Crystallization studies of the murine c-di-GMP sensor protein STING

Su, Yi-Che; Tu, Zhi‐Le; Yang, Chih‐Yung; Chin, Ko‐Hsin; Chuah, Mary Lay‐Cheng; Liang, Zhao‐Xun; Chou, Shan‐Ho

doi:10.1107/s1744309112024372

Cited by 9 publications

(6 citation statements)

References 49 publications

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“…To date, five structures of hSTING in apo and c-di-GMPbound forms have been reported (Huang et al, 2012;Ouyang et al, 2012;Shu et al, 2012;Su et al, 2012;Yin et al, 2012). Interesting, they all seem to adopt a similar set of open-form structures, which are different to the closed-form structures of mSTING that we have described in this manuscript.…”

Section: Why Do Hsting and Msting Adopt Different Apo And C-di-gmp-bomentioning

confidence: 63%

“…The sample preparation for truncated mSTING and its complexes with c-di-GMP have been reported in a previous communication (Su et al, 2012). In brief, the full-length Mus musculus (mouse) STING gene was synthesized using a twostep PCR-based DNA-synthesis method using Escherichia coli optimized codons (Xiong et al, 2008).…”

Section: Methodsmentioning

confidence: 99%

“…Screening for crystallization conditions for SeMetlabelled and native mSTING and their c-di-GMP-bound complexes were performed using sitting-drop vapour diffusion in 96-well plates (Hampton Research) at 277 K by mixing 0.5 ml protein solution with 0.5 ml reservoir solution and equilibrating against 50 ml reservoir solution. Pyramid-shaped crystals of mSTING 138-344 appeared in 7 d from drops equilibrated against 50 ml reservoir solution comprising 2% PEG 400, 1.6 M ammonium sulfate, 0.1 M MES monohydrate; pyramid-like crystals of SeMet-labelled mSTING 138-344 appeared in 7 d from drops equilibrated against 50 ml reservoir solution comprising 1.6 M potassium/sodium phosphate, 0.1 M Na HEPES pH 7.5 (Su et al, 2012). Crystals of both proteins suitable for diffraction experiments were grown from drops obtained by mixing 1.5 ml protein solution with 1.5 ml reservoir solution and equilibrated against 500 ml reservoir solution at 277 K.…”

Section: Crystallization Of Truncated Variants Of Mstingmentioning

confidence: 99%

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Novel c-di-GMP recognition modes of the mouse innate immune adaptor protein STING

Chin

et al. 2013

Acta Crystallogr D Biol Cryst

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The mammalian ER protein STING (stimulator of interferon genes; also known as MITA, ERIS, MPYS or TMEM173) is an adaptor protein that links the detection of cytosolic dsDNA to the activation of TANK-binding kinase 1 (TBK1) and its downstream transcription factor interferon regulatory factor 3 (IFN3). Recently, STING itself has been found to be the direct receptor of bacterial c-di-GMP, and crystal structures of several human STING C-terminal domain (STING-CTD) dimers in the apo form or in complex with c-di-GMP have been published. Here, a novel set of structures of mouse STING-CTD (mSTING(137-344)) in apo and complex forms determined from crystals obtained under different crystallization conditions are reported. These novel closed-form structures exhibited considerable differences from previously reported open-form human STING-CTD structures. The novel mSTING structures feature extensive interactions between the two monomers, a unique asymmetric c-di-GMP molecule with one guanine base in an unusual syn conformation that is well accommodated in the dimeric interface with many direct specific interactions and two unexpected equivalent secondary peripheral c-di-GMP binding sites. Replacement of the amino acids crucial for specific c-di-GMP binding in mSTING significantly changes the ITC titration profiles and reduces the IFN-β reporter luciferase activity. Taken together, these results reveal a more stable c-di-GMP binding mode of STING proteins that could serve as a template for rational drug design to stimulate interferon production by mammalian cells.

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Section: Why Do Hsting and Msting Adopt Different Apo And C-di-gmp-bomentioning

confidence: 63%

Section: Methodsmentioning

confidence: 99%

Section: Crystallization Of Truncated Variants Of Mstingmentioning

confidence: 99%

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Novel c-di-GMP recognition modes of the mouse innate immune adaptor protein STING

Chin

et al. 2013

Acta Crystallogr D Biol Cryst

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“…The availability of CDN-STING crystal structures along with recent results describing human STING allele-CDN dependent signaling relationships will facilitate structure-based studies to develop effective CDN compounds that activate innate immunity across genetically diverse human populations [Chin et al 2013; Huang et al 2012; Ouyang et al 2012; Shang et al 2012; Shu et al 2012; Su et al 2012]. Hydrogen bonding between CDN oxygen and phosphate atoms with amino acids at the interface of the STING homo-dimer binding cleft promotes its conformational transition and activate downstream signaling [Burdette and Vance, 2013].…”

Section: Cdn Synthesis and Design Of Compounds With Increased Potencymentioning

confidence: 99%

Rationale, progress and development of vaccines utilizing STING-activating cyclic dinucleotide adjuvants

Dubensky

Kanne

Leong

2013

Therapeutic Advances in Vaccines

163

152

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A principal barrier to the development of effective vaccines is the availability of adjuvants and formulations that can elicit both effector and long-lived memory CD4 and CD8 T cells. Cellular immunity is the presumptive immune correlate of protection against intracellular pathogens: a group composed of bacteria, viruses and protozoans that is responsible for a staggering level of morbidity and mortality on a global scale. T-cell immunity is also correlated with clinical benefit in cancer, and the development of therapeutic strategies to harness the immune system to treat diverse malignancies is currently undergoing a renaissance. Cyclic dinucleotides (CDNs) are ubiquitous small molecule second messengers synthesized by bacteria that regulate diverse processes and are a relatively new class of adjuvants that have been shown to increase vaccine potency. CDNs activate innate immunity by directly binding the endoplasmic reticulum-resident receptor STING (stimulator of interferon genes), activating a signaling pathway that induces the expression of interferon-β (IFN-β) and also nuclear factor-κB (NF-κB) dependent inflammatory cytokines. The STING signaling pathway has emerged as a central Toll-like receptor (TLR) independent mediator of host innate defense in response to sensing cytosolic nucleic acids, either through direct binding of CDNs secreted by bacteria, or, as shown recently, through binding of a structurally distinct CDN produced by a host cell receptor in response to binding cytosolic double-stranded (ds)DNA. Although this relatively new class of adjuvants has to date only been evaluated in mice, newly available CDN-STING cocrystal structures will likely intensify efforts in this field towards further development and evaluation in human trials both in preventive vaccine and immunotherapy settings.

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“…Transmembrane Protein 173 (TMEM173), also named stimulator of interferon genes (STING), is a transmembrane protein localized in endoplasmic reticulum[4]. Previous studies revealed that it played an important role in the innate immune response to viral and bacterial infections via regulating type-I IFN signaling and thereby innate immunity[5].…”

Section: Introductionmentioning

confidence: 99%

Decreased Expression of TMEM173 Predicts Poor Prognosis in Patients with Hepatocellular Carcinoma

et al. 2016

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Hepatocellular carcinoma (HCC) is one of the most lethal cancer types, and chronic infection with Hepatitis B Virus (HBV) is identified as the strongest risk factor for HCC. Transmembrane Protein 173 (TMEM173) is a pattern recognition receptor which functions as a major regulator of the innate immune response to viral and bacterial infections. However, the prognostic value of TMEM173 in HCC remains elusive. Thus, we aimed to evaluate the potential prognostic significance of TMEM173 expression in HCC patients following curative resection. Immunohistochemistry was used to detect TMEM173 expression in 96 HCC patients. We found that TMEM173 protein expression was remarkably decreased in tumor tissues compared to non-tumor tissues, and that TMEM173 staining intensity was inversely correlated with tumor size, tumor invasion TNM stage and overall survival (OS) in HCC patients. Multivariate analysis supported TMEM173 as an independent prognostic factor, and identified that combining TMEM173 expression with TNM stage showed better prognostic efficiency for OS in HCC patients. In summary, TMEM173 was discovered having an independent prognostic value and may serve as a potential immunotherapeutic target for HCC.

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Crystallization studies of the murine c-di-GMP sensor protein STING

Cited by 9 publications

References 49 publications

Novel c-di-GMP recognition modes of the mouse innate immune adaptor protein STING

Novel c-di-GMP recognition modes of the mouse innate immune adaptor protein STING

Rationale, progress and development of vaccines utilizing STING-activating cyclic dinucleotide adjuvants

Decreased Expression of TMEM173 Predicts Poor Prognosis in Patients with Hepatocellular Carcinoma

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