Protein dynamics are essential for protein function, and yet it has been challenging to access the underlying atomic motions in solution on nanosecond-to-microsecond time scales. We present a structural ensemble of ubiquitin, refined against residual dipolar couplings (RDCs), comprising solution dynamics up to microseconds. The ensemble covers the complete structural heterogeneity observed in 46 ubiquitin crystal structures, most of which are complexes with other proteins. Conformational selection, rather than induced-fit motion, thus suffices to explain the molecular recognition dynamics of ubiquitin. Marked correlations are seen between the flexibility of the ensemble and contacts formed in ubiquitin complexes. A large part of the solution dynamics is concentrated in one concerted mode, which accounts for most of ubiquitin's molecular recognition heterogeneity and ensures a low entropic complex formation cost.

Summary The BCL6 transcriptional repressor is the most frequently involved oncogene in diffuse large B cell lymphoma (DLBCL). We combined computer-aided drug design with functional assays to identify low molecular weight compounds that bind to the corepressor binding groove of the BCL6 BTB domain. One such compound disrupted BCL6/corepressor complexes in vitro and in vivo, and was observed by X-ray crystallography and NMR to bind the critical site within the BTB groove. This compound could induce expression of BCL6 target genes and kill BCL6-positive DLBCL cell lines. In xenotransplantation experiments, the compound was non-toxic and potently suppressed DLBCL tumors in vivo. The compound also killed primary DLBCLs from human patients.

SUMMARY Kae1 is a universally conserved ATPase and part of the essential gene set in bacteria. In archaea and eukaryotes, Kae1 is embedded within the protein kinase-containing KEOPS complex. Mutation of KEOPS subunits in yeast leads to striking telomere and transcription defects but the exact biochemical function of KEOPS is not known. As a first step to elucidating its function, we solved the atomic structure of archaea-derived KEOPS complexes involving Kae1, Bud32, Pcc1 and Cgi121 subunits. Our studies suggest that Kae1 is regulated at two levels by the primordial protein kinase Bud32, which is itself regulated by Cgi121. Moreover, Pcc1 appears to function as a dimerization module, perhaps suggesting that KEOPS may be a processive molecular machine. Lastly, as Bud32 lacks the conventional substrate-recognition infrastructure of eukaryotic protein kinases including an activation segment, Bud32 may provide a glimpse of the evolutionary history of the protein kinase family.

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Residual dipolar couplings (RDCs) provide information about the dynamic average orientation of internuclear vectors and amplitudes of motion up to milliseconds. They complement relaxation methods, especially on a time-scale window that we have called supra-s c (s c \ supra-s c \ 50 ls). Here we present a robust approach called Self-Consistent RDC-based Model-free analysis (SCRM) that delivers RDC-based order parametersindependent of the details of the structure used for alignment tensor calculation-as well as the dynamic average orientation of the inter-nuclear vectors in the protein structure in a self-consistent manner. For ubiquitin, the SCRM analysis yields an average RDC-derived order parameter of the NH vectors S 2 rdc ¼ 0:72 AE 0:02 compared to S 2 LS = 0.778 ± 0.003 for the Lipari-Szabo order parameters, indicating that the inclusion of the supra-s c window increases the averaged amplitude of mobility observed in the sub-s c window by about 34%. For the b-strand spanned by residues Lys48 to Leu50, an alternating pattern of backbone NH RDC order parameter S 2 rdc ðNHÞ = (0.59, 0.72, 0.59) was extracted. The backbone of Lys48, whose side chain is known to be involved in the poly-ubiquitylation process that leads to protein degradation, is very mobile on the supra-s c time scale (S 2 rdc ðNHÞ = 0.59 ± 0.03), while it is inconspicuous (S 2 LS ðNHÞ = 0.82) on the sub-s c as well as on ls-ms relaxation dispersion time scales. The results of this work differ from previous RDC dynamics studies of ubiquitin in the sense that the results are essentially independent of structural noise providing a much more robust assessment of dynamic effects that underlie the RDC data.

The hydrogenation of internal alkynes with [Cp*Ru]-based catalysts is distinguished by an unorthodox stereochemical course in that E-alkenes are formed by trans-delivery of the two H atoms of H. A combined experimental and computational study now provides a comprehensive mechanistic picture: a metallacyclopropene (η-vinyl complex) is primarily formed, which either evolves into the E-alkene via a concerted process or reacts to give a half-sandwich ruthenium carbene; in this case, one of the C atoms of the starting alkyne is converted into a methylene group. This transformation represents a formal gem-hydrogenation of a π-bond, which has hardly any precedent. The barriers for trans-hydrogenation and gem-hydrogenation are similar: whereas DFT predicts a preference for trans-hydrogenation, CCSD(T) finds gem-hydrogenation slightly more facile. The carbene, once formed, will bind a second H molecule and evolve to the desired E-alkene, a positional alkene isomer or the corresponding alkane; this associative pathway explains why double bond isomerization and over-reduction compete with trans-hydrogenation. The computed scenario concurs with para-hydrogen-induced polarization transfer (PHIP) NMR data, which confirm direct trans-delivery of H, the formation of carbene intermediates by gem-hydrogenation, and their evolution into product and side products alike. Propargylic -OR (R = H, Me) groups exert a strong directing and stabilizing effect, such that several carbene intermediates could be isolated and characterized by X-ray diffraction. The gathered information spurred significant preparative advances: specifically, highly selective trans-hydrogenations of propargylic alcohols are reported, which are compatible with many other reducible functional groups. Moreover, the ability to generate metal carbenes by gem-hydrogenation paved the way for noncanonical hydrogenative cyclopropanations, ring expansions, and cycloadditions.

Cytochrome P450 monooxygenases play a crucial role in the biosynthesis of many natural products and in the human metabolism of numerous pharmaceuticals. This has inspired synthetic organic and medicinal chemists to exploit them as catalysts in regio-and stereoselective CH-activating oxidation of structurally simple and complex organic compounds such as steroids. However, levels of regio-and stereoselectivity as well as activity are not routinely high enough for real applications. Protein engineering using rational design or directed evolution has helped in many respects, but simultaneous engineering of multiple catalytic traits such as activity, regioselectivity, and stereoselectivity, while overcoming tradeoffs and diminishing returns, remains a challenge. Here we show that the exploitation of information derived from mutability landscapes and molecular dynamics simulations for rationally designing iterative saturation mutagenesis constitutes a viable directed evolution strategy. This combined approach is illustrated by the evolution of P450 BM3 mutants which enable nearly perfect regio-and diastereoselective hydroxylation of five different steroids specifically at the C16-position with unusually high activity, while avoiding activity−selectivity trade-offs as well as keeping the screening effort relatively low. The C16 alcohols are of practical interest as components of biologically active glucocorticoids.

The activation of olefins for asymmetric chemical synthesis traditionally relies on transition metal catalysts. In contrast, biological enzymes with Brønsted acidic sites of appropriate strength can protonate olefins and thereby generate carbocations that ultimately react to form natural products. Although chemists have recently designed chiral Brønsted acid catalysts to activate imines and carbonyl compounds, mimicking these enzymes to protonate simple olefins that then engage in asymmetric catalytic reactions has remained a substantial synthetic challenge. Here, we show that a class of confined and strong chiral Brønsted acids enables the catalytic asymmetric intramolecular hydroalkoxylation of unbiased olefins. The methodology gives rapid access to biologically active 1,1-disubstituted tetrahydrofurans, including (-)-Boivinianin A.