Solid-state NMR enhanced by dynamic nuclear polarization as a novel tool for ribosome structural biology

The impact of Nuclear Magnetic Resonance (NMR) on studies of large macromolecular complexes hinges on improvements in sensitivity and resolution. Dynamic nuclear polarization (DNP) in the solid state can offer improved sensitivity, provided sample preparation is optimized to preserve spectral resolution. For a few nanomoles of intact ribosomes and an 800kDa ribosomal complex we demonstrate that the combination of DNP and magic-angle spinning NMR (MAS-NMR) allows one to overcome current sensitivity limitations so that homo- and heteronuclear 13C and 15N NMR correlation spectra can be recorded. Ribosome particles, directly pelleted and frozen into an NMR rotor, yield DNP signal enhancements on the order of ~25-fold and spectra that exhibit narrow linewidths, suitable for obtaining site-specific information. We anticipate that the same approach is applicable to other high molecular weight complexe

Solid-state NMR enhanced by dynamic nuclear polarization as a novel tool for ribosome structural biology

The impact of Nuclear Magnetic Resonance (NMR) on studies of large macromolecular complexes hinges on improvements in sensitivity and resolution. Dynamic nuclear polarization (DNP) in the solid state can offer improved sensitivity, provided sample preparation is optimized to preserve spectral resolution. For a few nanomoles of intact ribosomes and an 800 kDa ribosomal complex we demonstrate that the combination of DNP and magic-angle spinning NMR (MAS-NMR) allows one to overcome current sensitivity limitations so that homo- and heteronuclear C-13 and N-15 NMR correlation spectra can be recorded. Ribosome particles, directly pelleted and frozen into an NMR rotor, yield DNP signal enhancements on the order of similar to 25-fold and spectra that exhibit narrow linewidths, suitable for obtaining site-specific information. We anticipate that the same approach is applicable to other high molecular weight complexes

Molecular Mechanism of Protein Kinase Recognition and Sorting by the Hsp90 Kinome-Specific Cochaperone Cdc37

Despite the essential functions of Hsp90, little is known about the mechanism that controls substrate entry into its chaperone cycle. We show that the role of Cdc37 cochaperone reaches beyond that of an adaptor protein and find that it participates in the selective recruitment of only client kinases. Cdc37 recognizes kinase specificity determinants in both clients and nonclients and acts as a general kinase scanning factor. Kinase sorting within the client-to-nonclient continuum relies on the ability of Cdc37 to challenge the conformational stability of clients by locally unfolding them. This metastable conformational state has high affinity for Cdc37 and forms stable complexes through a multidomain cochaperone interface. The interaction with nonclients is not accompanied by conformational changes of the substrate and results in substrate dissociation. Collectively, Cdc37 performs a quality control of protein kinases, where induced conformational instability acts as a flag for Hsp90 dependence and stable cochaperone association

Molecular Mechanism of Protein Kinase Recognition and Sorting by the Hsp90 Kinome-Specific Cochaperone Cdc37

Despite the essential functions of Hsp90, little is known about the mechanism that controls substrate entry into its chaperone cycle. We show that the role of Cdc37 cochaperone reaches beyond that of an adaptor protein and find that it participates in the selective recruitment of only client kinases. Cdc37 recognizes kinase specificity determinants in both clients and nonclients and acts as a general kinase scanning factor. Kinase sorting within the client-to-nonclient continuum relies on the ability of Cdc37 to challenge the conformational stability of clients by locally unfolding them. This metastable conformational state has high affinity for Cdc37 and forms stable complexes through a multidomain cochaperone interface. The interaction with nonclients is not accompanied by conformational changes of the substrate and results in substrate dissociation. Collectively, Cdc37 performs a quality control of protein kinases, where induced conformational instability acts as a flag for Hsp90 dependence and stable cochaperone association

Molecular Insights into the Interaction of Hsp90 with Allosteric Inhibitors Targeting the C-terminal Domain

Unique to targeting the C-terminal domain of Hsp90 (C-Hsp90) is the ability to uncouple the cytotoxic and cytoprotective outcomes of Hsp90 modulation. After the identification of novobiocin as a C-Hsp90 interacting ligand a diverse gamut of novologues emerged, from which KU-32 and KU-596 exhibited strong neuroprotective activity. However, further development of these ligands is hampered by the difficulty to obtain structural information on their complexes with Hsp90. Using saturation transfer difference (STD) NMR spectroscopy, we found that the primary binding epitopes of KU-32 and KU596 map at the ring systems of the ligands and specifically the coumarin and biphenyl structures, respectively. Based on both relative and absolute STD effects, we identified KU-596 sites that can be explored to design novel third-generation novologues. In addition, chemical shift perturbations obtained by methyl-TROSY reveal that novologues bind at the cryptic, C-Hsp90 ATP-binding pocket and produce global, long-range structural rearrangements to dimeric Hsp90