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Insights into proteasome inhibition Proteasomes are large protein complexes that degrade and remove proteins to maintain proper cellular physiology and growth. Proteasomes are a validated target for anticancer therapy, but drug design has been hampered by poor understanding of how inhibitors interact with the active site. Schrader et al. succeeded in crystallizing various proteasome-inhibitor complexes. They subsequently obtained crystal structures for the native human proteasome and eight different inhibitor complexes at resolutions between 1.9 and 2.1 Å. The inhibitors sampled include drugs that are approved or in trial for cancer treatment. Science , this issue p. 594

In eukaryotes the nucleocytoplasmic transport of macromolecules is mainly mediated by soluble nuclear transport receptors of the karyopherin-β superfamily termed importins and exportins. The prototypical and highly versatile exportin CRM1 (chromosome region maintenance 1) is essential for nuclear depletion of numerous structurally and functionally unrelated protein and RNP cargoes. CRM1 has been shown to bind RanGTP (GTP bound RAs-related nuclear protein) and cargo proteins in an allosteric manner and to adopt a toroidal structure in several functional transport complexes. It was thought to maintain this conformation, with N- and C-terminal regions in close proximity, throughout the entire nucleocytoplasmic transport cycle.A recently solved structure of cargo-free CRM1 however revealed a superhelical, open conformation. Using molecular dynamics simulations, two distinct features of this conformation and their influence on the structural stability were investigated. One of those, the C-terminal helix, was identified as a major stabilising factor of the superhelical conformation.We furthermore showed that the overall configuration of CRM1 influences the local configuration of the cargo binding site. Based on these results we suggest a mechanism for the observed cooperative binding.

During the elongation cycle, after peptide-bonds are formed in the ribosome, transfer RNAs translocate to their new binding sites. Combining high-resolution crystal structures, cryo-EM data of multiple translocation intermediates of factorless retro-translocation, and atomistic molecular dynamics simulations, we have analysed collective motions, intrinsic time scales, and overall transition rates of ribosomal motions promoting tRNA translocation.From a Marcus-Theory type transition state analysis of fast molecular fluctuations, unexpectedly fast sub-microsecond intrinsic rates were resolved for body and head rotations as well as swiveling motions. The tRNA transitions between A,P, and E site are seen to be clearly slower than microseconds and seem to determine the overall transition rate between the intermediates. Remarkably, our kinetic analysis recovers the sequence of intermediates proposed in Fischer et al. that was purely based on structural similarity, thereby adding time information to these data.Together with the millisecond dynamics revealed from single molecule studies and the slow dynamics between pre and post states, a Frauenfelder-type hierarchy of time scales and corresponding free energy barriers emerges, underscoring the notion of the ribosome as a stochastic molecular machine.

We combined high-resolution crystal structures with cryo-EM maps of 13 intermediate states of ribosomal factorless spontaneous retro-translocation to obtain structures of the 70S ribosome in each of the 13 states in atomic detail. We then performed 100 ns all-atom, explicit-solvent molecular dynamics simulations for each of these states. Intrinsic rates for key ribosomal motions between the states were estimated from the short time fluctuations of the L1-stalk, the tRNAs and intersubunit rotations. The rates revealed rapid, sub-microsecond motions of the L1-stalk and the 30S subunit. Surprisingly, it is tRNA motions, rather than large-scale intersubunit rotations, that are rate limiting for most transitions. The interaction free energy profile of the L1-stalk with the tRNA obtained from additional umbrella sampling simulations of the L1-stalk/tRNA interactions revealed an active role of the L1-stalk in pulling the tRNA from the P to the E site. Further, by detailed analysis of the frequency of contacts between 50S ribosomal proteins L5 and L16 and the tRNAs, we identified specific residues which guide the tRNAs between the binding sites. A sequence analysis of the L1, L5 and L16 proteins revealed that the conservation score for contacting residues is significantly above average. Different types of contacts characterize the interplay of these proteins with the tRNAs and involve 1) sliding of L5 residues along the tRNA elbow 2) stepping of the tRNA between L16 contact patches 3) final pulling of the tRNA by the L1-stalk. These contact mechanisms can explain how both rapid translocation and a stable tRNA binding affinity can be achieved despite large-scale displacements.

Shape complementarity is a compulsory condition for molecular recognition. In our 3D ligand-based virtual screening approach called SQUIRREL, we combine shape-based rigid body alignment with fuzzy pharmacophore scoring. Retrospective validation studies demonstrate the superiority of methods which combine both shape and pharmacophore information on the family of peroxisome proliferator-activated receptors (PPARs). We demonstrate the real-life applicability of SQUIRREL by a prospective virtual screening study, where a potent PPARalpha agonist with an EC50 of 44 nM and 100-fold selectivity against PPARgamma has been identified...