Structural basis of competitive recognition of p53 and MDM2 by HAUSP/USP7: implications for the regulation of the p53-MDM2 pathway

Abstract

Herpesvirus-associated ubiquitin-specific protease (HAUSP, also known as USP7), a deubiquitylating enzyme of the ubiquitin-specific processing protease family, specifically deubiquitylates both p53 and MDM2, hence playing an important yet enigmatic role in the p53-MDM2 pathway. Here we demonstrate that both p53 and MDM2 specifically recognize the N-terminal tumor necrosis factor-receptor associated factor (TRAF)-like domain of HAUSP in a mutually exclusive manner. HAUSP preferentially forms a stable HAUSP-MDM2 complex even in the presence of excess p53. The HAUSP-binding elements were mapped to a peptide fragment in the carboxy-terminus of p53 and to a short-peptide region preceding the acidic domain of MDM2. The crystal structures of the HAUSP TRAF-like domain in complex with p53 and MDM2 peptides, determined at 2.3-A and 1.7-A resolutions, respectively, reveal that the MDM2 peptide recognizes the same surface groove in HAUSP as that recognized by p53 but mediates more extensive interactions. Structural comparison led to the identification of a consensus peptide-recognition sequence by HAUSP. These results, together with the structure of a combined substrate-binding-and-deubiquitylation domain of HAUSP, provide important insights into regulation of the p53-MDM2 pathway by HAUSP.

Figure 1. Structure of the HAUSP N-Terminal TRAF-Like Domain

(A) Structure of the HAUSP TRAF-like domain in a ribbon diagram (left) and a surface representation (right). Secondary structural elements (left) and the putative substrate-binding groove (right) are labeled. (B) Sequence alignment of the HAUSP TRAF-like domain with other TRAF family members. Conserved residues are shown in yellow. Residues that interact with p53 through hydrogen bonds and van der Waals contacts are identified by green arrow heads and green squares, respectively. Residues that interact with MDM2 through hydrogen bonds and van der Waals contacts are indicated by red arrow heads and red squares, respectively. Conserved residues that are involved in binding to peptides in other TRAF family proteins, but not in HAUSP, are colored red and indicated by purple background.

Figure 2. Structural Basis of p53 Recognition by HAUSP

(A) Identification of a short-peptide fragment in p53 (residues 359–362) as the HAUSP-binding element. Various p53 fragments were individually incubated with HAUSP protein (residues 53–206) and their interactions were examined by gel filtration. The results are summarized in the left panel. Two representative experiments, for WT p53 (residues 325–367, WT) and mutant p53 (residues 325–367, S362A), are shown on the right panel. Note that, because the elution volumes for free HAUSP (residues 53–206) and for free p53 (residues 325–367) correspond to fraction numbers 36 and 35, these two proteins appear to co-migrate on gel filtration—but they do not interact with each other. The protein concentrations for gel filtration were: HAUSP (53–206), 55 μM; p53 WT, 46 μM; p53 mutant, 51 μM. (B) Overall structure of the HAUSP TRAF-like domain bound to p53 peptide is shown in a surface representation (left) and in a ribbon diagram (right). Binding by the p53 peptide does not induce any significant conformational changes in HAUSP as shown by the structural comparison of the free and p53-bound TRAF-like domain (right panel). (C) A stereo view of the specific interactions between p53 and HAUSP. Hydrogen bonds are represented by red dashed lines. All interacting residues are labeled.

Figure 3. HAUSP Preferentially Forms a Stable HAUSP–MDM2 Complex in the Presence of Excess p53

(A) The TRAF-like domain of HAUSP is responsible for binding to MDM2. Various HAUSP fragments were individually incubated with MDM2 protein (residues 170–423) and their interactions were examined by gel filtration. The results are summarized here. (B) Identification of a minimal HAUSP-binding element in MDM2. Various MDM2 fragments were individually incubated with HAUSP TRAF-like domain (residues 53–206) and their interactions were examined by gel filtration. The results are summarized here. (C) HAUSP preferentially forms a stable HAUSP–MDM2 complex in the presence of excess p53. HAUSP (residues 1–206) interacts with both p53 (residues 351–382, upper panel) and MDM2 (residues 208–289, middle panel). However, in the presence of a 10-fold excess amount of p53, HAUSP formed a stable complex only with MDM2 (lower panel). The relevant peak fractions were visualized by SDS-PAGE followed by Coomassie staining. (D) Determination of binding affinities between the HAUSP TRAF-like domain (residues 53–206) and peptides derived from p53 and MDM2 by ITC. The p53 and MDM2 peptides contain residues 351–382 and 208–242, respectively. The binding affinities for the p53 and MDM2 peptides are 3 and 21 μM, respectively.

Figure 4. Structural Basis of MDM2 Recognition by HAUSP

(A) Overall structure of the HAUSP TRAF-like domain bound to MDM2 peptide is shown in a ribbon diagram (left) and in a surface representation (right). The important MDM2 residues are highlighted in yellow. (B) A stereo view of the specific interactions between MDM2 and HAUSP. These interactions are more extensive than those between p53 and HAUSP. Hydrogen bonds are represented by red dashed lines. All interacting residues are labeled.

Figure 5. Structural Comparison of Peptide Binding by HAUSP Reveals a Consensus Sequence

(A) MDM2 peptide (red) binds to the same surface groove as the p53 peptide (magenta). Residues from MDM2 and p53 are shown in yellow and green, respectively. (B) Superposition of three HAUSP-binding peptides derived from MDM2 (red), p53 (magenta), and EBNA1 (green). The HAUSP TRAF-like domain is shown in a transparent surface representation, with critical residues shown in brown. (C) Structural alignment of HAUSP-binding peptides reveals a consensus sequence. The HAUSP surface groove (in a transparent surface representation) for binding to the consensus tetrapeptide is shown in the left panel. The consensus sequence is shown in the right panel.

Figure 6. Structure of an Extended HAUSP Fragment

(A) Structure of a HAUSP fragment (residues 53–560) that contains both the substrate-binding (green) and the catalytic domains. Binding sites for ubiquitin and substrate are indicated. The linker sequences between these two domains have high-temperature factors and are flexible in the crystals. (B) A structure-based model showing HAUSP bound to an ubiquitylated MDM2. Only one ubiquitin moiety and the MDM2 peptide are shown in this model.