The single-component type-II NADH dehydrogenases (NDH-2s) serve as alternatives to the multisubunit respiratory complex I (type-I NADH dehydrogenase (NDH-1), also called NADH:ubiquinone oxidoreductase; EC 1.6.5.3) in catalysing electron transfer from NADH to ubiquinone in the mitochondrial respiratory chain. The yeast NDH-2 (Ndi1) oxidizes NADH on the matrix side and reduces ubiquinone to maintain mitochondrial NADH/NAD(+) homeostasis. Ndi1 is a potential therapeutic agent for human diseases caused by complex I defects, particularly Parkinson's disease, because its expression restores the mitochondrial activity in animals with complex I deficiency. NDH-2s in pathogenic microorganisms are viable targets for new antibiotics. Here we solve the crystal structures of Ndi1 in its substrate-free, NADH-, ubiquinone- and NADH-ubiquinone-bound states, to help understand the catalytic mechanism of NDH-2s. We find that Ndi1 homodimerization through its carboxy-terminal domain is critical for its catalytic activity and membrane targeting. The structures reveal two ubiquinone-binding sites (UQ(I) and UQ(II)) in Ndi1. NADH and UQ(I) can bind to Ndi1 simultaneously to form a substrate-protein complex. We propose that UQ(I) interacts with FAD to act as an intermediate for electron transfer, and that NADH transfers electrons through this FAD-UQ(I) complex to UQ(II). Together our data reveal the regulatory and catalytic mechanisms of Ndi1 and may facilitate the development or targeting of NDH-2s for potential therapeutic applications.
Protein ubiquitination is a multifaceted post-translational modification that controls almost every process in eukaryotic cells. Recently, the Legionella effector SdeA was reported to mediate a unique phosphoribosyl-linked ubiquitination through successive modifications of the Arg42 of ubiquitin (Ub) by its mono-ADP-ribosyltransferase (mART) and phosphodiesterase (PDE) domains. However, the mechanisms of SdeA-mediated Ub modification and phosphoribosyl-linked ubiquitination remain unknown. Here we report the structures of SdeA in its ligand-free, Ub-bound and Ub-NADH-bound states. The structures reveal that the mART and PDE domains of SdeA form a catalytic domain over its C-terminal region. Upon Ub binding, the canonical ADP-ribosyltransferase toxin turn-turn (ARTT) and phosphate-nicotinamide (PN) loops in the mART domain of SdeA undergo marked conformational changes. The Ub Arg72 might act as a 'probe' that interacts with the mART domain first, and then movements may occur in the side chains of Arg72 and Arg42 during the ADP-ribosylation of Ub. Our study reveals the mechanism of SdeA-mediated Ub modification and provides a framework for further investigations into the phosphoribosyl-linked ubiquitination process.
Directed evolution has long been a key strategy to generate enzymes with desired properties like high selectivity, but experimental barriers and analytical costs of screening enormous mutant libraries have limited such efforts. Here, we describe an ultrahigh-throughput dual-channel microfluidic droplet screening system that can be used to screen up to ~107 enzyme variants per day. As an example case, we use the system to engineer the enantioselectivity of an esterase to preferentially produce desired enantiomers of profens, an important class of anti-inflammatory drugs. Using two types of screening working modes over the course of five rounds of directed evolution, we identify (from among 5 million mutants) a variant with 700-fold improved enantioselectivity for the desired (S)-profens. We thus demonstrate that this screening platform can be used to rapidly generate enzymes with desired enzymatic properties like enantiospecificity, chemospecificity, and regiospecificity.
Fucosylated glycoconjugates are involved in a variety of physiological and pathological processes. However, economical production of fucosylated drugs and prebiotic supplements has been hampered by the poor catalytic efficiency of fucosyltransferases. Here, we developed a fluorescence-activated cell sorting system that enables the ultrahigh-throughput screening (>107 mutants/hour) of such enzymes and designed a companion strategy to assess the screening performance of the system. After three rounds of directed evolution, a mutant M32 of the α1,3-FucT from Helicobacter pylori was identified with 6- and 14-fold increases in catalytic efficiency (kcat/Km) for the synthesis of Lewis x and 3′-fucosyllactose, respectively. The structure of the M32 mutant revealed that the S45F mutation generates a clamp-like structure that appears to improve binding of the galactopyranose ring of the acceptor substrate. Moreover, molecular dynamic simulations reveal that helix α5, is more mobile in the M32 mutant, possibly explaining its high fucosylation activity.
Mitochondrial ClpP is a serine protease located in the mitochondrial matrix. This protease participates in mitochondrial protein quality control by degrading misfolded or damaged proteins, thus maintaining normal metabolic function. Mitochondrial ClpP is a stable heptamer ring with peptidase activity that forms a multimeric complex with the ATP-dependent unfoldase ClpX (ClpXP) leading to proteolytic activity. Emerging evidence demonstrates that ClpXP is over-expressed in hematologic malignancies and solid tumors and is necessary for the viability of a subset of tumors. In addition, both inhibition and hyperactivation of ClpXP leads to impaired respiratory chain activity and causes cell death in cancer cells. Therefore, targeting mitochondrial ClpXP could be a novel therapeutic strategy for the treatment of malignancy. Here, we review the structure and function of mitochondrial ClpXP as well as strategies to target this enzyme complex as a novel therapeutic approach for malignancy.
Staphylococcus aureus ( S. aureus ) pathogenesis is a complex process involving a diverse array of extracellular and cell wall components. ClfB, an MSCRAMM (Microbial Surface Components Recognizing Adhesive Matrix Molecules) family surface protein, described as a fibrinogen-binding clumping factor, is a key determinant of S. aureus nasal colonization, but the molecular basis for ClfB-ligand recognition remains unknown. In this study, we solved the crystal structures of apo-ClfB and its complexes with fibrinogen α (Fg α) and cytokeratin 10 (CK10) peptides. Structural comparison revealed a conserved glycine-serine-rich (GSR) ClfB binding motif (GSSGXGXXG) within the ligands, which was also found in other human proteins such as Engrailed protein, TCF20 and Dermokine proteins. Interaction between Dermokine and ClfB was confirmed by subsequent binding assays. The crystal structure of ClfB complexed with a 15-residue peptide derived from Dermokine revealed the same peptide binding mode of ClfB as identified in the crystal structures of ClfB-Fg α and ClfB-CK10. The results presented here highlight the multi-ligand binding property of ClfB, which is very distinct from other characterized MSCRAMMs to-date. The adherence of multiple peptides carrying the GSR motif into the same pocket in ClfB is reminiscent of MHC molecules. Our results provide a template for the identification of other molecules targeted by S. aureus during its colonization and infection. We propose that other MSCRAMMs like ClfA and SdrG also possess multi-ligand binding properties.
Glycosyltransferases (GTs) offer very attractive approaches to the synthesis of complex oligosaccharides. However, the limited number of available GTs, together with their instability and strict substrate specificity, have severely hampered the broad application of these enzymes. Previous attempts to broaden the range of substrate scope and to increase the activity of GTs via protein engineering have met with limited success, partially because of the lack of effective high-throughput screening methods. Recently, we reported an ultra-high-throughput screening method for sialyltransferases based on fluorescence-activated cell sorting (Aharoni et al. Nat. Methods 2006, 3, 609-614). Here, we considerably improve this method via the introduction of a two-color screening protocol to minimize the probability of false positive mutants and demonstrate its generality through directed evolution of a neutral sugar transferase, beta-1,3-galactosyltransferase CgtB. A variant with broader substrate tolerance than the wild-type enzyme and 300-fold higher activity was identified rapidly from a library of >10(7) CgtB mutants. Importantly, the variant effected much more efficient synthesis of G(M1a) and asialo G(M1) oligosaccharides, the building blocks of important therapeutic glycosphingolipids, than did the parent enzyme. This work not only establishes a new methodology for the directed evolution of galactosyltransferases, but also suggests a powerful strategy for the screening of almost all GT activities, thereby facilitating the engineering of glycosyltransferases.
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