Min-Kyu Cho

Structural characterization of α‐synuclein in an aggregation prone state

The relation of α‐synuclein (αS) aggregation to Parkinsons disease has long been recognized, but the pathogenic species and its molecular properties have yet to be identified. To obtain insight into the properties of αS in an aggregation‐prone state, we studied the structural properties of αS at acidic pH using NMR spectroscopy and computation. NMR demonstrated that αS remains natively unfolded at lower pH, but secondary structure propensities were changed in proximity to acidic residues. The ensemble of conformations of αS at acidic pH is characterized by a rigidification and compaction of the Asp and Glu‐rich C‐terminal region, an increased probability for proximity between the NAC‐region and the C‐terminal region and a lower probability for interactions between the N‐ and C‐terminal regions.

Identification and characterization of novel classes of macrophage migration inhibitory factor (MIF) inhibitors with distinct mechanisms of action

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is considered an attractive therapeutic target in multiple inflammatory and autoimmune disorders. In addition to its known biologic activities, MIF can also function as a tautomerase. Several small molecules have been reported to be effective inhibitors of MIF tautomerase activity in vitro. Herein we employed a robust activity-based assay to identify different classes of novel inhibitors of the catalytic and biological activities of MIF. Several novel chemical classes of inhibitors of the catalytic activity of MIF with IC50 values in the range of 0.2–15.5 μm were identified and validated. The interaction site and mechanism of action of these inhibitors were defined using structure-activity studies and a battery of biochemical and biophysical methods. MIF inhibitors emerging from these studies could be divided into three categories based on their mechanism of action: 1) molecules that covalently modify the catalytic site at the N-terminal proline residue, Pro1; 2) a novel class of catalytic site inhibitors; and finally 3) molecules that disrupt the trimeric structure of MIF. Importantly, all inhibitors demonstrated total inhibition of MIF-mediated glucocorticoid overriding and AKT phosphorylation, whereas ebselen, a trimer-disrupting inhibitor, additionally acted as a potent hyperagonist in MIF-mediated chemotactic migration. The identification of biologically active compounds with known toxicity, pharmacokinetic properties, and biological activities in vivo should accelerate the development of clinically relevant MIF inhibitors. Furthermore, the diversity of chemical structures and mechanisms of action of our inhibitors makes them ideal mechanistic probes for elucidating the structure-function relationships of MIF and to further determine the role of the oligomerization state and catalytic activity of MIF in regulating the function(s) of MIF in health and disease.

Mitogen‐activated protein kinases interacting kinases are autoinhibited by a reprogrammed activation segment

Autoinhibition is a recurring mode of protein kinase regulation and can be based on diverse molecular mechanisms. Here, we show by crystal structure analysis, nuclear magnetic resonance (NMR)‐based nucleotide affinity studies and rational mutagenesis that nonphosphorylated mitogen‐activated protein (MAP) kinases interacting kinase (Mnk) 1 is autoinhibited by conversion of the activation segment into an autoinhibitory module. In a Mnk1 crystal structure, the activation segment is repositioned via a Mnk‐specific sequence insertion at the N‐terminal lobe with the following consequences: (i) the peptide substrate binding site is deconstructed, (ii) the interlobal cleft is narrowed, (iii) an essential Lys–Glu pair is disrupted and (iv) the magnesium‐binding loop is locked into an ATP‐competitive conformation. Consistently, deletion of the Mnk‐specific insertion or removal of a conserved phenylalanine side chain, which induces a blockade of the ATP pocket, increase the ATP affinity of Mnk1. Structural rearrangements required for the activation of Mnks are apparent from the cocrystal structure of a Mnk2D228G–staurosporine complex and can be modeled on the basis of crystal packing interactions. Our data suggest a novel regulatory mechanism specific for the Mnk subfamily.

Conserved core of amyloid fibrils of wild type and A30P mutant α-synuclein

The major component of neural inclusions that are the pathological hallmark of Parkinsons disease are amyloid fibrils of the protein α‐synuclein (aS). Here we investigated if the disease‐related mutation A30P not only modulates the kinetics of aS aggregation, but also alters the structure of amyloid fibrils. To this end we optimized the method of quenched hydrogen/deuterium exchange coupled to NMR spectroscopy and performed two‐dimensional proton‐detected high‐resolution magic angle spinning experiments. The combined data indicate that the A30P mutation does not cause changes in the number, location and overall arrangement of β‐strands in amyloid fibrils of aS. At the same time, several residues within the fibrillar core retain nano‐second dynamics. We conclude that the increased pathogenicity related to the familial A30P mutation is unlikely to be caused by a mutation‐induced change in the conformation of aS aggregates.

Dissociation of Amyloid Fibrils of α‐Synuclein in Supercooled Water

Several neurodegenerative diseases, including Alzheimer s, Creutzfeldt–Jakob, and Parkinson s disease, are associated with the formation of amyloid fibrils. Amyloid fibrils have a b-sheet-rich molecular architecture called a cross-b structure. The b-sheet conformation imparts extremely high thermodynamic stability and remarkable physical properties to amyloid fibrils. They are highly resistant to hydrostatic pressure and high temperature, whereas protofibrils and earlier aggregates are more sensitive to these extreme conditions. The stability of mature amyloid fibrils exceeds that of globular proteins, thus suggesting that they may represent the global minimum in terms of free energy. In addition, they have a strength comparable to that of steel. Nature exploits these unusual properties of amyloidgenic structures for a variety of physiological functions. Moreover, fibrillar peptide structures might have great potential as structural or structuring elements in nanotechnology applications. The native state of proteins can be unfolded both by high temperature and by cooling. Cold denaturation is predicted by the Gibbs–Helmholtz Equation, and attributed to specific interactions between nonpolar protein groups and water: tightly packed structures unfold at sufficiently low temperature to expose internal nonpolar groups to the water. Direct observation of cold denaturation is generally hard to achieve in the absence of denaturant, extreme pH values, or mutations, as the transition temperature for most proteins is well below 0 8C. Freezing, however, can be avoided down to temperatures as low as 20 8C by careful supercooling of small sample volumes. Nevertheless, this is generally not sufficiently cold to induce denaturation in stable, native proteins. Here we demonstrate that amyloid fibrils of the protein asynuclein (aS), which constitute the insoluble aggregates found in brains of patients suffering from Parkinson s disease, are highly sensitive to low temperature. Despite their remarkable stability to hydrostatic pressure and high temperatures, mature amyloid fibrils of aS rapidly dissociate in supercooled water at 15 8C. N-Labeled aS amyloid fibrils were prepared in vitro by incubating 0.1 mm freshly prepared N-labeled aS in 20 mm tris(hydroxymethyl)aminomethane (Tris) and 100 mm NaCl at pH 7.4. Incubation was carried out under continuous stirring at 37 8C for up to 14 days until a steady state was reached, as judged by thioflavin-T (ThT) fluorescence. Matured fibrils were pelleted by centrifugation at 215000g for 2 h and then resuspended in 50 mm phosphate buffer. Transmission electron micrographs showed regular fibrils with a diameter of approximately 40 nm (Figure 1a). A strong ThT fluorescence signal was detected for the fibrils (see the Supporting Information). Previous X-ray diffraction and solid-state NMR measurements have shown that amyloid fibrils of aS adopt a cross-b structure. No cross-peaks were visible in the H-N HSQC spectra, which is in agreement with the large molecular weight of amyloid fibrils and their associated fast relaxation (Figure 1a).

Cold denaturation of a protein dimer monitored at atomic resolution

Protein folding and unfolding are crucial for a range of biological phenomena and human diseases. Defining the structural properties of the involved transient species is therefore of prime interest. Using a combination of cold denaturation with NMR spectroscopy, we reveal detailed insight into the unfolding of the homodimeric repressor protein CylR2. Seven three-dimensional structures of CylR2 at temperatures from 25 °C to -16 °C reveal a progressive dissociation of the dimeric protein into a native-like monomeric intermediate followed by transition into a highly dynamic, partially folded state. The core of the partially folded state seems critical for biological function and misfolding.

Cold-induced changes in the protein ubiquitin.

Conformational changes are essential for protein-protein and protein-ligand recognition. Here we probed changes in the structure of the protein ubiquitin at low temperatures in supercooled water using NMR spectroscopy. We demonstrate that ubiquitin is well folded down to 263 K, although slight rearrangements in the hydrophobic core occur. However, amide proton chemical shifts show non-linear temperature dependence in supercooled solution and backbone hydrogen bonds become weaker in the region that is most prone to cold-denaturation. Our data suggest that the weakening of the hydrogen bonds in the β-sheet of ubiquitin might be one of the first events that occur during cold-denaturation of ubiquitin. Interestingly, the same region is strongly involved in ubiquitin-protein complexes suggesting that this part of ubiquitin more easily adjusts to conformational changes required for complex formation.