Biochemistry and Pharmacology - Research Publications
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ItemThe structure of the extracellular domains of human interleukin 11? receptor reveals mechanisms of cytokine engagement(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2020-06-12)Interleukin (IL) 11 activates multiple intracellular signaling pathways by forming a complex with its cell surface α-receptor, IL-11Rα, and the β-subunit receptor, gp130. Dysregulated IL-11 signaling has been implicated in several diseases, including some cancers and fibrosis. Mutations in IL-11Rα that reduce signaling are also associated with hereditary cranial malformations. Here we present the first crystal structure of the extracellular domains of human IL-11Rα and a structure of human IL-11 that reveals previously unresolved detail. Disease-associated mutations in IL-11Rα are generally distal to putative ligand-binding sites. Molecular dynamics simulations showed that specific mutations destabilize IL-11Rα and may have indirect effects on the cytokine-binding region. We show that IL-11 and IL-11Rα form a 1:1 complex with nanomolar affinity and present a model of the complex. Our results suggest that the thermodynamic and structural mechanisms of complex formation between IL-11 and IL-11Rα differ substantially from those previously reported for similar cytokines. This work reveals key determinants of the engagement of IL-11 by IL-11Rα that may be exploited in the development of strategies to modulate formation of the IL-11-IL-11Rα complex.
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ItemINPHARMA-Based Determination of Ligand Binding Modes at α1-Adrenergic Receptors Explains the Molecular Basis of Subtype Selectivity(WILEY-V C H VERLAG GMBH, 2020-09-10)The structural poses of ligands that bind weakly to protein receptors are challenging to define. In this work we have studied ligand interactions with the adrenoreceptor (AR) subtypes, α1A -AR and α1B -AR, which belong to the G protein-coupled receptor (GPCR) superfamily, by employing the solution-based ligand-observed NMR method interligand NOEs for pharmacophore mapping (INPHARMA). A lack of receptor crystal structures and of subtype-selective drugs has hindered the definition of the physiological roles of each subtype and limited drug development. We determined the binding pose of the weakly binding α1A -AR-selective agonist A-61603 relative to an endogenous agonist, epinephrine, at both α1A -AR and α1B -AR. The NMR experimental data were quantitatively compared, by using SpINPHARMA, to the back-calculated spectra based on ligand poses obtained from all-atom molecular dynamics simulations. The results helped mechanistically explain the selectivity of (R)-A-61603 towards α1A -AR, thus demonstrating an approach for targeting subtype selectivity in ARs.
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ItemProbing the correlation between ligand efficacy and conformational diversity at the ?(1A)-adrenoreceptor reveals allosteric coupling of its microswitches(American Society for Biochemistry and Molecular Biology, 2020-05-22)G protein–coupled receptors (GPCRs) use a series of conserved microswitches to transmit signals across the cell membrane via an allosteric network encompassing the ligand-binding site and the G protein-binding site. Crystal structures of GPCRs provide snapshots of their inactive and active states, but poorly describe the conformational dynamics of the allosteric network that underlies GPCR activation. Here, we analyzed the correlation between ligand binding and receptor conformation of the α1A-adrenoreceptor, a GPCR that stimulates smooth muscle contraction in response to binding noradrenaline. NMR of [13CϵH3]methionine-labeled α1A-adrenoreceptor variants, each exhibiting differing signaling capacities, revealed how different classes of ligands modulate the conformational equilibria of this receptor. [13CϵH3]Methionine residues near the microswitches exhibited distinct states that correlated with ligand efficacies, supporting a conformational selection mechanism. We propose that allosteric coupling among the microswitches controls the conformation of the α1A-adrenoreceptor and underlies the mechanism of ligand modulation of GPCR signaling in cells.
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ItemCo-regulation of the transcription controlling ATF2 phosphoswitch by JNK and p38(NATURE RESEARCH, 2020-11-13)Transcription factor phosphorylation at specific sites often activates gene expression, but how environmental cues quantitatively control transcription is not well-understood. Activating protein 1 transcription factors are phosphorylated by mitogen-activated protein kinases (MAPK) in their transactivation domains (TAD) at so-called phosphoswitches, which are a hallmark in response to growth factors, cytokines or stress. We show that the ATF2 TAD is controlled by functionally distinct signaling pathways (JNK and p38) through structurally different MAPK binding sites. Moreover, JNK mediated phosphorylation at an evolutionarily more recent site diminishes p38 binding and made the phosphoswitch differently sensitive to JNK and p38 in vertebrates. Structures of MAPK-TAD complexes and mechanistic modeling of ATF2 TAD phosphorylation in cells suggest that kinase binding motifs and phosphorylation sites line up to maximize MAPK based co-regulation. This study shows how the activity of an ancient transcription controlling phosphoswitch became dependent on the relative flux of upstream signals.