The unfolded protein response (UPR) is an evolutionarily conserved mechanism by which all eukaryotic cells adapt to the accumulation of unfolded proteins in the endoplasmic reticulum (ER). Inositolrequiring kinase 1 (IRE1) and PKR-related ER kinase (PERK) are two type I transmembrane ER-localized protein kinase receptors that signal the UPR through a process that involves homodimerization and autophosphorylation. To elucidate the molecular basis of the ER transmembrane signaling event, we determined the x-ray crystal structure of the luminal domain of human IRE1␣. The monomer of the luminal domain comprises a unique fold of a triangular assembly of ␤-sheet clusters. Structural analysis identified an extensive dimerization interface stabilized by hydrogen bonds and hydrophobic interactions. Dimerization creates an MHClike groove at the interface. However, because this groove is too narrow for peptide binding and the purified luminal domain forms high-affinity dimers in vitro, peptide binding to this groove is not required for dimerization. Consistent with our structural observations, mutations that disrupt the dimerization interface produced IRE1␣ molecules that failed to either dimerize or activate the UPR upon ER stress. In addition, mutations in a structurally homologous region within PERK also prevented dimerization. Our structural, biochemical, and functional studies in vivo altogether demonstrate that IRE1 and PERK have conserved a common molecular interface necessary and sufficient for dimerization and UPR signaling. endoplasmic reticulum ͉ protein structure ͉ signal transduction ͉ protein kinase ͉ endoplasmic reticulum stress T he endoplasmic reticulum (ER) of eukaryotic cells is the cellular compartment where secretory and transmembrane proteins fold into their native conformations and undergo posttranslational modifications that are important for their structure and function. When protein folding in the ER is perturbed, a set of signal transduction pathways is activated to reduce the proteinfolding load and increase folding capacity. These pathways are collectively termed the unfolded protein response (UPR) (1-4). To increase the folding capacity, synthesis of ER resident chaperones and folding catalysts is induced. To decrease the folding load in the ER, global mRNA translation is attenuated and clearance of misfolded proteins through ER-associated degradation is increased. UPR signaling is mediated by three ER resident transmembrane proteins: IRE1, PERK, and ATF6.IRE1 is a type I transmembrane protein kinase receptor that also has a site-specific RNase activity that, upon activation, initiates a site-specific unconventional splicing reaction (5, 6). The substrate for IRE1 RNase in metazoans is Xbp1 mRNA, which encodes a basic leucine zipper transcription factor of the ATF͞CREB family. XBP1 controls expression of genes containing an X-box element or a UPR element in their promoter regions (7-10). The IRE1-mediated splicing reaction introduces into XBP1 an alternative C terminus, thereby generating an X...

Bioactive natural products have evolved to inhibit specific cellular targets and have served as lead molecules for health and agricultural applications for the past century. The post-genomics era has brought a renaissance in the discovery of natural products using synthetic-biology tools. However, compared to traditional bioactivity-guided approaches, genome mining of natural products with specific and potent biological activities remains challenging. Here we present the discovery and validation of a potent herbicide that targets a critical metabolic enzyme that is required for plant survival. Our approach is based on the co-clustering of a self-resistance gene in the natural-product biosynthesis gene cluster, which provides insight into the potential biological activity of the encoded compound. We targeted dihydroxy-acid dehydratase in the branched-chain amino acid biosynthetic pathway in plants; the last step in this pathway is often targeted for herbicide development. We show that the fungal sesquiterpenoid aspterric acid, which was discovered using the method described above, is a sub-micromolar inhibitor of dihydroxy-acid dehydratase that is effective as a herbicide in spray applications. The self-resistance gene astD was validated to be insensitive to aspterric acid and was deployed as a transgene in the establishment of plants that are resistant to aspterric acid. This herbicide-resistance gene combination complements the urgent ongoing efforts to overcome weed resistance. Our discovery demonstrates the potential of using a resistance-gene-directed approach in the discovery of bioactive natural products.

The MVB pathway plays essential roles in several eukaryotic cellular processes. Proper function of the MVB pathway requires reversible membrane association of the ESCRTs, a process catalyzed by Vps4 ATPase. Vta1 regulates the Vps4 activity, but its mechanism of action was poorly understood. We report the high-resolution crystal structures of the Did2- and Vps60-binding N-terminal domain and the Vps4-binding C-terminal domain of S. cerevisiae Vta1. The C-terminal domain also mediates Vta1 dimerization and both subunits are required for its function as a Vps4 regulator. Emerging from our analysis is a mechanism of regulation by Vta1 in which the C-terminal domain stabilizes the ATP-dependent double ring assembly of Vps4. In addition, the MIT motif-containing N-terminal domain, projected by a long disordered linker, allows contact between the Vps4 disassembly machinery and the accessory ESCRT-III proteins. This provides an additional level of regulation and coordination for ESCRT-III assembly and disassembly.

he directed evolution of enzymes promises to eliminate the long-standing limitations of biocatalysis in organic chemistry and biotechnology-the often-observed limited substrate scope, insufficient activity, and poor regioselectivity or stereoselectivity. Saturation mutagenesis at sites lining the binding pocket with formation of focused libraries has emerged as the technique of choice, but choosing the optimal size of the randomization site and reduced amino acid alphabet for minimizing the labor-determining screening effort remains a challenge. Here, we introduce structure-guided triple-code saturation mutagenesis (TCSM) by encoding three rationally chosen amino acids as building blocks in the randomization of large multiresidue sites. In contrast to conventional NNK codon degeneracy encoding all 20 canonical amino acids and requiring the screening of more than 10(15) transformants for 95% library coverage, TCSM requires only small libraries not exceeding 200800 transformants in one library. The triple code utilizes structural (X-ray) and consensus-derived sequence data, and is therefore designed to match the steric and electrostatic characteristics of the particular enzyme. Using this approach, limonene epoxide hydrolase has been successfully engineered as stereoselective catalysts in the hydrolytic desymmetrization of meso-type epoxides with formation of either (R,R)- or (S,S)-configurated diols on an optional basis and kinetic resolution of chiral substrates. Crystal structures and docking computations support the source of notably enhanced and inverted enantioselectivity

Catalytic asymmetric reduction of prochiral ketones with the formation of enantio-pure secondary alcohols is of fundamental importance in organic chemistry, chiral man-made transition-metal catalysts, or organocatalysts and enzymes of the alcohol dehydrogenase (ADH) type. A distinct limitation is the traditional requirement that the αand α′-moieties flanking the carbonyl function differ sterically and/or electronically. Difficultto-reduce ketones such as tetrahydrofuran-3-one and tetrahydrothiofuran-3one and related substrates are particularly challenging, irrespective of the catalyst type. The ADH from Thermoethanolicus brockii (TbSADH) is an attractive industrial biocatalyst, because of its high thermostability, but it also fails in the reduction of such ketones. We have successfully applied directed evolution using the previously developed concept of triple-code saturation mutagenesis at sites lining the TbSADH binding pocket with tetrahydrofuran-3-one serving as the model compound. Highly (R)-and (S)-selective variants were evolved (95%−99% ee) with minimal screening. These robust catalysts also proved to be effective in the asymmetric reduction of tetrahydrothiofuran-3-one and other challenging prochiral ketones as well. The chiral products, which are generally prepared by multistep routes, serve as synthons in the preparation of several important therapeutic drugs.

Directed evolution based on saturation mutagenesis at sites lining the binding pocket is a commonly practiced strategy for enhancing or inverting the stereoselectivity of enzymes for use in organic chemistry or biotechnology. However, as the number of residues in a randomization site increases to five or more, the screening effort for 95 % library coverage increases astronomically until it is no longer feasible. We propose the use of a single amino acid for saturation mutagenesis at superlarge randomization sites comprising 10 or more residues. When used to reshape the binding pocket of limonene epoxide hydrolase, this strategy, which drastically reduces the search space and thus the screening effort, resulted in R,R- and S,S-selective mutants for the hydrolytic desymmetrization of cyclohexene oxide and other epoxides. X-ray crystal structures and docking studies of the mutants unveiled the source of stereoselectivity and shed light on the mechanistic intricacies of this enzyme.

SecB is a bacterial chaperone involved in directing pre-protein to the translocation pathway by its specific interaction with the peripheral membrane ATPase SecA. The SecB-binding site on SecA is located at its C terminus and consists of a stretch of highly conserved residues. The crystal structure of SecB in complex with the C-terminal 27 amino acids of SecA from Haemophilus influenzae shows that the SecA peptide is structured as a CCCH zinc-binding motif. One SecB tetramer is bound by two SecA peptides, and the interface involves primarily salt bridges and hydrogen bonding interactions. The structure explains the importance of the zinc-binding motif and conserved residues at the C terminus of SecA in its high-affinity binding with SecB. It also suggests a model of SecB-SecA interaction and its implication for the mechanism of pre-protein transfer in bacterial protein translocation.

Enzymatic stereodivergent synthesis to access all possible product stereoisomers bearing multiple stereocenters is relatively undeveloped, although enzymes are being increasingly used in both academic and industrial areas. When two stereocenters and thus four stereoisomeric products are involved, obtaining stereodivergent enzyme mutants for individually accessing all four stereoisomers would be ideal. Although significant success has been achieved in directed evolution of enzymes in general, stereodivergent engineering of one enzyme into four highly stereocomplementary variants for obtaining the full complement of stereoisomers bearing multiple stereocenters remains a challenge. Using Candida antarctica lipase B (CALB) as a model, we report the protein engineering of this enzyme into four highly stereocomplementary variants needed for obtaining all four stereoisomers in transesterification reactions between racemic acids and racemic alcohols in organic solvents. By generating and screening less than 25 variants each isomer, we achieved >90% selectivity for all of the four possible stereoisomers in the model reaction. This difficult feat was accomplished by developing a strategy dubbed "focused rational iterative site-specific mutagenesis" (FRISM) at sites lining the enzyme's binding pocket. The accumulation of single mutations by iterative site-specific mutagenesis using a restricted set of rationally chosen amino acids allows the formation of ultra-small mutant libraries requiring minimal screening for stereoselectivity. The crystal structure of all stereodivergent CALB variants, flanked by MD simulations, uncovered the source of selectivity.