Table 1.
Crystallographic data, phasing and refinement statistics.
Figure 1.
Vps33, Vps16CTD–Vps33, rat Munc18–1 (PDB entry 3PUJ), and Sly1 (1MQS) are shown with cylinders representing α-helices. The different position of domain 1 in Vps33, relative to Munc18–1 and Sly1, is especially notable. The tip of domain 3a shows varying degrees of disorder, as discussed in the text.
Figure 2.
Sequence alignment for Vps33 and the C-terminal region of Vps16.
Intermolecular contacts (<4 Å) are indicated using boxes. For Vps33, these boxes are yellow except for those residues depicted in magenta in Fig. 3B. For Vps16, boxes are color coded to match the Vps33 domain(s) contacted by a given residue. The distal tip of Vps33 domain 3a is highlighted with a blue box. The ‘hinge’ proline (see Fig. 6 legend) is highlighted with a red box. Secondary structural assignments for Vps33 are based on [16] and are colored by domain as in Fig. 1; helix α12 is shown in light blue to indicate that it is ordered in Vps33 but not in Vps16CTD–Vps33 (see text for details). Sequence alignments were performed using CLUSTALW [68] on 15 Vps33 and 15 Vps16 orthologs; for clarity, only 5 orthologs are shown here. The orthologs shown (with percentage sequence identity for Vps33/Vps16 listed in parentheses) are: Homo sapiens (37/33), Drosophila melanogaster (30/27), Aspergillus niger (61/58), and Saccharomyces cerevisiae (19/20).
Figure 3.
Interaction between Vps16CTD and Vps33.
(A) Vps16CTD and Vps33, oriented as in Fig. 1, are separated and rotated to reveal the contact surfaces. (B) In magenta are shown the positions of Vps33 residue substitutions engineered to disrupt the complex. Also indicated is Phe-656, one of three residues near the C-terminus of Vps33 (and therefore located in domain 2) that is well-ordered only in the Vps16CTD–Vps33 complex. (C) Size exclusion chromatography was used to analyze wild-type Vps33, full-length Vps16, and the combination of the two. Shown for comparison is the sum of the chromatograms for the individual proteins. The Vps16–Vps33 complex elutes earlier from the column, consistent with its larger size. (D) As in panel C, but with Vps33 A411D/H451D in place of wild-type Vps33. The binding reaction is indistinguishable from the sum of the individual protein chromatograms, indicating the absence of a detectable interaction. The same result was obtained for Vps33 A411D/L454E (not shown).
Figure 4.
The position of Vps33 domain 1 is unique among known SM proteins.
(A) All known SM protein structures (PDB entries 1EPU, 1FVF, 1MQS, 2XHE, 3C98, 3PUJ, and 3PUK), including multiple molecules within the asymmetric unit (whenever present), were aligned with Vps33 based on domains 2 and 3. Vps33 is shown in ribbon representation, colored as in Fig. 1; all other SM proteins are shown in simple representation and colored gray. (B) Using the structure of Munc18–1 in complex with syntaxin 1 (2XHE), a ternary SNARE complex (1SFC) was modeled into the central cleft of Vps33. (C) As in panel B, but with the tip of Vps33 modeled in a closed conformation. For model generation, see Materials and Methods.
Figure 5.
Alterations in Vps33 domain 1 eliminate the N-peptide binding site.
(A) Arg-4 plays a key role in the binding of the N-peptide of syntaxin 1A to domain 1 of Munc18–1 (PDB entry 3C98) [12], forming a network of hydrogen bonds and salt bridges denoted by dashed orange lines. (B) The same peptide, overlaid on the corresponding surface of Vps33, clashes with Vps33 residue Arg-115 (purple). (C) A different view of the complex shown in panel A highlights the hydrophobic pocket into which Leu-8 of syntaxin 1A packs. (D) The corresponding view of the model shown in panel B illustrates that Vps33 residue Leu-129 fills the hydrophobic residue binding pocket.
Figure 6.
Domain 3a displays an open conformation featuring conserved surface-exposed residues.
(A) Highly conserved residues were determined by comparing the sequences of fifteen Vps33 orthologs from yeast to human and are shown on the C. thermophilum structure as spheres. (B) A surface representation reveals that a majority of the conserved surface-exposed residues map to domain 3a. Except in domain 3a, few surface-exposed conserved residues are visible on the ‘back’ side of Vps33 (not shown). (C) The two Vps33 monomers present in the asymmetric unit (chains A and B), while highly similar overall, show significant structural divergence in domain 3a. Pro-355, a potential hinge residue [14], is highlighted. The tip of loop 3a was not visible in the Vps16CTD–Vps33 complex. (D) Superposition with open and closed Munc18 structures reveals that Vps33 domain 3a adopts an open conformation. Also shown are the relevant regions of open rat Munc18–1 (PDB entry 3PUJ, which includes the N-peptide of syntaxin 4) and closed M. brevicollis Munc18 (2XHE, which includes syntaxin 1).