Glycan Stability and Flexibility: Thermodynamic and Kinetic Characterization of Nonconventional Hydrogen Bonding in Lewis Antigens.

We provide evidence for CH-based nonconventional hydrogen bonds (H-bonds) for 10 Lewis antigens and two of their rhamnose analogues. We also characterize the thermodynamics and kinetics of the H-bonds in these molecules and present a plausible explanation for the presence of nonconventional H-bonds in Lewis antigens. Using an alternative method to simultaneously fit a series of temperature-dependent fast exchange nuclear magnetic resonance (NMR) spectra, we determined that the H-bonded conformation is favored by ∼1 kcal/mol over the non-H-bonded conformation. Additionally, a comparison of temperature-dependent 13C linewidths in various Lewis antigens and the two rhamnose analogues reveals H-bonds between the carbonyl oxygen of the N-acetyl group of N-acetylglucosamine and the OH2 group of galactose/fucose. The data presented herein provide insight into the contribution of nonconventional H-bonding to molecular structure and could therefore be used for the rational design of therapeutics.

Quantification of reaction cycle parameters for an essential molecular switch in an auxin-responsive transcription circuit in rice.

Protein-based molecular switches play critical roles in biological processes. The importance of the prolyl cis-trans switch is underscored by the ubiquitous presence of peptidyl prolyl isomerases such as cyclophilins that accelerate the intrinsically slow isomerization rate. In rice, a tryptophan-proline (W-P) cis-trans switch in transcription repressor protein OsIAA11 along with its associated cyclophilin LRT2 are essential components in a negative feedback gene regulation circuit that controls lateral root initiation in response to the plant hormone auxin. Importantly, no quantitative characterizations of the individual (microscopic) thermodynamic and kinetic parameters for any cyclophilin-catalyzed W-P isomerization have been reported. Here we present NMR studies that determine and independently validate these parameters for LRT2 catalysis of the W-P motif in OsIAA11, providing predictive power for understanding the role of this switch in the auxin-responsive circuit and the resulting lateral rootless phenotype in rice. We show that the observed isomerization rate is linearly dependent on LRT2 concentration but is independent of OsIAA11 concentration over a wide range, and LRT2 is optimally tuned to maintain OsIAA11 at its cis-trans equilibrium to supply the slower downstream cis-specific proteasomal degradation with maximal OsIAA11 substrate. This indicates that accelerating the LRT2-catalyzed isomerization would not accelerate OsIAA degradation, whereas decreasing this rate via targeted mutation could reveal relationships between circuit dynamics and lateral root development. Moreover, we show that sequences flanking the highly conserved Aux/IAA W-P motif do not impact LRT2 catalysis, suggesting that the parameters determined here are broadly applicable across highly conserved cyclophilins and their Aux/IAA targets.

Isomerase-catalyzed binding of interleukin-1 receptor-associated kinase 1 to the EVH1 domain of vasodilator-stimulated phosphoprotein.

Interleukin-1 receptor-associated kinase 1 (IRAK1) is a crucial signaling kinase in the immune system, involved in Toll-like receptor signaling. Vasodilator-stimulated phosphoprotein (VASP) is a central player in cell migration that regulates actin polymerization and connects signaling events to cytoskeletal remodeling. A VASP­IRAK1 interaction is thought to be important in controlling macrophage migration in response to protein kinase C-ε activation. We show that the monomeric VASP EVH1 domain directly binds to the 168WPPPP172 motif in the IRAK1 undefined domain (IRAK1-UD) with moderate affinity (KDApp = 203 ± 3 µM). We further show that this motif adopts distinct cis and trans isomers for the Trp168­Pro169 peptide bond with nearly equal populations, and that binding to the VASP EVH1 domain is specific for the trans isomer, coupling binding to isomerization. Nuclear magnetic resonance line shape analysis and tryptophan fluorescence experiments reveal the complete kinetics and thermodynamics of the binding reaction, showing diffusion-limited binding to the trans isomer followed by slow, isomerization-dependent binding. We further demonstrate that the peptidyl-prolyl isomerase cyclophilin A (CypA) catalyzes isomerization of the Trp168­Pro169 peptide bond and accelerates binding of the IRAK1-UD to the VASP EVH1 domain. We propose that binding of IRAK1 to tetrameric VASP is regulated by avidity through the assembly of IRAK1 onto receptor-anchored signaling complexes and that an isomerase such as CypA may modulate IRAK1 signaling in vivo. These studies demonstrate a direct interaction between IRAK1 and VASP and suggest a potential mechanism for how this interaction might be regulated by both assembly of IRAK1 onto an activated signaling complex and PPIase enzymes.

The IL-33-PIN1-IRAK-M axis is critical for type 2 immunity in IL-33-induced allergic airway inflammation.

Interleukin 33 (IL-33) is among the earliest-released cytokines in response to allergens that orchestrate type 2 immunity. The prolyl cis-trans isomerase PIN1 is known to induce cytokines for eosinophil survival and activation by stabilizing cytokines mRNAs, but the function of PIN1 in upstream signaling pathways in asthma is unknown. Here we show that interleukin receptor associated kinase M (IRAK-M) is a PIN1 target critical for IL-33 signaling in allergic asthma. NMR analysis and docking simulations suggest that PIN1 might regulate IRAK-M conformation and function in IL-33 signaling. Upon IL-33-induced airway inflammation, PIN1 is activated for binding with and isomerization of IRAK-M, resulting in IRAK-M nuclear translocation and induction of selected proinflammatory genes in dendritic cells. Thus, the IL-33-PIN1-IRAK-M is an axis critical for dendritic cell activation, type 2 immunity and IL-33 induced airway inflammation.

Neighboring phosphoSer-Pro motifs in the undefined domain of IRAK1 impart bivalent advantage for Pin1 binding.

The peptidyl prolyl isomerase Pin1 has two domains that are considered to be its binding (WW) and catalytic (PPIase) domains, both of which interact with phosphorylated Ser/Thr-Pro motifs. This shared specificity might influence substrate selection, as many known Pin1 substrates have multiple sequentially close phosphoSer/Thr-Pro motifs, including the protein interleukin-1 receptor-associated kinase-1 (IRAK1). The IRAK1 undefined domain (UD) contains two sets of such neighboring motifs (Ser131/Ser144 and Ser163/Ser173), suggesting possible bivalent interactions with Pin1. Using a series of NMR titrations with 15N-labeled full-length Pin1 (Pin1-FL), PPIase, or WW domain and phosphopeptides representing the Ser131/Ser144 and Ser163/Ser173 regions of IRAK1-UD, bivalent interactions were investigated. Binding studies using singly phosphorylated peptides showed that individual motifs displayed weak affinities (> 100 µm) for Pin1-FL and each isolated domain. Analysis of dually phosphorylated peptides binding to Pin1-FL showed that inclusion of bivalent states was necessary to fit the data. The resulting complex model and fitted parameters were applied to predict the impact of bivalent states at low micromolar concentrations, demonstrating significant affinity enhancement for both dually phosphorylated peptides (3.5 and 24 µm for peptides based on the Ser131/Ser144 and Ser163/Ser173 regions, respectively). The complementary technique biolayer interferometry confirmed the predicted affinity enhancement for a representative set of singly and dually phosphorylated Ser131/Ser144 peptides at low micromolar concentrations, validating model predictions. These studies provide novel insights regarding the complexity of interactions between Pin1 and activated IRAK1, and more broadly suggest that phosphorylation of neighboring Ser/Thr-Pro motifs in proteins might provide competitive advantage at cellular concentrations for engaging with Pin1.