Structure and evolution of ENTH and VHS/ENTH-like domains in tepsin

Abstract

Tepsin is currently the only accessory trafficking protein identified in adaptor-related protein 4 (AP4)-coated vesicles originating at the trans-Golgi network (TGN). The molecular basis for interactions between AP4 subunits and motifs in the tepsin C-terminus have been characterized, but the biological role of tepsin remains unknown. We determined X-ray crystal structures of the tepsin epsin N-terminal homology (ENTH) and VHS/ENTH-like domains. Our data reveal unexpected structural features that suggest key functional differences between these and similar domains in other trafficking proteins. The tepsin ENTH domain lacks helix0, helix8 and a lipid binding pocket found in epsin1/2/3. These results explain why tepsin requires AP4 for its membrane recruitment and further suggest ENTH domains cannot be defined solely as lipid binding modules. The VHS domain lacks helix8 and thus contains fewer helices than other VHS domains. Structural data explain biochemical and biophysical evidence that tepsin VHS does not mediate known VHS functions, including recognition of dileucine-based cargo motifs or ubiquitin. Structural comparisons indicate the domains are very similar to each other, and phylogenetic analysis reveals their evolutionary pattern within the domain superfamily. Phylogenetics and comparative genomics further show tepsin within a monophyletic clade that diverged away from epsins early in evolutionary history (~1500 million years ago). Together, these data provide the first detailed molecular view of tepsin and suggest tepsin structure and function diverged away from other epsins. More broadly, these data highlight the challenges inherent in classifying and understanding protein function based only on sequence and structure.

Keywords: evolution; membrane trafficking; protein structure; vesicle coats.

Figure 1. Tepsin architecture and crystal structure of tepsin ENTH domain

(A) Overall domain architecture of human tepsin, which contains an N-terminal ENTH (tENTH) and internal VHS/ENTH-like (tVHS) domains. The unstructured C-terminus contains two motifs for binding C-terminal appendage domains of AP4 ε and β4 subunits. (B) Crystal structure of human tENTH residues 1–136 at 1.38 Å resolution. tENTH contains only seven α-helices; it lacks both helix0 and helix8 found in other ENTH domains. (C) View of key interactions that facilitate packing between helices α1 and α3 in tENTH, including an ion pair (Arg10 and Glu55) and multiple hydrophobic interactions.

Figure 2. tENTH is a multimer in crystals but a monomer in solution

(A) Top panel: The tENTH 1–136 construct crystallized as a dimer (labeled A and B) in the asymmetric unit. Bottom panel: The tetramer observed in the crystal lattice of the tENTH 1–153 structure. (B) Gel filtration profile of the tENTH domain (blue trace) with standards (grey trace), which are consistent with tENTH existing as a monomer in solution.

Figure 3. Structural and functional comparison of epsin1 and tepsin ENTH domains

(A) Overlay of epsin1 ENTH (yellow; PDB: 1H0A) and tENTH (green) domains. Tepsin lacks the amphipathic helix0 found in other ENTHs and instead contains an elongated helix1. (B) Equivalent electrostatic surface views of epsin1 ENTH (left) and tENTH (right). Epsin1 contains a basic binding pocket to accommodate a phosphoinositide head group, while tepsin lacks this binding pocket because of the absence of helix0. Biochemical data using recombinant ENTH domains on PIP strips confirms the structural predictions that epsin1 ENTH recognizes phosphoinositides, while tepsin ENTH does not.

Figure 4. Crystal structure of the tepsin VHS/ENTH-like domain

(A) Ribbon diagrams of tVHS domain structure determined at 1.8 Å resolution; the views are rotated by 90 degrees. Unlike other VHS domains, tVHS contains only six α-helices. For consistency with other VHS domains, the helices are labelled α1–4, α5/6 (see Results), and α7. (B) Electrostatic surface representations of tVHS, shown in the same orientations as (A). tVHS contains a deep acidic pocket or groove on one surface.

Figure 5. Structural comparison of VHS domains

(A) Overlay of VHS domain structures from tepsin, GGA3 (PDB: 1JPL), STAM1 (PDB: 3LDZ), and TOM1 (PDB: 1ELK). (B) Overlay of tepsin and GG3 VHS domains to highlight key structural differences; view is rotated 90 degrees relative to (A). tVHS aligns well with other VHS domains at helices 1–4. tVHS contains a bent α-helix (α5/6), while other VHS domains contain two separate helices (α5, α6). Helix8 is absent in tVHS.

Figure 6. tVHS structure explains functional divergence from other VHS domains

(A) Top panel: overlay of tepsin (blue) and GGA3 (grey) VHS domains. The acidic dileucine motif recognized by GGA3 is shown in grey stick figures. GGA3 VHS helix8 contributes key surface area to accommodate dileucine motif binding; loss of this helix prevents binding by tVHS. Bottom panel: ITC run with recombinant tVHS protein and 10x molar excess of a model acidic dileucine motif (DSVIL), demonstrating no binding. (B) tVHS ribbon and transparent electrostatic surface modeled by overlaying the STAM1-Ub structure (PDB: 3LDZ). tVHS lacks key residues that form a Ub binding surface, and an arginine present in tVHS would clash with ubiquitin. Bottom panel: HSQC experiment of ¹⁵N-labeled ubiquitin (black) spectrum overlaid with a spectrum collected in the presence of a ten times molar excess of tVHS domain. We observe no chemical shifts, suggesting that tVHS cannot bind ubiquitin.

Figure 7. Evolution of ENTH and VHS domains

(A) Structural overlay of tENTH, tVHS, epsin1 ENTH, and GGA3 VHS domains. All four domains demonstrate a conserved structural core containing helices α1–7. (B) Phylogenetic tree of ENTH, tepsin ENTH (tENTH), VHS, and tepsin VHS-like (tVHS) domains, with ANTH domains as an outgroup.

Figure 8. Epsin family phylogenetic tree

Tepsin forms a monophyletic clade that likely diverged early in the evolutionary history of eukaryotes. (CALM sequences, which contain ANTH domains, were used as an outgroup.)