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Binding-Pocket and Lid-Region Substitutions Render Human STING Sensitive to the Species-Specific Drug DMXAA

SUMMARY The drug DMXAA (5,6-dimethylxanthenone-4-acetic acid) showed therapeutic promise against solid tumors in mouse models but subsequently failed in human clinical trials. DMXAA was later discovered to activate mouse, but not human, STING, an adaptor protein in the cyclic dinucleotide cGAMP-mediated signaling pathway, inducing type I interferon expression. To facilitate the development of compounds that target human STING, we combined structural, biophysical, and cellular assays to study mouse and human chimeric proteins and their interaction with DMXAA. We identified a single substitution (G230I) that enables a DMXAA-induced conformational transition of hSTING from an inactive “open” to an active “closed” state. We also identified a substitution within the binding pocket (Q266I) that cooperates with G230I and the previously identified S162A binding-pocket point substitution, rendering hSTING highly sensitive to DMXAA. These findings should facilitate the reciprocal engineering of DMXAA analogs that bind and stimulate wild-type hSTING and their exploitation for vaccine-adjuvant and anti-cancer drug development.

(D) Intermolecular contacts in the complex of DMXAA bound to hSTING S162A/Q266I (aa 155-341). The bound DMXAA is shown in biscuit color, with individual STING subunits in the symmetrical dimer shown in yellow and magenta.
(E) BMDCs were infected with retroviruses (same as in Figure 4F). Cells were incubated with 50 µg/ml of DMXAA and supernatants were collected 18 hours after treatment. CXCL10 protein levels were determined by ELISA. Data shown are means ± SEM (n=3), representative of two independent experiments.

SUPPLEMENTAL EXPERIMENTAL PROCEDURES Protein Expression and Purification
The sequences corresponding to residues aa 140-379 and 155-341 of hSTING were inserted into a modified pRSFDuet-1 vector (Novagen), in which the target protein was separated from the preceding His 6 -SUMO tag by an ubiquitin-like protease (ULP1) cleavage site. The gene sequences were subsequently confirmed by sequencing. The fusion proteins were expressed in BL21 (DE3) RIL cell strain. The cells were grown at 37 °C until OD600 reached approx. 0.6. The temperature was then shifted to 18 °C and the cells were induced by addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to the culture medium at a final concentration of 0.3 mM. After induction, the cells were grown overnight. The fusion proteins were purified over a Ni-NTA affinity column. The His 6 -SUMO tag was removed by ULP1 cleavage during dialysis against buffer containing 40 mM Tris-HCl, 300 mM NaCl, pH 7.5. After dialysis, the His 6 -SUMO tag was removed by Ni-NTA affinity column and the sample was further fractionated over a gel filtration 16/60 G200 Superdex column. The final sample of hSTING contains about 16 mg/ml protein, 20 mM Tris-HCl, 150 mM NaCl, pH 7.5. All the mutants were cloned and purified using the same protocol as used for preparation of the wt protein.

Crystallization
For crystallization of DMXAA with hSTING mutants (aa 155-341), the protein samples were mixed and incubated with DMXAA (1:2 molar ratio) for 0.5 h at room temperature before setting up crystals. The crystals were generated by sitting drop vapor diffusion

Structure Determination
All the diffraction data sets were collected at the Advanced Photon Source, Argonne National laboratories, and were indexed, integrated and scaled using the RAPD online server. All the structures were solved using molecular replacement method in PHASER (McCoy et al., 2007) using the structure of hSTING H232 complex bound to c[G(2',5')pA(3',5')p] (PDB: 4LOH) as the search model. The model building was conducted using the program COOT (Emsley et al., 2010) and structural refinement was conducted using the program PHENIX (Adams et al., 2010). The statistics of data collection and refinement are shown in Table S1.

Isothermal Titration Calorimetry Binding Assay
The dissociation constants (Kd) and thermodynamic parameters of binding reactions of hSTING wt and mutants (aa 140-379) with DMXAA were measured by isothermal titration calorimetry using a MicroCal ITC200 calorimeter at 25°C. First, wt and mutant protein samples were dialyzed overnight against working buffer (100 mM NaCl, 30 mM HEPES, pH 7.5) at 4 °C. Then, the protein samples were diluted with working buffer and the lyophilized DMXAA were dissolved in working buffer. The titration was carried out with 16 successive injections of 2.4 µL DMXAA, spaced 180s apart, into the sample cell containing the protein solution. The data were fit using the program Origin 7.0 software.

STING-Plasmids for Eukaryotic Expression and Generation of Point Mutants
pMAX-Flag-hSTING and pMAX-Flag-mSTING were described previously (Gao et al, 2013b). Point mutants were generated by site-directed PCR mutagenesis via the Quikchange Primer Design method (Agilent, Santa Clara, CA) using PfuUltra HotStart DNA Polymerase (Agilent) and suitable primers. For combination of multiple distant substitutions and subcloning of constructs, STING fragments were PCR-amplified using Q5 DNA Polymerase (NEB, Ipswich, MA) and full-length constructs were assembled using Gibson Assembly (NEB). Constructs were verified by restriction digestion and Sanger sequencing (Seqlab, Göttingen, Germany). Reporter constructs were described previously (Gao et al, 2013a). Name DNA Sequence

Generation of Bone Marrow-derived Dendritic Cells
The protocol for generation of bone marrow-derived dendritic cells was described before (Dai et al., 2014). Bone marrow-derived dendritic cells were generated by culturing bone marrow cells from the tibia and femur of STING Gt/Gt mice in complete medium (CM) in the presence of GM-CSF (30 ng/ml, produced by the Monoclonal Antibody Core facility at the Sloan Kettering Institute) for 10 days. CM is RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin, 0.1 mM essential and nonessential amino acids, 2 mM L-glutamine, 1 mM sodium pyruvate, and 10 mM HEPES buffer. Cells were fed every 2-3 days by replacing 50% of the old medium with fresh medium. Cells were plated into 6-well plate (1 x 10 6 cells per well) at day 10, the day before retroviral infection.

Retroviruses Transduction and DMXAA Treatment
hSTING cDNAs were cloned into pQCXIP-HA vector. In vitro packaging system was used to generate retroviruses according to protocol (Clontech). BMDCs (1x10 6 cells/well) were seeded into 6-well plates and infected with retroviruses. 48 hours after retroviral infection, cells were stimulated with DMXAA at a final concentration of 50 µg/ml (Sigma).
For real-time PCR analysis, cells were collected at 3 hours post DMXAA treatment.
Supernatants were collected at 18 hours post DMXAA treatment. CXCL10 protein level was determined by ELISA.

RNA Isolation and Real-time Quantitative PCR
RNA was extracted from cell pellets with an RNeasy Mini kit (Qiagen) and was reverse

Western Blot Analysis
BMDCs (1 x 10 6 cells) were infected with retroviruses carrying WT hSTING and various hSTING mutants. Cells were collected at 2 days post retroviral infection. Whole-cell lysates were prepared by lysing cells in RIPA buffer. Equal amounts of proteins were subjected to SDS-PAGE and transferred to a nitrocellulose membrane. The levels of HA-hSTING in transduced cells were determined by using a mouse monoclonal antibody specific for HA tag (Covance). Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control, and was detected using a polyclonal antibody against GAPDH (Cell Signaling).