The phosphorylation-specific peptidyl-prolyl isomerase Pin1 catalyzes the isomerization of the peptide bond preceding a proline residue between cis and trans isomers. To best understand the mechanisms of Pin1 regulation, rigorous enzymatic assays of isomerization are required. However, most measures of isomerase activity require significant constraints on substrate sequence and only yield rate constants for the cis isomer, kcatcis and apparent Michaelis constants, KMApp. By contrast, NMR lineshape analysis is a powerful tool for determining microscopic rates and populations of each state in a complex binding scheme. The isolated catalytic domain of Pin1 was employed as a first step towards elucidating the reaction scheme of the full-length enzyme. A 24-residue phosphopeptide derived from the amyloid precurser protein intracellular domain (AICD) phosphorylated at Thr668 served as a biologically-relevant Pin1 substrate. Specific 13C labeling at the Pin1-targeted proline residue provided multiple reporters sensitive to individual isomer binding and on-enzyme catalysis. We have performed titration experiments and employed lineshape analysis of phosphopeptide 13C-1H constant time HSQC spectra to determine Kcatcis, Kcattrans, KDcis, and KDtrans for the catalytic domain of Pin1 acting on this AICD substrate. The on-enzyme equilibrium value of [E·trans]/[E·cis] = 3.9 suggests that the catalytic domain of Pin1 is optimized to operate on this substrate near equilibrium in the cellular context. This highlights the power of lineshape analysis for determining the microscopic parameters of enzyme catalysis, and demonstrates the feasibility of future studies of Pin1-PPIase mutants to gain insights on the catalytic mechanism of this important enzyme.
Expert pathologists commonly perform visual interpretation of histology slides for cervix tissue abnormality diagnosis. We investigated an automated, localized, fusion-based approach for cervix histology image analysis for squamous epithelium classification into Normal, CIN1, CIN2, and CIN3 grades of cervical intraepithelial neoplasia (CIN). The epithelium image analysis approach includes medial axis determination, vertical segment partitioning as medial axis orthogonal cuts, individual vertical segment feature extraction and classification, and image-based classification using a voting scheme fusing the vertical segment CIN grades. Results using 61 images showed at least 15.5% CIN exact grade classification improvement using the localized vertical segment fusion versus global image features.
DNA binding by the ETS transcriptional repressor ETV6 (or TEL) is auto-inhibited ~ 50-fold due to an α-helix that sterically blocks its ETS domain binding interface. Using NMR spectroscopy, we demonstrate that this marginally-stable helix is unfolded, and not displaced to a non-inhibitory position, when ETV6 is bound to DNA containing a consensus 5’GGAA3’ recognition site. Although significantly lower in affinity, binding to non-specific DNA is auto-inhibited ~ 5-fold and also accompanied by helix unfolding. Based on NMR chemical shift perturbations, both specific and non-specific DNA are bound via the same canonical ETS domain interface. However, spectral perturbations are smaller for the non-specific complex, suggesting weaker and less well-defined interactions than in the specific complex. In parallel, the crystal structure of ETV6 bound to a specific DNA duplex was determined. The structure of this complex reveals that a non-conserved histidine residue in the ETS domain recognition helix helps establish the specificity of ETV6 for DNA-binding sites containing 5’GGAA3’ versus 5’GGAT3’. These studies provide a unified steric mechanism for attenuating ETV6 binding to both specific and non-specific DNA and expand the repertoire of characterized auto-inhibitory strategies utilized to regulate ETS factors.
Although conformational changes in TCRs and pMHC molecules often occur upon binding, their relationship to intrinsic flexibility and role in ligand selectivity are poorly understood. Here we used NMR to study TCR-pMHC binding, examining recognition of the QL9/H-2Ld complex by the 2C TCR. Although the majority of the CDR loops of the 2C TCR rigidify upon binding, the CDR3β loop remains mobile within the TCR-pMHC interface. Remarkably, the region of the QL9 peptide that interfaces with CDR3β is also mobile in the free pMHC and in the TCR-pMHC complex. Determination of conformational exchange kinetics revealed that the motions of CDR3β and QL9 are closely matched. The matching of conformational exchange in the free proteins and its persistence in the complex enhances the thermodynamic and kinetic stability of the TCR-pMHC complex and provides a mechanism for facile binding. We thus propose that matching of structural fluctuations is a component of how TCRs scan amongst potential ligands for those that can bind with sufficient stability to enable T cell signaling.
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