[Show abstract][Hide abstract] ABSTRACT: NMR studies of multi-domain protein complexes provide unique insight into their molecular interactions and dynamics in solution. For large proteins domain-selective isotope labeling is desired to reduce signal overlap, but available methods require extensive optimization and often give poor ligation yields. We present an optimized strategy for segmental labeling of multi-domain proteins using the S. aureus transpeptidase Sortase A. Critical improvements compared to existing protocols are (1) the efficient removal of cleaved peptide fragments by centrifugal filtration and (2) a strategic design of cleavable and non-cleavable affinity tags for purification. Our approach enables routine production of milligram amounts of purified segmentally labeled protein for NMR and other biophysical studies.
Journal of Biomolecular NMR 08/2015; DOI:10.1007/s10858-015-9981-0 · 3.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Epstein-Barr virus (EBV) is a γ-herpesvirus that may cause infectious mononucleosis in young adults. In addition, epidemiological and molecular evidence links EBV to the pathogenesis of lymphoid and epithelial malignancies. EBV has the unique ability to transform resting B cells into permanently proliferating, latently infected lymphoblastoid cell lines. Epstein-Barr virus nuclear antigen 2 (EBNA-2) is a key regulator of viral and cellular gene expression for this transformation process. The N-terminal region of EBNA-2 comprising residues 1-58 appears to mediate multiple molecular functions including self-association and transactivation. However, it remains to be determined if the N-terminus of EBNA-2 directly provides these functions or if these activities merely depend on the dimerization involving the N-terminal domain. To address this issue, we determined the three-dimensional structure of the EBNA-2 N-terminal dimerization (END) domain by heteronuclear NMR-spectroscopy. The END domain monomer comprises a small fold of four β-strands and an α-helix which form a parallel dimer by interaction of two β-strands from each protomer. A structure-guided mutational analysis showed that hydrophobic residues in the dimer interface are required for self-association in vitro. Importantly, these interface mutants also displayed severely impaired self-association and transactivation in vivo. Moreover, mutations of solvent-exposed residues or deletion of the α-helix do not impair dimerization but strongly affect the functional activity, suggesting that the EBNA-2 dimer presents a surface that mediates functionally important intra- and/or intermolecular interactions. Our study shows that the END domain is a novel dimerization fold that is essential for functional activity. Since this specific fold is a unique feature of EBNA-2 it might provide a novel target for anti-viral therapeutics.
[Show abstract][Hide abstract] ABSTRACT: Kinase inhibition is considered to be an important therapeutic target for LRRK2 mediated Parkinson's disease (PD). Many LRRK2 kinase inhibitors have been reported, but have yet to be optimized in order to qualify as drug candidates for the treatment of the disease. In order to start a structure-function analysis of such inhibitors we mutated the active site of Dictyostelium Roco4 kinase to resemble LRRK2. Here, we show Saturation Transfer Difference (STD) NMR and the first co-crystal structures of two potent in vitro inhibitors, LRRK2-IN-1 and Compound19, with mutated Roco4. Our data demonstrate that this system can serve as excellent tool for the structural characterization and optimization of LRRK2 inhibitors using X-ray crystallography and NMR spectroscopy.
[Show abstract][Hide abstract] ABSTRACT: One function of the glucocorticoid receptor (GR) in corticotroph cells is to suppress the transcription of the gene encoding proopiomelanocortin (POMC), the precursor of the stress hormone adrenocorticotropin (ACTH). Cushing disease is a neuroendocrine condition caused by partially glucocorticoid-resistant corticotroph adenomas that excessively secrete ACTH, which leads to hypercortisolism. Mutations that impair GR function explain glucocorticoid resistance only in sporadic cases. However, the proper folding of GR depends on direct interactions with the chaperone heat shock protein 90 (HSP90, refs. 7,8). We show here that corticotroph adenomas overexpress HSP90 compared to the normal pituitary. N- and C-terminal HSP90 inhibitors act at different steps of the HSP90 catalytic cycle to regulate corticotroph cell proliferation and GR transcriptional activity. C-terminal inhibitors cause the release of mature GR from HSP90, which promotes its exit from the chaperone cycle and potentiates its transcriptional activity in a corticotroph cell line and in primary cultures of human corticotroph adenomas. In an allograft mouse model, the C-terminal HSP90 inhibitor silibinin showed anti-tumorigenic effects, partially reverted hormonal alterations, and alleviated symptoms of Cushing disease. These results suggest that the pathogenesis of Cushing disease caused by overexpression of heat shock proteins and consequently misregulated GR sensitivity may be overcome pharmacologically with an appropriate HSP90 inhibitor.
Nature Medicine 02/2015; 21(3). DOI:10.1038/nm.3776 · 27.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Der Reaktionszyklus des molekularen Chaperons Hsp90 ist durch ATP-getriebene Konformationsänderungen charakterisiert, die der ATP-Hydrolyse vorgeschaltet sind. Es gibt bereits niedermolekulare Inhibitoren von Hsp90, die mit der ATP-Bindung konkurrieren. Wir fragten uns, ob Verbindungen existieren, die den konformationellen Zyklus beschleunigen können. In einem FRET-basierten Screen, der Konformationsänderungen in Hsp90 detektiert, identifizierten wir zwei Verbindungen und analysierten ihre Wirkungsweise. Wir können zeigen, dass die intrinsische Inhibition von Hsp90, welche konformationelle Umlagerungen verhindert, durch diese Substanzen überwunden wird. Diese Wirkungsweise ähnelt der des Cochaperons Aha1 von Hsp90, das die ATPase-Aktivität beschleunigt. Während beide Verbindungen Konformationsänderungen beeinflussen, so steuern sie jeweils verschiedene Aspekte der Strukturübergänge. Zudem unterscheiden sich ihre durch NMR-Spektroskopie bestimmten Bindestellen. Unsere Studie zeigt, dass kleine Moleküle in der Lage sind, spezifische geschwindigkeitsbestimmende Übergänge in Hsp90 in ähnlicher Weise zu beschleunigen wie dies durch Protein-Cofaktoren geschieht.
[Show abstract][Hide abstract] ABSTRACT: The molecular chaperone Hsp90 undergoes an ATP-driven cycle of conformational changes in which large structural rearrangements precede ATP hydrolysis. Well-established small-molecule inhibitors of Hsp90 compete with ATP-binding. We wondered whether compounds exist that can accelerate the conformational cycle. In a FRET-based screen reporting on conformational rearrangements in Hsp90 we identified compounds. We elucidated their mode of action and showed that they can overcome the intrinsic inhibition in Hsp90 which prevents these rearrangements. The mode of action is similar to that of the co-chaperone Aha1 which accelerates the Hsp90 ATPase. However, while the two identified compounds influence conformational changes, they target different aspects of the structural transitions. Also, the binding site determined by NMR spectroscopy is distinct. This study demonstrates that small molecules are capable of triggering specific rate-limiting transitions in Hsp90 by mechanisms similar to those in protein cofactors.
[Show abstract][Hide abstract] ABSTRACT: Genetic equality between males and females is established by chromosome-wide dosage-compensation mechanisms. In the fruitfly Drosophila melanogaster, the dosage-compensation complex promotes twofold hypertranscription of the single male X-chromosome and is silenced in females by inhibition of the translation of msl2, which codes for the limiting component of the dosage-compensation complex1, 2. The female-specific protein Sex-lethal (Sxl) recruits Upstream-of-N-ras (Unr) to the 3′ untranslated region of msl2 messenger RNA, preventing the engagement of the small ribosomal subunit3. Here we report the 2.8 Å crystal structure, NMR and small-angle X-ray and neutron scattering data of the ternary Sxl–Unr–msl2 ribonucleoprotein complex featuring unprecedented intertwined interactions of two Sxl RNA recognition motifs, a Unr cold-shock domain and RNA. Cooperative complex formation is associated with a 1,000-fold increase of RNA binding affinity for the Unr cold-shock domain and involves novel ternary interactions, as well as non-canonical RNA contacts by the α1 helix of Sxl RNA recognition motif 1. Our results suggest that repression of dosage compensation, necessary for female viability, is triggered by specific, cooperative molecular interactions that lock a ribonucleoprotein switch to regulate translation. The structure serves as a paradigm for how a combination of general and widespread RNA binding domains expands the code for specific single-stranded RNA recognition in the regulation of gene expression.