important outcome of the SDSL studies performed in our laboratory is that fibrils of amyloid beta, islet amyloid polypeptide, alpha-synuclein, and tau have their beta-strands aligned in an in-register, parallel fashion. Future studies promise to yield molecular information about fibril topography and protofilament arrangement and can be extended to include oligomeric structures.

Amyloid↗

PubMed2004

Template-assisted filament growth by parallel stacking of tau.

Tau filaments are found in >20 neurodegenerative diseases. Yet, because of their enormous molecular weights and poor tendency to form highly ordered 3D crystal lattices, they have evaded high-resolution structure determination. Here, we studied 25 derivatized tau mutants by using electron paramagnetic resonance and fluorescence spectroscopy to report structural details of tau filaments. Based on strong spin exchange and pyrene excimer formation of core residues, we find that individual tau proteins form single molecule layers along the fiber axis that perfectly stack on top of each other by in-register, parallel alignment of beta-strands. We suggest a model of filament growth wherein the existing filament serves as a template for the incoming, unfolded tau molecule, resulting in a new structured layer with maximized hydrogen-bonded contact surface and side-chain stacking.

Amino Acid Sequence↗

PubMed2004

Determinants of liposome fusion mediated by synaptic SNARE proteins.

Synaptic exocytosis requires the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins syntaxin 1, SNAP-25, and synaptobrevin (VAMP). Assembly of the SNAREs into a stable core complex is supposed to catalyze membrane fusion, and proteoliposomes reconstituted with synaptic SNARE proteins spontaneously fuse with each other. We now show that liposome fusion mediated by synaptic SNAREs is inhibited by botulinum neurotoxin E (BoNT/E) but can be rescued by supplementing the C-terminal portion of SNAP-25. Furthermore, fusion is prevented by a SNAP-25-specific antibody known to block exocytosis in chromaffin cells, and it is competed for by soluble fragments of the R-SNAREs synaptobrevin 2, endobrevin/VAMP-8, and tomosyn. No accumulation of clustered vesicles is observed during the reaction. Rapid artificial clustering of SNARE-containing proteoliposomes enhances the fusion rate at low but not at saturating liposome concentrations. We conclude that the rate of liposome fusion is dominated by the intrinsic properties of the SNAREs rather than by the preceding docking step.

Animals↗

PubMed2003

A transient N-terminal interaction of SNAP-25 and syntaxin nucleates SNARE assembly.

The SNARE proteins syntaxin, SNAP-25, and synaptobrevin play a central role during Ca(2+)-dependent exocytosis at the nerve terminal. Whereas syntaxin and SNAP-25 are located in the plasma membrane, synaptobrevin resides in the membrane of synaptic vesicles. It is thought that gradual assembly of these proteins into a membrane-bridging ternary SNARE complex ultimately leads to membrane fusion. According to this model, syntaxin and SNAP-25 constitute an acceptor complex for synaptobrevin. In vitro, however, syntaxin and SNAP-25 form a stable complex that contains two syntaxin molecules, one of which is occupying and possibly obstructing the binding site of synaptobrevin. To elucidate the assembly pathway of the synaptic SNAREs, we have now applied a combination of fluorescence and CD spectroscopy. We found that SNARE assembly begins with the slow and rate-limiting interaction of syntaxin and SNAP-25. Their interaction was prevented by N-terminal but not by C-terminal truncations, suggesting that for productive assembly all three participating helices must come together simultaneously. This suggests a complicated nucleation process that might be the reason for the observed slow assembly rate. N-terminal truncations of SNAP-25 and syntaxin also prevented the formation of the ternary complex, whereas neither N- nor C-terminal shortened synaptobrevin helices lost their ability to interact. This suggests that binding of synaptobrevin occurs after the establishment of the syntaxin-SNAP-25 interaction. Moreover, binding of synaptobrevin was inhibited by an excess of syntaxin, suggesting that a 1:1 interaction of syntaxin and SNAP-25 serves as the on-pathway SNARE assembly intermediate.

Binding Sites↗

PubMed2003

The Habc domain and the SNARE core complex are connected by a highly flexible linker.

Syntaxin 1a is a member of the SNARE superfamily of small, mostly membrane-bound proteins that mediate membrane fusion in all eukaryotic cells. Upon membrane fusion, syntaxin 1 forms a stable complex with its partner SNAREs. Syntaxin contains a C-terminal transmembrane domain, an adjacent SNARE motif that interacts with its partner SNAREs, and an N-terminal Habc domain. The Habc domain reversibly folds back upon the SNARE motif, resulting in a "closed" conformation that is stabilized by binding to the protein munc18. The SNARE motif and the Habc domain are separated by a linker region of about 40 amino acids. When syntaxin is complexed with munc18, the linker is structured and consists of a mix of turns and small alpha-helices. When syntaxin is complexed with its partner SNAREs, the Habc domain is dissociated, but the structure of the linker region is not known. Here we used site-directed spin labeling and EPR spectroscopy to determine the structure of the linker region of syntaxin in the SNARE complex. We found that the entire linker region of syntaxin is unstructured except for three residues at the N-terminal and six residues at the C-terminal boundary whereas the structures of the flanking regions in the Habc domain and the SNARE motif correspond to the high-resolution structures of the isolated fragments. We conclude that the linker region exhibits a high degree of conformational flexibility.

Amino Acid Motifs↗

PubMed2002

SNAREs in native plasma membranes are active and readily form core complexes with endogenous and exogenous SNAREs.

During neuronal exocytosis, the vesicle-bound soluble NSF attachment protein (SNAP) receptor (SNARE) synaptobrevin 2 forms complexes with the plasma membrane-bound SNAREs syntaxin 1A and SNAP25 to initiate the fusion reaction. However, it is not known whether in the native membrane SNAREs are constitutively active or whether they are unable to enter SNARE complexes unless activated before membrane fusion. Here we used binding of labeled recombinant SNAREs to inside-out carrier supported plasma membrane sheets of PC12 cells to probe for the activity of endogenous SNAREs. Binding was specific, saturable, and depended on the presence of membrane-resident SNARE partners. Our data show that virtually all of the endogenous syntaxin 1 and SNAP-25 are highly reactive and readily form SNARE complexes with exogenously added SNAREs. Furthermore, complexes between endogenous SNAREs were not detectable when the membranes are freshly prepared, but they slowly form upon prolonged incubation in vitro. We conclude that the activity of membrane-resident SNAREs is not downregulated by control proteins but is constitutively active even if not engaged in fusion events.

Animals↗

PubMed2001

Rapid and selective binding to the synaptic SNARE complex suggests a modulatory role of complexins in neuroexocytosis.

The Ca(2+)-triggered release of neurotransmitters is mediated by fusion of synaptic vesicles with the plasma membrane. The molecular machinery that translates the Ca(2+) signal into exocytosis is only beginning to emerge. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins syntaxin, SNAP-25, and synaptobrevin are central components of the fusion apparatus. Assembly of a membrane-bridging ternary SNARE complex is thought to initiate membrane merger, but the roles of other factors are less understood. Complexins are two highly conserved proteins that modulate the Ca(2+) responsiveness of neurotransmitter release. In vitro, they bind in a 1:1 stoichiometry to the assembled synaptic SNARE complex, making complexins attractive candidates for controlling the exocytotic fusion apparatus. We have now performed a detailed structural, kinetic, and thermodynamic analysis of complexin binding to the SNARE complex. We found that no major conformational changes occur upon binding and that the complexin helix is aligned antiparallel to the four-helix bundle of the SNARE complex. Complexins bound rapidly (approximately 5 x 10(7) m(-1) s(-1)) and with high affinity (approximately 10 nm), making it one of the fastest protein-protein interactions characterized so far in membrane trafficking. Interestingly, neither affinity nor binding kinetics was substantially altered by Ca(2+) ions. No interaction of complexins was detectable either with individual SNARE proteins or with the binary syntaxin x SNAP-25 complex. Furthermore, complexin did not promote the formation of SNARE complex oligomers. Together, our data suggest that complexins modulate neuroexocytosis after assembly of membrane-bridging SNARE complexes.

Adaptor Proteins, Vesicular Transport↗