Structure-guided design of substrate-binding pocket inversed the stereoselectivity of an NADH-dependent medium-chain alcohol dehydrogenase (MDR) from Prelog to anti-Prelog. The pocket-forming amino ...acids, especially the unconserved residues as hotspots, play critical roles in directing MDRs' stereoselectivity.
Polyethylene glycol (PEG) is widely used in industry and medicine. Anti-PEG antibodies have been developed for characterizing PEGylated drugs and other applications. However, the underlying mechanism ...for specific PEG binding has not been elucidated.
The Fab of two cognate anti-PEG antibodies 3.3 and 2B5 were each crystallized in complex with PEG, and their structures were determined by X-ray diffraction. The PEG-Fab interactions in these two crystals were analyzed and compared with those in a PEG-containing crystal of an unrelated anti-hemagglutinin 32D6-Fab. The PEG-binding stoichiometry was examined by using analytical ultracentrifuge (AUC).
A common PEG-binding mode to 3.3 and 2B5 is seen with an S-shaped core PEG fragment bound to two dyad-related Fab molecules. A nearby satellite binding site may accommodate parts of a longer PEG molecule. The core PEG fragment mainly interacts with the heavy-chain residues D31, W33, L102, Y103 and Y104, making extensive contacts with the aromatic side chains. At the center of each half-circle of the S-shaped PEG, a water molecule makes alternating hydrogen bonds to the ether oxygen atoms, in a similar configuration to that of a crown ether-bound lysine. Each satellite fragment is clamped between two arginine residues, R52 from the heavy chain and R29 from the light chain, and also interacts with several aromatic side chains. In contrast, the non-specifically bound PEG fragments in the 32D6-Fab crystal are located in the elbow region or at lattice contacts. The AUC data suggest that 3.3-Fab exists as a monomer in PEG-free solution but forms a dimer in the presence of PEG-550-MME, which is about the size of the S-shaped core PEG fragment.
The differing amino acids in 3.3 and 2B5 are not involved in PEG binding but engaged in dimer formation. In particular, the light-chain residue K53 of 2B5-Fab makes significant contacts with the other Fab in a dimer, whereas the corresponding N53 of 3.3-Fab does not. This difference in the protein-protein interaction between two Fab molecules in a dimer may explain the temperature dependence of 2B5 in PEG binding, as well as its inhibition by crown ether.
Escherichia coli phytase (EcAppA) which hydrolyzes phytate has been widely applied in the feed industry, but the need to improve the enzyme activity and thermostability remains. Here, we conduct ...rational design with two strategies to enhance the EcAppA performance. First, residues near the substrate binding pocket of EcAppA were modified according to the consensus sequence of two highly active Citrobacter phytases. One out of the eleven mutants, V89T, exhibited 17.5% increase in catalytic activity, which might be a result of stabilized protein folding. Second, the EcAppA glycosylation pattern was modified in accordance with the Citrobacter phytases. An N-glycosylation motif near the substrate binding site was disrupted to remove spatial hindrance for phytate entry and product departure. The de-glycosylated mutants showed 9.6% increase in specific activity. On the other hand, the EcAppA mutants that adopt N-glycosylation motifs from CbAppA showed improved thermostability that three mutants carrying single N-glycosylation motif exhibited 5.6-9.5% residual activity after treatment at 80°C (1.8% for wild type). Furthermore, the mutant carrying all three glycosylation motifs exhibited 27% residual activity. In conclusion, a successful rational design was performed to obtain several useful EcAppA mutants with better properties for further applications.
Proteins can spontaneously tie a variety of intricate topological knots through twisting and threading of the polypeptide chains. Recently developed artificial intelligence algorithms have predicted ...several new classes of topological knotted proteins, but the predictions remain to be authenticated experimentally. Here, we showed by X-ray crystallography and solution-state NMR spectroscopy that Q9PR55, an 89-residue protein from Ureaplasma urealyticum, possesses a novel 71 knotted topology that is accurately predicted by AlphaFold 2, except for the flexible N terminus. Q9PR55 is monomeric in solution, making it the smallest and most complex knotted protein known to date. In addition to its exceptional chemical stability against urea-induced unfolding, Q9PR55 is remarkably robust to resist the mechanical unfolding-coupled proteolysis by a bacterial proteasome, ClpXP. Our results suggest that the mechanical resistance against pulling-induced unfolding is determined by the complexity of the knotted topology rather than the size of the molecule.
Abstract
Mammalian cyclic GMP-AMP synthase (cGAS) and its homologue dinucleotide cyclase in Vibrio cholerae (VcDncV) produce cyclic dinucleotides (CDNs) that participate in the defense against viral ...infection. Recently, scores of new cGAS/DncV-like nucleotidyltransferases (CD-NTases) were discovered, which produce various CDNs and cyclic trinucleotides (CTNs) as second messengers. Here, we present the crystal structures of EcCdnD, a CD-NTase from Enterobacter cloacae that produces cyclic AMP-AMP-GMP, in its apo-form and in complex with ATP, ADP and AMPcPP, an ATP analogue. Despite the similar overall architecture, the protein shows significant structural variations from other CD-NTases. Adjacent to the donor substrate, another nucleotide is bound to the acceptor binding site by a non-productive mode. Isothermal titration calorimetry results also suggest the presence of two ATP binding sites. GTP alone does not bind to EcCdnD, which however binds to pppApG, a possible intermediate. The enzyme is active on ATP or a mixture of ATP and GTP, and the best metal cofactor is Mg2+. The conserved residues Asp69 and Asp71 are essential for catalysis, as indicated by the loss of activity in the mutants. Based on structural analysis and comparison with VcDncV and RNA polymerase, a tentative catalytic pathway for the CTN-producing EcCdnD is proposed.
Isoprenyl diphosphate synthases (IDSs) catalyze condensation reactions of isoprene units to produce isoprenoids or terpenoids, the largest class of natural products on earth. IDSs are divided into ...trans- and cis-IDS superfamilies depending on the configuration of the resulting carbon–carbon double bonds. Compared with trans-IDSs, cis-IDS family members exhibit more variable active site structure and versatile function. The archetypal cis-IDSs are homodimers and produce linear isoprenoids via head-to-tail condensation. Recently, heterodimeric cis-IDSs containing a noncatalytic subunit that also belongs to the cis-IDS superfamily have been identified. Moreover, several cis-IDS-fold members have been found to produce nonlinear isoprenoids via “head-to-middle” condensation. In this Review, we summarize the structural features and catalytic mechanism of the versatile cis-IDS superfamily. This information provides important guidance for research regarding biosynthesis of important natural products and medical intermediates, cellular metabolism, and disease control.
The bifunctional enzyme UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE) plays a key role in sialic acid production. It is different from the non-hydrolyzing enzymes for bacterial cell wall biosynthesis, ...and it is feed-back inhibited by the downstream product CMP-Neu5Ac. Here the complex crystal structure of the N-terminal epimerase part of human GNE shows a tetramer in which UDP binds to the active site and CMP-Neu5Ac binds to the dimer-dimer interface. The enzyme is locked in a tightly closed conformation. By comparing the UDP-binding modes of the non-hydrolyzing and hydrolyzing UDP-GlcNAc epimerases, we propose a possible explanation for the mechanistic difference. While the epimerization reactions of both enzymes are similar, Arg113 and Ser302 of GNE are likely involved in product hydrolysis. On the other hand, the CMP-Neu5Ac binding mode clearly elucidates why mutations in Arg263 and Arg266 can cause sialuria. Moreover, full-length modelling suggests a channel for ManNAc trafficking within the bifunctional enzyme.
K1 capsular polysaccharide (CPS)-associated Klebsiella pneumoniae is the primary cause of pyogenic liver abscesses (PLA) in Asia. Patients with PLA often have serious complications, ultimately ...leading to a mortality of ~ 5%. This K1 CPS has been reported as a promising target for development of glycoconjugate vaccines against K. pneumoniae infection. The pyruvylation and O-acetylation modifications on the K1 CPS are essential to the immune response induced by the CPS. To date, however, obtaining the fragments of K1 CPS that contain the pyruvylation and O-acetylation for generating glycoconjugate vaccines still remains a challenge.
We analyzed the digested CPS products with NMR spectroscopy and mass spectrometry to reveal a bacteriophage-derived polysaccharide depolymerase specific to K1 CPS. The biochemical and biophysical properties of the enzyme were characterized and its crystal structures containing bound CPS products were determined. We also performed site-directed mutagenesis, enzyme kinetic analysis, phage absorption and infectivity studies, and treatment of the K. pneumoniae-infected mice with the wild-type and mutant enzymes.
We found a bacteriophage-derived polysaccharide lyase that depolymerizes the K1 CPS into fragments of 1-3 repeating trisaccharide units with the retention of the pyruvylation and O-acetylation, and thus the important antigenic determinants of intact K1 CPS. We also determined the 1.46-Å-resolution, product-bound crystal structure of the enzyme, revealing two distinct carbohydrate-binding sites in a trimeric β-helix architecture, which provide the first direct evidence for a second, non-catalytic, carbohydrate-binding site in bacteriophage-derived polysaccharide depolymerases. We demonstrate the tight interaction between the pyruvate moiety of K1 CPS and the enzyme in this second carbohydrate-binding site to be crucial to CPS depolymerization of the enzyme as well as phage absorption and infectivity. We also demonstrate that the enzyme is capable of protecting mice from K1 K. pneumoniae infection, even against a high challenge dose.
Our results provide insights into how the enzyme recognizes and depolymerizes the K1 CPS, and demonstrate the potential use of the protein not only as a therapeutic agent against K. pneumoniae, but also as a tool to prepare structurally-defined oligosaccharides for the generation of glycoconjugate vaccines against infections caused by this organism.
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•A high-quality cryo-EM structure of malate synthase G (MSG) at 2.89 Å.•A complete structural comparison of a 723-residue MSG determined by cryo-EM and X-ray crystallography.•Cryo-EM ...revealed the structural dynamics of MSG.•The structural dynamics unraveled by cryo-EM, crystallography and NMR are highly correlated.
Determination of sub-100 kDa (kDa) structures by cryo-electron microscopy (EM) is a longstanding but not straightforward goal. Here, we present a 2.9-Å cryo-EM structure of a 723-amino acid apo-form malate synthase G (MSG) from Escherichia coli. The cryo-EM structure of the 82-kDa MSG exhibits the same global folding as structures resolved by crystallography and nuclear magnetic resonance (NMR) spectroscopy, and the crystal and cryo-EM structures are indistinguishable. Analyses of MSG dynamics reveal consistent conformational flexibilities among the three experimental approaches, most notably that the α/β domain exhibits structural heterogeneity. We observed that sidechains of F453, L454, M629, and E630 residues involved in hosting the cofactor acetyl-CoA and substrate rotate differently between the cryo-EM apo-form and complex crystal structures. Our work demonstrates that the cryo-EM technique can be used to determine structures and conformational heterogeneity of sub-100 kDa biomolecules to a quality as high as that obtained from X-ray crystallography and NMR spectroscopy.
We report the first structure of heptaprenyl diphosphate synthase from Staphylococcus aureus (SaHepPPS), together with an investigation of its mechanism of action and inhibition. The protein is ...involved in the formation of menaquinone, a key electron transporter in many bacteria, including pathogens. SaHepPPS consists of a “catalytic ” subunit (SaHepPPS‐2) having two “DDXXD” motifs and a “regulatory” subunit (SaHepPPS‐1) that lacks these motifs. High concentrations of the substrates, isopentenyl diphosphate and farnesyl diphosphate, inhibit the enzyme, which is also potently inhibited by bisphosphonates. The most active inhibitors (Ki∼200 nm) were N‐alkyl analogues of zoledronate containing ∼C6 alkyl side chains. They were modestly active against S. aureus cell growth, and growth inhibition was partially “rescued” by the addition of menaquinone‐7. Because SaHepPPS is essential for S. aureus cell growth, its structure is of interest in the context of the development of menaquinone biosynthesis inhibitors as potential antibiotic leads.
New drug target: Staphylococcus aureus heptaprenyl diphosphate synthase (HepPPS), involved in menaquinone biosynthesis, functions as a heterodimer and is inhibited by bisphosphonates (IC50=0.83 μm), which also inhibit S. aureus cell growth, an effect that is rescued by menaquinone. HepPPS is also partially inhibited by its substrates FPP as well as IPP, most likely due to disruption of the heterodimeric complex structure.
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