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Changes in Signal Transducer and Activator of Transcription 3 (STAT3) Dynamics Induced by Complexation with Pharmacological Inhibitors of Src Homology 2 (SH2) Domain Dimerization*

  • Diana Resetca
    Affiliations
    Center for Research in Mass Spectrometry, Department of Chemistry, York University, Toronto, Ontario M3J 1P3, Canada
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  • Sina Haftchenary
    Footnotes
    Affiliations
    Department of Chemical and Physical Sciences, University of Toronto Mississauga, Mississauga, Ontario L5L 1C6, Canada
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  • Patrick T. Gunning
    Footnotes
    Affiliations
    Department of Chemical and Physical Sciences, University of Toronto Mississauga, Mississauga, Ontario L5L 1C6, Canada
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  • Derek J. Wilson
    Correspondence
    To whom correspondence should be addressed: York University Chemistry Dept., 4700 Keele St., Toronto, Ontario M3J 1P3, Canada. Tel.: 416-736-2100 (ext. 20786); Fax: 416-736-5936
    Affiliations
    Center for Research in Mass Spectrometry, Department of Chemistry, York University, Toronto, Ontario M3J 1P3, Canada
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  • Author Footnotes
    * This work was supported in part by National Sciences and Engineering Research Council (NSERC) Discovery Grant 257588 and an Ontario Ministry of Research and Innovation early researcher award (to D. J. W.).
    This article contains supplemental Fig. S1.
    1 Supported by an NSERC postgraduate scholarship-doctoral.
    2 Supported by the NSERC, Canadian Institutes of Health Research, and Canadian Breast Cancer Research Foundation.
Open AccessPublished:October 06, 2014DOI:https://doi.org/10.1074/jbc.M114.595454
      The activity of the transcription factor signal transducer and activator of transcription 3 (STAT3) is dysregulated in a number of hematological and solid malignancies. Development of pharmacological STAT3 Src homology 2 (SH2) domain interaction inhibitors holds great promise for cancer therapy, and a novel class of salicylic acid-based STAT3 dimerization inhibitors that includes orally bioavailable drug candidates has been recently developed. The compounds SF-1-066 and BP-1-102 are predicted to bind to the STAT3 SH2 domain. However, given the highly unstructured and dynamic nature of the SH2 domain, experimental confirmation of this prediction was elusive. We have interrogated the protein-ligand interaction of STAT3 with these small molecule inhibitors by means of time-resolved electrospray ionization hydrogen-deuterium exchange mass spectrometry. Analysis of site-specific evolution of deuterium uptake induced by the complexation of STAT3 with SF-1-066 or BP-1-102 under physiological conditions enabled the mapping of the in silico predicted inhibitor binding site to the STAT3 SH2 domain. The binding of both inhibitors to the SH2 domain resulted in significant local decreases in dynamics, consistent with solvent exclusion at the inhibitor binding site and increased rigidity of the inhibitor-complexed SH2 domain. Interestingly, inhibitor binding induced hot spots of allosteric perturbations outside of the SH2 domain, manifesting mainly as increased deuterium uptake, in regions of STAT3 important for DNA binding and nuclear localization.

      Introduction

      Signal transducer and activator of transcription 3 (STAT3), an Src homology 2 (SH2)
      The abbreviations used are: SH2
      Src homology 2
      HDX
      hydrogen-deuterium exchange
      TRESI
      time-resolved electrospray ionization
      MBP
      maltose-binding protein
      Csk
      C-terminal Src kinase.
      domain-containing protein (molecular weight, 88,000), mediates intracellular signaling downstream of a number of cytokine and growth factor receptors and is involved in regulating cell proliferation, survival, differentiation, angiogenesis, cell migration, and inflammatory signaling (
      • Yu H.
      • Pardoll D.
      • Jove R.
      STATs in cancer inflammation and immunity: a leading role for STAT3.
      ). Dysregulated STAT3 activity promotes the progression of a multitude of hematological and solid malignancies (
      • Bromberg J.
      • Darnell Jr., J.E.
      The role of STATs in transcriptional control and their impact on cellular function.
      ). STAT3 is a transcription factor whose canonical activation pathway is modulated by tyrosine phosphorylation. Phosphorylation at Tyr-705 drives STAT3 homodimerization or heterodimerization with other STAT family members mediated by its SH2 domain (
      • Haan S.
      • Hemmann U.
      • Hassiepen U.
      • Schaper F.
      • Schneider-Mergener J.
      • Wollmer A.
      • Heinrich P.C.
      • Grötzinger J.
      Characterization and binding specificity of the monomeric STAT3-SH2 domain.
      ), which binds the phosphotyrosine peptide with nanomolar affinity (
      • Ladbury J.E.
      • Lemmon M.A.
      • Zhou M.
      • Green J.
      • Botfield M.C.
      • Schlessinger J.
      Measurement of the binding of tyrosyl phosphopeptides to SH2 domains: a reappraisal.
      ). Inhibition of SH2 domain function is thus a robust strategy to antagonize its biological activity by inhibiting STAT3 dimerization (
      • Page B.D.
      • Ball D.P.
      • Gunning P.T.
      Signal transducer and activator of transcription 3 inhibitors: a patent review.
      ).
      The SH2 domain is a structurally conserved feature of many intracellular signaling transducers and is capable of recognizing and binding to phosphorylated tyrosine residues presented in the context of specific protein sequences (
      • Moran M.F.
      • Koch C.A.
      • Anderson D.
      • Ellis C.
      • England L.
      • Martin G.S.
      • Pawson T.
      Src homology region 2 domains direct protein-protein interactions in signal transduction.
      ). Comprising minimal secondary structure, the SH2 domain and the phosphopeptide binding interface are highly unstructured and dynamic (
      • Finerty Jr., P.J.
      • Mittermaier A.K.
      • Muhandiram R.
      • Kay L.E.
      • Forman-Kay J.D.
      NMR dynamics-derived insights into the binding properties of a peptide interacting with an SH2 domain.
      ), a property that makes the development of small molecule inhibitors targeted at this important domain class a challenge. Gunning and co-workers (
      • Zhang X.
      • Yue P.
      • Page B.D.
      • Li T.
      • Zhao W.
      • Namanja A.T.
      • Paladino D.
      • Zhao J.
      • Chen Y.
      • Gunning P.T.
      • Turkson J.
      Orally bioavailable small-molecule inhibitor of transcription factor Stat3 regresses human breast and lung cancer xenografts.
      ,
      • Fletcher S.
      • Page B.D.
      • Zhang X.
      • Yue P.
      • Li Z.H.
      • Sharmeen S.
      • Singh J.
      • Zhao W.
      • Schimmer A.D.
      • Trudel S.
      • Turkson J.
      • Gunning P.T.
      Antagonism of the Stat3-Stat3 protein dimer with salicylic acid based small molecules.
      ) have recently developed a number of pharmacological, orally bioavailable inhibitors of STAT3 based on the salicylic acid pharmacophore through a series of quantitative structure-activity relationship studies. Two such inhibitors, SF-1-066 and BP-1-102, demonstrate an IC50 of 35 and 19.7 μm, respectively, for the inhibition of STAT3 DNA binding activity in vitro and inhibit STAT3 dimerization (
      • Page B.D.
      • Fletcher S.
      • Yue P.
      • Li Z.
      • Zhang X.
      • Sharmeen S.
      • Datti A.
      • Wrana J.L.
      • Trudel S.
      • Schimmer A.D.
      • Turkson J.
      • Gunning P.T.
      Identification of a non-phosphorylated, cell permeable, small molecule ligand for the Stat3 SH2 domain.
      ). Additionally, BP-1-102 has demonstrated potent antitumor effects in vivo in human breast and lung cancer xenograft studies in mice (
      • Zhang X.
      • Yue P.
      • Page B.D.
      • Li T.
      • Zhao W.
      • Namanja A.T.
      • Paladino D.
      • Zhao J.
      • Chen Y.
      • Gunning P.T.
      • Turkson J.
      Orally bioavailable small-molecule inhibitor of transcription factor Stat3 regresses human breast and lung cancer xenografts.
      ). Given the demonstration that both SF-1-066 and BP-1-102 inhibit the binding of the phosphotyrosine peptide by the SH2 domain of STAT3 (
      • Zhang X.
      • Yue P.
      • Page B.D.
      • Li T.
      • Zhao W.
      • Namanja A.T.
      • Paladino D.
      • Zhao J.
      • Chen Y.
      • Gunning P.T.
      • Turkson J.
      Orally bioavailable small-molecule inhibitor of transcription factor Stat3 regresses human breast and lung cancer xenografts.
      ,
      • Zhang X.
      • Yue P.
      • Fletcher S.
      • Zhao W.
      • Gunning P.T.
      • Turkson J.
      A novel small-molecule disrupts Stat3 SH2 domain-phosphotyrosine interactions and Stat3-dependent tumor processes.
      ), a proposed mechanism of action involves inhibitor binding to the SH2 domain that disrupts its function or otherwise blocks the binding of the phosphopeptide regulatory region. In silico docking studies identified a putative binding site for the two inhibitors comprising all three phosphopeptide-binding subpockets of the SH2 domain (
      • Page B.D.
      • Fletcher S.
      • Yue P.
      • Li Z.
      • Zhang X.
      • Sharmeen S.
      • Datti A.
      • Wrana J.L.
      • Trudel S.
      • Schimmer A.D.
      • Turkson J.
      • Gunning P.T.
      Identification of a non-phosphorylated, cell permeable, small molecule ligand for the Stat3 SH2 domain.
      ), supporting this mechanism. However, little experimental evidence exists corroborating that the putative binding site of SF-1-066 and BP-1-102 is indeed in the SH2 domain, and the mechanism of action of these molecules is not yet fully understood.
      Although a number of x-ray crystal structures of STAT3 are available (
      • Nkansah E.
      • Shah R.
      • Collie G.W.
      • Parkinson G.N.
      • Palmer J.
      • Rahman K.M.
      • Bui T.T.
      • Drake A.F.
      • Husby J.
      • Neidle S.
      • Zinzalla G.
      • Thurston D.E.
      • Wilderspin A.F.
      Observation of unphosphorylated STAT3 core protein binding to target dsDNA by PEMSA and x-ray crystallography.
      ,
      • Ren Z.
      • Mao X.
      • Mertens C.
      • Krishnaraj R.
      • Qin J.
      • Mandal P.K.
      • Romanowski M.J.
      • McMurray J.S.
      • Chen X.
      Crystal structure of unphosphorylated STAT3 core fragment.
      ), including that of the phosphorylated STAT3 dimer (
      • Becker S.
      • Groner B.
      • Müller C.W.
      Three-dimensional structure of the Stat3β homodimer bound to DNA.
      ), derivation of x-ray crystal structures of STAT3 complexed with SF-1-066 and BP-1-102 has proven exceptionally challenging, and to date, no inhibitor-STAT3 structure has been resolved. Unlike these static images, dynamic representations of protein structures can offer unparalleled mechanistic insights into biological processes and interactions by depicting the time evolution of protein conformational ensembles (
      • Resetca D.
      • Wilson D.J.
      Characterizing rapid, activity-linked conformational transitions in proteins via sub-second hydrogen deuterium exchange mass spectrometry.
      ). Hydrogen-deuterium exchange (HDX) mass spectrometry (MS) when coupled with microfluidic sample processing enables the interrogation of protein dynamics of the native protein structures in solution, permitting the in situ observation of enzyme-catalyzed reactions (
      • Liuni P.
      • Jeganathan A.
      • Wilson D.J.
      Conformer selection and intensified dynamics during catalytic turnover in chymotrypsin.
      ) and protein-ligand interactions (
      • Rob T.
      • Gill P.K.
      • Golemi-Kotra D.
      • Wilson D.J.
      An electrospray ms-coupled microfluidic device for sub-second hydrogen/deuterium exchange pulse-labelling reveals allosteric effects in enzyme inhibition.
      ). HDX implemented on a microfluidic device for rapid mixing, quenching, and proteolytic digestion of the protein analyte offers substantial advantages over other techniques, including easy implementation, unlimited protein analyte size, time scales compatible with protein breathing motions, and site-specific resolution of up to a few amino acids (
      • Rob T.
      • Liuni P.
      • Gill P.K.
      • Zhu S.
      • Balachandran N.
      • Berti P.J.
      • Wilson D.J.
      Measuring dynamics in weakly structured regions of proteins using microfluidics-enabled subsecond H/D exchange mass spectrometry.
      ).
      We have applied a time-resolved electrospray ionization MS HDX (TRESI-MS/HDX) approach to the study of protein-ligand interactions involving the highly unstructured SH2 ligand-binding domain in the context of the near full-length STAT3 protein. TRESI-MS/HDX is particularly useful for interrogating weakly structured protein regions where short deuterium labeling pulses are essential for accurately assessing the magnitudes of dynamic perturbations (
      • Rob T.
      • Liuni P.
      • Gill P.K.
      • Zhu S.
      • Balachandran N.
      • Berti P.J.
      • Wilson D.J.
      Measuring dynamics in weakly structured regions of proteins using microfluidics-enabled subsecond H/D exchange mass spectrometry.
      ). Given that weakly structured regions experience moderate protection from exchange, conformational changes induced by ligand binding to such regions can be readily detected by TRESI-MS/HDX with a heightened degree of sensitivity. In the present work, TRESI-MS/HDX was applied to (i) experimentally identify the STAT3 binding site of the salicylic acid-based inhibitors SF-1-066 and BP-1-102 and (ii) probe the changes in protein dynamics induced in STAT3 upon complexation with these inhibitors.

      DISCUSSION

      This study highlights the utility of structural mass spectrometry based on TRESI-MS/HDX in probing protein-ligand interactions and understanding dynamic changes and mechanisms associated with ligand binding. The “bottom-up” HDX approach as implemented here enables one to generate accurate, subsecond time scale kinetic profiles of site-specific exchange in the native protein structures. This structural technique is particularly useful for interrogating protein domains with minimal secondary structure or a high degree of intrinsic disorder. Although NMR has been used to probe disordered proteins to a degree, it is highly limited by protein size in applications involving protein-ligand interactions (
      • Resetca D.
      • Wilson D.J.
      Characterizing rapid, activity-linked conformational transitions in proteins via sub-second hydrogen deuterium exchange mass spectrometry.
      ,
      • Montelione G.T.
      • Zheng D.
      • Huang Y.J.
      • Gunsalus K.C.
      • Szyperski T.
      Protein NMR spectroscopy in structural genomics.
      ).
      Here we applied the microfluidics-integrated TRESI-MS/HDX workflow to derive averaged dynamic structures of free and inhibitor-bound STAT3. Our examination of the dynamic changes in this protein largely confirmed in silico predictions with respect to the inhibitor binding regions as well as revealed additional regions inside the STAT3 SH2 domain that exhibit strong protection from exchange due to the interaction with the inhibitors. SF-1-066 and BP-1-102 represent a novel, state-of-the-art class of salicylic acid-based SH2-targeted STAT3 inhibitors with low micromolar and nanomolar range affinity for STAT3, respectively; however, no experimental evidence directly describes their interaction with the SH2 domain (
      • Zhang X.
      • Yue P.
      • Page B.D.
      • Li T.
      • Zhao W.
      • Namanja A.T.
      • Paladino D.
      • Zhao J.
      • Chen Y.
      • Gunning P.T.
      • Turkson J.
      Orally bioavailable small-molecule inhibitor of transcription factor Stat3 regresses human breast and lung cancer xenografts.
      ,
      • Page B.D.
      • Fletcher S.
      • Yue P.
      • Li Z.
      • Zhang X.
      • Sharmeen S.
      • Datti A.
      • Wrana J.L.
      • Trudel S.
      • Schimmer A.D.
      • Turkson J.
      • Gunning P.T.
      Identification of a non-phosphorylated, cell permeable, small molecule ligand for the Stat3 SH2 domain.
      ).
      It has been proposed that these inhibitors bind to the SH2 domain of STAT3 and preclude the binding of its ligand, the phospho-Tyr705-containing peptide of another STAT3 molecule, which in turn inhibits the ability of STAT3 to dimerize. This mechanism is supported by the observation that both SF-1-066 and BP-1-102 can inhibit the binding of phospho-Tyr-705-containing peptide to STAT3 in vitro (
      • Zhang X.
      • Yue P.
      • Page B.D.
      • Li T.
      • Zhao W.
      • Namanja A.T.
      • Paladino D.
      • Zhao J.
      • Chen Y.
      • Gunning P.T.
      • Turkson J.
      Orally bioavailable small-molecule inhibitor of transcription factor Stat3 regresses human breast and lung cancer xenografts.
      ,
      • Zhang X.
      • Yue P.
      • Fletcher S.
      • Zhao W.
      • Gunning P.T.
      • Turkson J.
      A novel small-molecule disrupts Stat3 SH2 domain-phosphotyrosine interactions and Stat3-dependent tumor processes.
      ) and inhibit the biological activity of STAT3 both in vitro and in vivo. It is unclear whether the ability of these compounds to inhibit phosphopeptide binding is a consequence of their direct complexation with the SH2 domain and competitive inhibition as predicted by in silico docking studies (
      • Page B.D.
      • Fletcher S.
      • Yue P.
      • Li Z.
      • Zhang X.
      • Sharmeen S.
      • Datti A.
      • Wrana J.L.
      • Trudel S.
      • Schimmer A.D.
      • Turkson J.
      • Gunning P.T.
      Identification of a non-phosphorylated, cell permeable, small molecule ligand for the Stat3 SH2 domain.
      ) or a consequence of their interaction with other regions of the STAT3 protein. Our data for the first time provide an experimental proof that the SF-1-066 and BP-1-102 compounds bind exclusively to the SH2 domain on the STAT3 molecule and complete the evidence set supporting the originally proposed mechanism for their biological activity. Additionally, our data reveal a series of increases in HDX outside the STAT3 SH2 domain due to inhibitor binding, suggesting allosteric effects that are potentially destabilizing to the protein.
      Peptides ISKERERAIL (589–598), LRFSE (608–612), and YKIMDATN (657–664), which exhibited significant decreases in deuterium uptake upon inhibitor binding, map to the surface of the inhibitor-binding pocket predicted by docking studies (Fig. 6, A and B). The side chain of Arg-609, mapping near the peptide LRFSE (608–612), was predicted to hydrogen bond with the salicylic acid moiety common to both inhibitors, greatly stabilizing the binding energy of the molecules (
      • Zhang X.
      • Yue P.
      • Page B.D.
      • Li T.
      • Zhao W.
      • Namanja A.T.
      • Paladino D.
      • Zhao J.
      • Chen Y.
      • Gunning P.T.
      • Turkson J.
      Orally bioavailable small-molecule inhibitor of transcription factor Stat3 regresses human breast and lung cancer xenografts.
      ,
      • Fletcher S.
      • Singh J.
      • Zhang X.
      • Yue P.
      • Page B.D.
      • Sharmeen S.
      • Shahani V.M.
      • Zhao W.
      • Schimmer A.D.
      • Turkson J.
      • Gunning P.T.
      Disruption of transcriptionally active Stat3 dimers with non-phosphorylated, salicylic acid-based small molecules: potent in vitro and tumor cell activities.
      ). This is consistent with large decreases in backbone deuteration observed in the LRFSE (608–612) peptide given the tight predicted occupancy of this subpocket by the salicylic acid moiety for both inhibitors.
      The peptide YKIMDATN (657–664), lining the cyclohexylbenzyl-binding subpocket, only experienced a significant decrease in deuterium uptake in the case of the higher affinity inhibitor BP-1-102 (Fig. 6, A and B). The binding of the cyclohexylbenzyl moiety to the predicted subpocket is dominated by relatively weak van der Waals interactions (
      • Page B.D.
      • Fletcher S.
      • Yue P.
      • Li Z.
      • Zhang X.
      • Sharmeen S.
      • Datti A.
      • Wrana J.L.
      • Trudel S.
      • Schimmer A.D.
      • Turkson J.
      • Gunning P.T.
      Identification of a non-phosphorylated, cell permeable, small molecule ligand for the Stat3 SH2 domain.
      ). This observation with respect to the YKIMDATN (657–664) peptide, which lines the deepest end of the cyclohexylbenzyl subpocket, is consistent with the tighter binding of the cyclohexylbenzyl moiety of BP-1-102 compared with SF-1-066 as predicted by in silico docking (Fig. 1D). The only structural difference between the two inhibitors is the substituent at the sulfonamide end of the molecule, a tosyl sulfonamide for SF-1-066 and a pentafluorobenzyl sulfonamide for BP-1-102. Despite this, in the peptide lining the relevant subpocket, ISKERERAIL (589–598), a significant but similar decrease in deuteration was observed that is not reflective of any differential effect on the backbone amides in this region.
      Other regions of the SH2 domain not immediately lining the surface of the predicted inhibitor-binding site also exhibited significant decreases in deuterium uptake upon inhibitor binding. Some of these segments interact directly with specific regions of the binding pocket. For example, the peptide SKEGGV (614–619) is located in proximity to LRFSE (608–612), which lines the salicylic acid moiety-binding subpocket, with the backbone amide of the Ser-611 making a hydrogen bond with the backbone carbonyl of Gly-618. Both inhibitors induce a significant decrease in deuterium uptake in the SKEGGV (614–619) peptide.
      Overall, the higher affinity inhibitor, BP-1-102, induced more decreases in deuterium uptake compared with SF-1-066 (Fig. 6C). This and other observations of significant decreases in deuterium uptake in the SH2 domain suggest that either (i) the inhibitors are able to bind to regions of the SH2 domain outside of the in silico predicted binding pocket or (ii) the binding of the inhibitors to the predicted binding pocket results in an overall stabilization of and possibly the induction of additional secondary structure in a large portion of the SH2 domain. We tend to favor the latter possibility given the lack of clustered HDX decreases elsewhere in the molecule outside of the SH2 domain that would point to nonspecific binding by the inhibitors to STAT3.
      Our data suggest that the core of the STAT3 SH2 domain represents a highly dynamic network potentially able to transmit local perturbations in dynamics to distal regions of the domain. Indeed, studies of dynamics of SH2 domains of other proteins reveal a general, global reduction or stabilization in dynamics in a large portion of the SH2 domain upon peptide ligand binding, resulting in increased rigidity that propagates throughout the entire domain (
      • Engen J.R.
      • Gmeiner W.H.
      • Smithgall T.E.
      • Smith D.L.
      Hydrogen exchange shows peptide binding stabilizes motions in Hck SH2.
      ,
      • Shoelson S.E.
      • Sivaraja M.
      • Williams K.P.
      • Hu P.
      • Schlessinger J.
      • Weiss M.A.
      Specific phosphopeptide binding regulates a conformational change in the PI 3-kinase SH2 domain associated with enzyme activation.
      ). Overall, the clustering of significant decreases in deuterium uptake in the SH2 domain of inhibitor-complexed STAT3 provides experimental evidence supporting the SH2 domain being the target site of the salicylic acid-based inhibitors SF-1-066 and BP-1-102. This finding corroborates in silico docking predictions as well as other experimental evidence. In fluorescence polarization assays with STAT3 and a fluorescently labeled phosphotyrosine peptide (5-carboxyfluorescein-GpYLPQTV-NH2 where pY is phosphotyrosine) that mimics the natural SH2 domain target substrate, both SF-1-066 and BP-1-102 inhibitors competed strongly with substrate binding with an IC50 of 20 and 4.1 μm, respectively (
      • Zhang X.
      • Yue P.
      • Page B.D.
      • Li T.
      • Zhao W.
      • Namanja A.T.
      • Paladino D.
      • Zhao J.
      • Chen Y.
      • Gunning P.T.
      • Turkson J.
      Orally bioavailable small-molecule inhibitor of transcription factor Stat3 regresses human breast and lung cancer xenografts.
      ,
      • Zhang X.
      • Yue P.
      • Fletcher S.
      • Zhao W.
      • Gunning P.T.
      • Turkson J.
      A novel small-molecule disrupts Stat3 SH2 domain-phosphotyrosine interactions and Stat3-dependent tumor processes.
      ).
      Interestingly, both inhibitors induced a burst of dynamic changes, mainly manifesting as significant relative increases in deuterium uptake, outside of the SH2 domain of STAT3 (Fig. 6, A and B). These allosteric changes induced in the inhibitor-complexed structures propagate into STAT3 domains and regions involved in DNA binding (β-barrel and connector domains) and nuclear localization of the protein (four-helix bundle and β-barrel domains) (
      • Becker S.
      • Groner B.
      • Müller C.W.
      Three-dimensional structure of the Stat3β homodimer bound to DNA.
      ,
      • Ma J.
      • Zhang T.
      • Novotny-Diermayr V.
      • Tan A.L.
      • Cao X.
      A novel sequence in the coiled-coil domain of Stat3 essential for its nuclear translocation.
      ). Interestingly, the majority of allosteric changes mapped to (or near) regions important to the DNA binding activity of STAT3 (DNA-binding domain, residues 406–514) (
      • Horvath C.M.
      • Wen Z.
      • Darnell Jr., J.E.
      A STAT protein domain that determines DNA sequence recognition suggests a novel DNA-binding domain.
      ). The backbone amide of Val-432 for instance is directly involved in DNA binding, and Glu-435 forms a hydrogen bond that is important for maintaining the structural rigidity of one of the DNA binding loops (
      • Becker S.
      • Groner B.
      • Müller C.W.
      Three-dimensional structure of the Stat3β homodimer bound to DNA.
      ). We observed a significant increase in deuterium uptake in the peptide IVTEEL (431–436) with both inhibitors. This suggests the possibility that the salicylic acid-based inhibitors SF-1-066 and BP-1-102 could also be acting via an allosteric mechanism to modulate non-canonical DNA binding of STAT3, a prediction that will need to be explored experimentally with the use of STAT3 mutants.
      The vast majority of allosteric changes induced by inhibitor binding (Fig. 6, A and B) mapped to regions predicted to have α-helical secondary structure (Fig. 2). Increases in HDX in α-helical regions most likely result from the loss or remodeling of secondary structure, affecting the hydrogen bonding network. STAT3 complexed with SF-1-066 exhibited more allosteric effects (Fig. 6C) compared with the tighter bound BP-1-102 complex, which may reflect the need for a degree of conformational freedom in the SH2 domain to efficiently “transmit” the allosteric signal. Nevertheless, this observation is qualitative and speculative and would necessitate further investigation. Allosteric changes were not observed in any peptides derived from the MBP protein that was fused via a polyasparagine linker to the N terminus of the STAT3 in our construct (data not shown). This confirms that changes induced by the binding of SF-1-066 and BP-1-102 inhibitors to STAT3 outside the SH2 domain are not a result of experimental error.
      Allosteric modulation is an important mechanism of communication between distinct domains within the protein, and exactly how the information is transmitted between domains is an area of active investigation (
      • Ma B.
      • Tsai C.J.
      • Haliloğlu T.
      • Nussinov R.
      Dynamic allostery: linkers are not merely flexible.
      ). The ability of the SH2 domain to induce conformational changes in other domains in the same protein has been demonstrated for the C-terminal Src kinase (Csk) (
      • Wong L.
      • Lieser S.A.
      • Miyashita O.
      • Miller M.
      • Tasken K.
      • Onuchic J.N.
      • Adams J.A.
      • Woods Jr., V.L.
      • Jennings P.A.
      Coupled motions in the SH2 and kinase domains of Csk control Src phosphorylation.
      ), a protein-tyrosine kinase that phosphorylates other kinases of the Src family. In Csk, the binding of the SH2 domain target peptide, Csk-binding protein, appears to induce conformational changes that are transmitted through the SH2 linker to the active site and other regions of the protein. Conversely, binding of Csk nucleotide substrates to the kinase domain was shown to impact HDX protection in other regions of the protein, including the SH2 domain, depending on the nature of the nucleotide present (
      • Hamuro Y.
      • Wong L.
      • Shaffer J.
      • Kim J.S.
      • Stranz D.D.
      • Jennings P.A.
      • Woods Jr., V.L.
      • Adams J.A.
      Phosphorylation driven motions in the COOH-terminal Src kinase, CSK, revealed through enhanced hydrogen-deuterium exchange and mass spectrometry (DXMS).
      ). Our study adds to the work of others examining allosteric modulation by HDX MS (
      • Zhang J.
      • Adrián F.J.
      • Jahnke W.
      • Cowan-Jacob S.W.
      • Li A.G.
      • Iacob R.E.
      • Sim T.
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      • Engen J.R.
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      • Warmuth M.
      • Gray N.S.
      Targeting Bcr-Abl by combining allosteric with ATP-binding-site inhibitors.
      ,
      • Iacob R.E.
      • Pene-Dumitrescu T.
      • Zhang J.
      • Gray N.S.
      • Smithgall T.E.
      • Engen J.R.
      Conformational disturbance in Abl kinase upon mutation and deregulation.
      ,
      • Panjarian S.
      • Iacob R.E.
      • Chen S.
      • Wales T.E.
      • Engen J.R.
      • Smithgall T.E.
      Enhanced SH3/linker interaction overcomes Abl kinase activation by gatekeeper and myristic acid binding pocket mutations and increases sensitivity to small molecule inhibitors.
      ), highlighting the vast utility of this structural technique in probing the evolution of allosteric changes in intact proteins under physiological conditions.
      In summary, in silico predicted interactions of SF-1-066 and BP-1-102 inhibitors with regions of the STAT3 SH2 domain were experimentally confirmed by analyzing changes in site-specific deuterium uptake upon STAT3 complexation with the inhibitors. Showing a large contiguous decrease in deuterium uptake around the putative SH2 domain-binding site, our data support a mechanism of inhibition driven by “pacification” of the SH2 domain through complexation. This compliments prior biochemical evidence that points to disruption of STAT3 dimerization as the primary inhibitory mechanism. However, inhibitor binding was also found to induce substantial changes in the dynamics of the DNA-binding and nuclear localization domains, which could reflect additional modes of inhibition targeting non-canonical activation pathways (
      • Nkansah E.
      • Shah R.
      • Collie G.W.
      • Parkinson G.N.
      • Palmer J.
      • Rahman K.M.
      • Bui T.T.
      • Drake A.F.
      • Husby J.
      • Neidle S.
      • Zinzalla G.
      • Thurston D.E.
      • Wilderspin A.F.
      Observation of unphosphorylated STAT3 core protein binding to target dsDNA by PEMSA and x-ray crystallography.
      ,
      • Inghirami G.
      • Chiarle R.
      • Simmons W.J.
      • Piva R.
      • Schlessinger K.
      • Levy D.E.
      New and old functions of STAT3: a pivotal target for individualized treatment of cancer.
      ). Our study highlights the incremental benefits of dynamic structure analysis to understanding protein-ligand interactions via the examination of conformational ensembles rather than static representations of the protein complex structures.

      Acknowledgments

      We thank Dr. Rob C. Laister, Princess Margaret Cancer Center (Toronto, Ontario, Canada), for generously providing the STAT3 cDNA.

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