Successful treatment of chronic myelogenous leukemia is based on inhibitors binding to the ATP site of the deregulated breakpoint cluster region (Bcr)-Abelson tyrosine kinase (Abl) fusion protein.Recently, a new type of allosteric inhibitors targeting the Abl myristoyl pocket was shown in preclinical studies to overcome ATP-site inhibitor resistance arising in some patients. Using NMR and smallangle X-ray scattering, we have analyzed the solution conformations of apo Abelson tyrosine kinase (c-Abl) and c-Abl complexes with ATPsite and allosteric inhibitors. Binding of the ATP-site inhibitor imatinib leads to an unexpected open conformation of the multidomain SH3-SH2-kinase c-Abl core, whose relevance is confirmed by cellular assays on Bcr-Abl. The combination of imatinib with the allosteric inhibitor GNF-5 restores the closed, inactivated state. Our data provide detailed insights on the poorly understood combined effect of the two inhibitor types, which is able to overcome drug resistance.CML | heteronuclear NMR | protein dynamics

Modulation of protein–protein interactions (PPIs) by small molecules has emerged as a valuable approach in drug discovery. Compared to direct inhibition, PPI stabilization is vastly underexplored but has strong advantages, including the ability to gain selectivity by targeting an interface formed only upon association of proteins. Here, we present the application of a site-directed screening technique based on disulfide trapping (tethering) to select for fragments that enhance the affinity between protein partners. We target the phosphorylation-dependent interaction between the hub protein 14-3-3σ and a peptide derived from Estrogen Receptor α (ERα), an important breast cancer target that is negatively regulated by 14-3-3σ. We identify orthosteric stabilizers that increase 14-3-3/ERα affinity up to 40-fold and propose the mechanism of stabilization based on X-ray crystal structures. These fragments already display partial selectivity toward ERα-like motifs over other representative 14-3-3 clients. This first of its kind study illustrates the potential of the tethering approach to overcome the hurdles in systematic PPI stabilizer discovery.

For a wide range of proteins of high interest, the major obstacle for NMR studies is the lack of an affordable eukaryotic expression system for isotope labeling. Here, a simple and affordable protocol is presented to produce uniform labeled proteins in the most prevalent eukaryotic expression system for structural biology, namely Spodoptera frugiperda insect cells. Incorporation levels of 80% can be achieved for (15)N and (13)C with yields comparable to expression in full media. For (2)H,(15)N and (2)H,(13)C,(15)N labeling, incorporation is only slightly lower with 75 and 73%, respectively, and yields are typically twofold reduced. The media were optimized for isotope incorporation, reproducibility, simplicity and cost. High isotope incorporation levels for all labeling patterns are achieved by using labeled algal amino acid extracts and exploiting well-known biochemical pathways. The final formulation consists of just five commercially available components, at costs 12-fold lower than labeling media from vendors. The approach was applied to several cytosolic and secreted target proteins.

Prion diseases are caused by the aggregation of the native alpha-helical prion protein PrP(C) into its pathological beta-sheet-rich isoform PrP(Sc). In current models of PrP(Sc), helix1 is assumed to be preferentially converted into beta-sheet during aggregation of PrP(C). This was supported by the NMR structure of PrP(C) since, in contrast to the isolated helix1, helix2 and helix3 are connected by a small loop and are additionally stabilized by an interhelical disulfide bond. However, helix1 is extremely hydrophilic and has a high helix propensity. This prompted us to investigate the role of helix1 in prion aggregation using humPrP(23-159) including helix1 (144-156) compared with the C-terminal-truncated isoform humPrP(23-144) corresponding to the pathological human stop mutations Q160Stop and Y145Stop, respectively. Most unexpectedly, humPrP(23-159) aggregated significantly faster compared with the truncated fragment humPrP(23-144), clearly demonstrating that helix1 is involved in the aggregation process. However, helix1 is not resistant to digestion with proteinase K in fibrillar humPrP(23-159), suggesting that helix1 is not converted to beta-sheet. This is confirmed by Fourier transformation infrared spectroscopy since there is almost no difference in beta-sheet content of humPrP(23-159) fibrils compared with humPrP(23-144). In conclusion, we provide strong direct evidence that in contrast to earlier assumptions helix1 is not converted into beta-sheet during aggregation of PrP(C) to PrP(Sc).

Proteins typically interact with multiple binding partners, and often different parts of their surfaces are employed to establish these protein–protein interactions (PPIs). Members of the class of 14-3-3 adapter proteins bind to several hundred other proteins in the cell. Multiple small molecules for the modulation of 14-3-3 PPIs have been disclosed; however, they all target the conserved phosphopeptide binding channel, so that selectivity is difficult to achieve. Here we report on the discovery of two individual secondary binding sites that have been identified by combining nuclear magnetic resonance-based fragment screening and X-ray crystallography. The two pockets that these fragments occupy are part of at least three physiologically relevant and structurally characterized 14-3-3 PPI interfaces, including those with serotonin N-acetyltransferase and plant transcription factor FT. In addition, the high degree of conservation of the two sites implies their relevance for 14-3-3 PPIs. This first identification of secondary sites on 14-3-3 proteins bound by small molecule ligands might facilitate the development of new chemical tool compounds for more selective PPI modulation.

Background:In human prion diseases, the phenotype is modified by a methionine/valine polymorphism at codon 129. Results: Prion stop mutants have a conserved amyloid core comprising residue 129. Conclusion:The polymorphic residue 129 is buried in the amyloid core structure. Significance: The data support a critical role of the methionine/valine 129 polymorphism in human prion diseases.

Misfolding intermediates play a key role in defining aberrant protein aggregation and amyloid formation in more than 15 different human diseases. However, their experimental characterization is challenging due to the transient nature and conformational heterogeneity of the involved states. Here, we demonstrate that direct carbon-detected NMR experiments allow observation, assignment, and structural analysis of molten globule amyloid intermediates that are severely broadened by conformational exchange. The method is used to characterize the structure and dynamics of partially unfolded intermediates of the 99-residue protein -2-microglobulin, which is the major component of insoluble aggregates occurring in dialysis-related amyloidosis. Comparison of the conformational properties of the molten globule-like intermediates with levels of deuterium incorporation into amyloid fibrils of -2-microglobulin revealed a close relationship between the conformational properties of the metastable intermediates and the -sheet-rich insoluble aggregates of -2-microglobulin.Misfolding intermediates play a key role in defining aberrant protein aggregation and amyloid formation in more than 15 different human diseases. 1,2 However, the experimental characterization of the conformation of amyloid intermediates is challenging due to their transient nature and conformational heterogeneity. 3,4 This severely limits our knowledge about the relation of the conformation of the precursor states to the structure of amyloid fibrils.Dialysis-related amyloidosis is a protein misfolding disease resulting from deposition of amyloid aggregates in skeletal tissue that contain fibrils of the 99-residue-long protein -2-microglobulin (b2m). 5 Amyloid formation of b2m is strongly enhanced in conditions that destabilize its globular structure. 6 This can be achieved in Vitro by acid denaturation, with two distinct intermediate states being formed under acidic conditions. The highest population of the partially unfolded intermediate occurs at pH 3.6, where also the rate of fibril formation reaches a maximum. 7 However, long and straight amyloid fibrils resembling those extracted from patients are formed from a precursor state formed at pH 2.5. 8,9 Here, we compare the conformational properties of these two partially unfolded intermediates with single-residue resolution and demonstrate that a close relationship exists between the structure and dynamics of the amyloid precursor species and the morphology of mature fibrils of b2m.At pH 2.5 b2m is highly unfolded, the majority of resonances are observed in 1 H-15 N HSQC spectra (Supporting Information, Figure 1) and can be assigned to individual residues. 10 In contrast, only about 40-60 resolved peaks on top of a broad hump of unresolved signal intensity (visible at lower contour levels) could be observed at pH 3.6 ( Figure 1a). 11 To obtain sequence-specific information about the pH 3.6 intermediate, we tested direct 13 C detection. 12-14 Direct carbon detection was previously successfully used fo...

Intrinsically disordered or unstructured proteins (IUPs) play a key role in normal and pathological biochemical processes. Despite their importance for function, this category of proteins remains beyond the reach of classical structural biology because of their inherent conformational heterogeneity. Measurements of global dimensions strongly suggested a random coil-like behavior of IUPs.[1] In contrast to these findings, NMR spectroscopy detected significant amounts of local structure in denatured and unfolded states. Recently, these two apparently contradicting behaviors were reconciled when it was shown that the local conformational behavior of IUPs can be described by a simple model based on residuespecific f/f propensities. [2,3] In particular, residual dipolar couplings (RDCs), which can be measured for proteins that are weakly aligned in dilute liquid-crystalline media, could be predicted from this model by assuming a steric interaction between the protein and the alignment medium. Furthermore, we showed that RDCs are also sensitive detectors of transient long-range interactions in IUPs.