Three-dimensional structure of the rSly1 N-terminal domain reveals a conformational change induced by binding to syntaxin 5.

Sec1/Mun18-like (SM) proteins and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) play central roles in intracellular membrane fusion. Diverse modes of interaction between SM proteins and SNAREs from the syntaxin family have been described. However, the observation that the N-terminal domains of Sly1 and Vps45, the SM proteins involved in traffic at the endoplasmic reticulum, the Golgi, the trans-Golgi network and the endosomes, bind to similar N-terminal sequences of their cognate syntaxins suggested a unifying theme for SM protein/SNARE interactions in most internal membrane compartments. To further understand this mechanism of SM protein/SNARE coupling, we have elucidated the structure in solution of the isolated N-terminal domain of rat Sly1 (rSly1N) and analyzed its complex with an N-terminal peptide of rat syntaxin 5 by NMR spectroscopy. Comparison with the crystal structure of a complex between Sly1p and Sed5p, their yeast homologues, shows that syntaxin 5 binding requires a striking conformational change involving a two-residue shift in the register of the C-terminal beta-strand of rSly1N. This conformational change is likely to induce a significant alteration in the overall shape of full-length rSly1 and may be critical for its function. Sequence analyses indicate that this conformational change is conserved in the Sly1 family but not in other SM proteins, and that the four families represented by the four SM proteins found in yeast (Sec1p, Sly1p, Vps45p and Vps33p) diverged early in evolution. These results suggest that there are marked distinctions between the mechanisms of action of each of the four families of SM proteins, which may have arisen from different regulatory requirements of traffic in their corresponding membrane compartments.

Munc18-1 binds directly to the neuronal SNARE complex.

Both SM proteins (for Sec1/Munc18-like proteins) and SNARE proteins (for soluble NSF-attachment protein receptors) are essential for intracellular membrane fusion, but the general mechanism of coupling between their functions is unclear, in part because diverse SM protein/SNARE binding modes have been described. During synaptic vesicle exocytosis, the SM protein Munc18-1 is known to bind tightly to the SNARE protein syntaxin-1, but only when syntaxin-1 is in a closed conformation that is incompatible with SNARE complex formation. We now show that Munc18-1 also binds tightly to assembled SNARE complexes containing syntaxin-1. The newly discovered Munc18-1/SNARE complex interaction involves contacts of Munc18-1 with the N-terminal H(abc) domain of syntaxin-1 and the four-helical bundle of the assembled SNARE complex. Together with earlier studies, our results suggest that binding of Munc18-1 to closed syntaxin-1 is a specialization that evolved to meet the strict regulatory requirements of neuronal exocytosis, whereas binding of Munc18-1 to assembled SNARE complexes reflects a general function of SM proteins involved in executing membrane fusion.

A Munc13/RIM/Rab3 tripartite complex: from priming to plasticity?

alpha-RIMs and Munc13s are active zone proteins that control priming of synaptic vesicles to a readily releasable state, and interact with each other via their N-terminal sequences. The alpha-RIM N-terminal sequence also binds to Rab3s (small synaptic vesicle GTPases), an interaction that regulates presynaptic plasticity. We now demonstrate that alpha-RIMs contain adjacent but separate Munc13- and Rab3-binding sites, allowing formation of a tripartite Rab3/RIM/Munc13 complex. Munc13 binding is mediated by the alpha-RIM zinc-finger domain. Elucidation of the three-dimensional structure of this domain by NMR spectroscopy facilitated the design of a mutation that abolishes alpha-RIM/Munc13 binding. Selective disruption of this interaction in the calyx of Held synapse decreased the size of the readily releasable vesicle pool. Our data suggest that the ternary Rab3/RIM/Munc13 interaction approximates synaptic vesicles to the priming machinery, providing a substrate for presynaptic plasticity. The modular architecture of alpha-RIMs, with nested binding sites for Rab3 and other targets, may be a general feature of Rab effectors that share homology with the alpha-RIM N-terminal sequence.

Three-dimensional structure of an independently folded extracellular domain of human amyloid-beta precursor protein.

Cleavage of amyloid-beta precursor protein (APP) by site-specific proteases generates amyloid-beta peptides (Abetas), which are thought to induce Alzheimer's disease. We have identified an independently folded extracellular domain of human APP localized proximal to the Abeta sequence, and determined the three-dimensional structure of this domain by NMR spectroscopy. The domain is composed of four alpha-helices, three of which form a tight antiparallel bundle, and constitutes the C-terminal half of the central extracellular region of APP that has been implicated in the regulation of APP cleavage. Sequence comparisons demonstrate that the domain is highly conserved among all members of the APP family, including invertebrate homologues, suggesting an important role for this region in the biological function of APP. The identification of this domain and the availability of its atomic structure will facilitate analysis of APP function and of the role of the extracellular region in the regulation of APP cleavage.

How Tlg2p/syntaxin 16 'snares' Vps45.

Soluble N-ethylmaleimide sensitive factor-attachment protein receptors (SNAREs) and Sec1p/Munc18-homologs (SM proteins) play key roles in intracellular membrane fusion. The SNAREs form tight four-helix bundles (core complexes) that bring the membranes together, but it is unclear how this activity is coupled to SM protein function. Studies of the yeast trans-Golgi network (TGN)/endosomal SNARE complex, which includes the syntaxin-like SNARE Tlg2p, have suggested that its assembly requires activation by binding of the SM protein Vps45p to the cytoplasmic region of Tlg2p folded into a closed conformation. Nuclear magnetic resonance and biochemical experiments now show that Tlg2p and Pep12p, a late- endosomal syntaxin that interacts functionally but not directly with Vps45p, have a domain structure characteristic of syntaxins but do not adopt a closed conformation. Tlg2p binds tightly to Vps45p via a short N-terminal peptide motif that is absent in Pep12p. The Tlg2p/Vps45p binding mode is shared by the mammalian syntaxin 16, confirming that it is a Tlg2p homolog, and resembles the mode of interaction between the SM protein Sly1p and the syntaxins Ufe1p and Sed5p. Thus, this mechanism represents the most widespread mode of coupling between syntaxins and SM proteins.

Convergence and divergence in the mechanism of SNARE binding by Sec1/Munc18-like proteins.

Sec1Munc18-like (SM) proteins functionally interact with soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) in membrane fusion, but the mechanisms of these interactions differ. In vertebrates, SM proteins that mediate exocytosis (Munc18-1, 18-2, and 18c) bind to the closed conformation of syntaxins 1-4, which requires the N-terminal H(abc) domains and SNARE motifs of these syntaxins. In contrast, SM proteins that mediate Golgi and endoplasmic reticulum fusion (Sly1 and Vps45) bind only to short N-terminal sequences of syntaxins 5, 16, or 18, independently of their H(abc) domains and SNARE motifs. We now show that Munc18-1, Sly1, and Vps45 interact with cognate syntaxins via similar, autonomously folded N-terminal domains, but the syntaxin 5-binding surface of the Sly1 N-terminal domain is opposite to the syntaxin 1-binding surface of the Munc18-1 N-terminal domain. In transfected cells, the N-terminal domain of Sly1 specifically disrupts the structure of the Golgi complex, supporting the notion that the interaction of Sly1 with syntaxin 5 is essential for fusion. These data, together with previous results, suggest that a relatively small N-terminal domain of SM proteins is dedicated to mechanistically distinct interactions with SNAREs, leaving the remaining large parts of SM proteins free to execute their as yet unknown function as effector domains.