Interferon-inducible protein 16: insight into the interaction with tumor suppressor p53

Abstract

IFI16 is a member of the interferon-inducible HIN-200 family of nuclear proteins. It has been implicated in transcriptional regulation by modulating protein-protein interactions with p53 tumor suppressor protein and other transcription factors. However, the mechanisms of interaction remain unknown. Here, we report the crystal structures of both HIN-A and HIN-B domains of IFI16 determined at 2.0 and 2.35 Å resolution, respectively. Each HIN domain comprises a pair of tightly packed OB-fold subdomains that appear to act as a single unit. We show that both HIN domains of IFI16 are capable of enhancing p53-DNA complex formation and transcriptional activation via distinctive means. HIN-A domain binds to the basic C terminus of p53, whereas the HIN-B domain binds to the core DNA-binding region of p53. Both interactions are compatible with the DNA-bound state of p53 and together contribute to the effect of full-length IFI16 on p53-DNA complex formation and transcriptional activation.

Figure 1. The HIN Domains of IFI16

(A) Overall domain architecture of the HIN-200 proteins in human (IFI16, MNDA, AIM2, IFIX) and mouse (p202a/b, p203, p204, p205). The PYRIN and HIN domains are indicated by the cyan and yellow shadings, respectively. Red shadings highlight S/T- rich region. (B) Structure of HIN-A (left) or HIN-B (right) domain of IFI16 in cartoon representation. N and C termini are indicated. See also Figure S1.

Figure 2. Subdomain/Subdomain Interactions of IFI16 HIN Domains

Close-up view of residues involved in subdomain/subdomain interaction for IFI16 HIN-A (left) and HIN-B (right).

Figure 3. Interaction of p53 C Terminus with IFI16 HIN-A or HIN-B Domain

(A) An equimolar mixture of His-tagged IFI16 HIN-A or HIN-B domain and a GST fusion protein containing the indicated p53 construct was mixed with glutathione-sepharose (L). After washing, protein was eluted with glutathione and visualized by western (E) using antibody against His-tag. (B) Increasing amounts of p53 (355–393) were incubated with IFI16 HIN-A (open triangle) or HIN-B (filled circle) domain, and binding was quantified by changes in tyrosine fluorescence. Tyrosine side chains are shown in red on the surface representation of HIN-A and HIN-B crystal structures.

Figure 4. EMSA of p53 (82–393) with p53-Consensus DNA Sequence in the Presence of IFI16 HIN-A Domain

(A) Polyacrylamide EMSA reactions without p53 (lanes 1–5) or with constant amount (20 μM) of p53 (lanes 6–10), in the presence of increasing protein concentration (0, 10, 20, 40, 80 μM) of HIN-A. (B) Agarose EMSA of constant amount (0.2 μM) of p53 in the presence of increasing protein concentration (0, 0.07, 0.2, 0.33 μM) of HIN-A (lanes 2–5) or HIN-B (lanes 6–9).

Figure 5. p53 C Terminus-Binding Surface of IFI16 HIN-A Domain

(A) Transparent electrostatic surface representation of IFI16 HIN-A (left) and HIN-B (right) crystal structures. White, red, and blue colors correspond to neutral, negatively, and positively charged surfaces, respectively. (B) Agarose EMSA of p53 (82–393) with wild-type or mutant IFI16 HIN-A (left). Molar ratio of p53 to HIN-A is 1:3. Surface representation of IFI16 HIN-A (right). Residues involved in binding are colored in magenta.

Figure 6. IFI16 HIN-B Domain Enhances p53-DNA Binding by Associating with p53 Core Domain

(A) Polyacrylamide EMSA reactions without p53 (82–360) (lanes 1–5) or with constant amount (2 μM) of p53 (lanes 6–10), in the presence of increasing protein concentration (0, 10, 20, 40, 80 μM) of HIN-B. (B) GST-pull-down of p53 and HIN-B domain. An equimolar mixture of His-tagged p53 (82–360) and GST-tagged IFI16 HIN-B, GST-tagged IFI16 HIN-A, or GST protein alone was mixed with glutathione-sepharose (L). After washing (Washes 1–3), protein was eluted (E1, E2) with reduced glutathione and visualized by Coomassie blue staining. (C) Agarose EMSA of constant amount (0.2 μM) of p53(82–393) in the presence of increasing protein concentration (0, 0.07, 0.2 μM) of full-length IFI16.

Figure 7. IFI16 Enhances p53-Mediated Transcription of the p21 Promoter

H1299 cells were cotransfected with the luciferase reporter vector containing the full-length p21 promoter, p21-Luc, and with pcDNA3-p53 and/or pcDNA3-IFI116 (full-length [FL], HIN-A or HIN-B). The cells were lysed and assayed for luciferase activity 48 hr after transfection. Standard error was calculated using three independent experimental readings. See also Figure S2.

Figure 8. Model of Full-Length IFI16

An averaged bead model/molecular envelope of the full-length IFI16 calculated from SAXS data, with dimension measurements and putative arrangement of its three folded domains: PYRIN, HIN-A, and HIN-B. See also Figure S3.