Structural basis for ESCRT-III CHMP3 recruitment of AMSH

Abstract

Endosomal sorting complexes required for transport (ESCRT) recognize ubiquitinated cargo and catalyze diverse budding processes including multivesicular body biogenesis, enveloped virus egress, and cytokinesis. We present the crystal structure of an N-terminal fragment of the deubiquitinating enzyme AMSH (AMSHΔC) in complex with the C-terminal region of ESCRT-III CHMP3 (CHMP3ΔN). AMSHΔC folds into an elongated 90 Å long helical assembly that includes an unusual MIT domain. CHMP3ΔN is unstructured in solution and helical in complex with AMSHΔC, revealing a novel MIT domain interacting motif (MIM) that does not overlap with the CHMP1-AMSH binding site. ITC and SPR measurements demonstrate an unusual high-affinity MIM-MIT interaction. Structural analysis suggests a regulatory role for the N-terminal helical segment of AMSHΔC and its destabilization leads to a loss of function during HIV-1 budding. Our results indicate a tight coupling of ESCRT-III CHMP3 and AMSH functions and provide insight into the regulation of ESCRT-III.

Figure 1

Crystal structure of AMSH in complex with CHMP3. (A) Stereo image of the experimental electron density map obtained after SAD phasing. (B) Ribbon representation of AMSHΔC (blue) in complex with CHMP3ΔN residues 200–222 (yellow). (C) Overlay of the Cα atoms of AMSHΔC (blue) with the VPS4 MIT domain (pdb 2JQ9) (green ribbon). See also Figure S2.

Figure 2

The CHMP3N- AMSHΔC interaction is dominated by polar contacts. (A) Close-up of the CHMP3-AMSH interactions. Hydrogen bonds and salt bridges along the CHMP3 helical segment mediate high affinity interaction. (B) Sequence alignment of the C-terminal regions of CHMP3, CHMP1A, CHMP1B and CHMP2A. CHMP3 residues contacting AMSH are indicated by red asterisks (polar contact), blue asterisks (main chain polar contacts) and black asterisks (hydrophobic contacts). (C) Pull down of CHMP3 by wild type and mutant His-AMSHΔC confirms the essential role of polar interactions. The left panel of the SDS-PAGE shows the input proteins and the right panel the pull down of CHMP3 by wild type and mutant His-AMSHΔC, as indicated. The K88A mutant reveals a strongly reduced interaction with CHMP3 and the E104 and K107A mutants show a complete loss of interaction in this assay. See also Figures S1 and S3.

Figure 3

Superpositioning of the Cα atoms of AMSH and UBPY reveal a regulatory role of the N-terminal regions of AMSH and UBPY. (A) Ribbon representation of the Cα overlay of AMSHΔC (blue) and UBPY (green)(pdb code 2A9U). (B) Close up of the two main contacts between AMSH helix 1 and helices 3 and 5 of the core.

Figure 4

Sequence and secondary structure alignment of AMSH, AMSH-LP and UBPY. Human AMSH (accession code AAD05037) residues 1 to 146 were aligned to the corresponding fragments of AMSH-LP (accession code BAC77766) and UBPY (accession code AAI10591). Residues contacting CHMP3 are indicated with blue triangles. Note that the CHMP3 contact residues are conserved between AMSH and AMSH-LP but not UBPY. Secondary structures are shown for AMSH and UBPY (pdb code 2A9U).

Figure 5

Catalytic inactive AMSH and CHMP3 expression inhibit HIV-1 budding. (A) (Left panel) Expression of catalytically inactive GFP-AMSH(D₃₄₈A) exerts a strong effect on HIV-1 budding when coexpressed with CHMP3 (lanes 3 and 4). In contrast expression of GFP-AMSH(D₃₄₈A-H₄A-R₁₄A) alone or in combination with CHMP3 has no effect on budding (lanes 5 and 6) comparable to vector expression (lane 1) or CHMP3 expression alone (lane 2). (Right panel) Western blot revealing the intracellular Gag processing. Only expression of GFP-AMSH(D₃₄₈A) alone and together with CHMP3 show a defect in intracellular Gag processing (lanes 3 and 4), while the N-terminal mutant GFP-AMSH(D₃₄₈A-H₄A-R₁₄A) resembles the negative control (lane 1) and CHMP3 expression (lane 2). (Lower panel) Western blot showing the expression levels of the AMSH constructs. (B) Pull down of GFP-AMSH(D₃₄₈A) (lanes 1 and 3) and GFP-AMSH(D₃₄₈A-H₄R₁₄A)- HA (lanes 2 and 4) with GST (lanes 1 and 2) and with GST-CHMP3(151-222) (lanes 3 and 4). The western blot below shows the expression pattern of both GFP-AMSH constructs and the panel on the side shows the protein input used for the pull down. (C) (Left panel) Expression of catalytically inactive AMSH(HH_335,337QQ)-HA exerts a mild effect on HIV-1 budding when coexpressed with CHMP3 (lanes 3 and 4). In contrast expression of AMSH(HH_335,337QQ-H₄R₁₄A)-HA alone or in combination with CHMP3 has no effect on budding (lanes 5 and 6). (Right panel) Western blot revealing the intracellular Gag processing. Only expression of AMSH(HH_335,337QQ)-HA and CHMP3 show a defect in intracellular Gag processing (lane 4), while the N-terminal mutant resembles that of AMSH(HH_335,337QQ)-HA expression (lane 3) or the vector control (lane 1). (Lower panel) Western blot revealing similar expression levels for the different AMSH constructs. See also Figure S4.

Figure 6

The H4, R14 mutations in combination with GFP alter the cellular localization of AMSH when co-expressed with CHMP3. (A) CHMP3-Flag and AMSH-HA. (B) CHMP3-Flag and catalytic inactive GFP-AMSH(D348A). (C) CHMP3-Flag and catalytic inactive GFP-AMSH(D348A-H4A-R14A). (D) CHMP3-Flag and inactive AMSH-HA carrying the mutations H335Q and H337Q, which render it catalytic inactive as well as the N-terminal mutations H4A and R14A. The scale bar is 10 μM.

Figure 7

Molecular model of AMSH in complex with membrane associated CHMP3. AMSH can be recruited to membranes early in the ESCRT pathway via ESCRT-0 Stam. This might be independent of ESCRT-III CHMP3 interaction or both processes could be linked. If AMSH interacts with activated CHMP3 present in ESCRT-III polymers on membranes, the DUB activity could have an action radius of > 20 nm due to the long helical arm of AMSH and the flexible linkage of the CHMP3 MIM to the core of CHMP3 mediating ESCRT-III polymerization.