Crystal structure of decaprenylphosphoryl‐β‐ D‐ribose 2'‐epimerase from Mycobacterium smegmatis

Decaprenylphosphoryl‐β‐D ‐ribose 2'‐epimerase (DprE1) is an essential enzyme in the biosynthesis of cell wall components and a target for development of anti‐tuberculosis drugs. We determined the crystal structure of a truncated form of DprE1 from Mycobacterium smegmatis in two crystal forms to up to 2.35 Å resolution. The structure extends from residue 75 to the C‐terminus and shares homology with FAD‐dependent oxidoreductases of the vanillyl‐alcohol oxidase family including the DprE1 homologue from M. tuberculosis. The M. smegmatis DprE1 structure reported here provides further insights into the active site geometry of this tuberculosis drug target. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Structural basis for binding and transfer of heme in bacterial heme‐acquisition systems

Periplasmic heme‐binding proteins (PBPs) in Gram‐negative bacteria are components of the heme acquisition system. These proteins shuttle heme across the periplasmic space from outer membrane receptors to ATP‐binding cassette (ABC) heme importers located in the inner‐membrane. In the present study, we characterized the structures of PBPs found in the pathogen Burkholderia cenocepacia (BhuT) and in the thermophile Roseiflexus sp. RS‐1 (RhuT) in the heme‐free and heme‐bound forms. The conserved motif, in which a well‐conserved Tyr interacts with the nearby Arg coordinates on heme iron, was observed in both PBPs. The heme was recognized by its surroundings in a variety of manners including hydrophobic interactions and hydrogen bonds, which was confirmed by isothermal titration calorimetry. Furthermore, this study of 3 forms of BhuT allowed the first structural comparison and showed that the heme‐binding cleft of BhuT adopts an “open” state in the heme‐free and 2‐heme‐bound forms, and a “closed” state in the one‐heme‐bound form with unique conformational changes. Such a conformational change might adjust the interaction of the heme(s) with the residues in PBP and facilitate the transfer of the heme into the translocation channel of the importer.     

C‐terminal β‐strand swapping in a consensus‐derived fibronectin Type III scaffold

The crystal structures of six different fibronectin Type III consensus‐derived Tencon domains, whose solution properties exhibit no, to various degrees of, aggregation according to SEC, have been determined. The structures of the five variants showing aggregation reveal 3D domain swapped dimers. In all five cases, the swapping involves the C‐terminal β‐strand resulting in the formation of Tencon dimers in which the target‐binding surface is blocked. All of the variants differ in sequence in the FG loop, which is the hinge loop in the β‐strand‐swapped dimers. The six tencon variants have between 0 and 5 residues inserted between positions 77 and 78 in the FG loop. Analysis of the structures suggests that a non‐glycine residue at position 77 and insertions of <4 residues may destabilize the β‐turn in the FG loop promoting β‐strand swapping. Swapped dimers with an odd number of inserted residues may be less stable, particularly if they contain proline residues, because they cannot form perfect β‐bridges in the FG regions that link the swapped dimers. The Tencon β‐swapped variants with the longest FG sequences are observed to form higher order hexameric or helical oligomeric structures in the crystal correlating well with the aggregation properties of these domains observed in solution. Understanding the structural basis for domain‐swapped dimerization and oligomerization will support engineering efforts of the Tencon domain to produce variants with desired biophysical properties. Proteins 2014; 82:1359–1369. © 2013 Wiley Periodicals, Inc.     

Structural and SAXS analysis of Tle5–Tli5 complex reveals a novel inhibition mechanism of H2‐T6SS in Pseudomonas aeruginosa

Widely spread in Gram‐negative bacteria, the type VI secretion system (T6SS) secretes many effector‐immunity protein pairs to help the bacteria compete against other prokaryotic rivals, and infect their eukaryotic hosts. Tle5 and Tle5B are two phospholipase effector protein secreted by T6SS of Pseudomonas aeruginosa. They can facilitate the bacterial internalization process into human epithelial cells by interacting with Akt protein of the PI3K‐Akt signal pathway. Tli5 and PA5086‐5088 are cognate immunity proteins of Tle5 and Tle5B, respectively. They can interact with their cognate effector proteins to suppress their virulence. Here, we report the crystal structure of Tli5 at 2.8Å resolution and successfully fit it into the Small angle X‐ray scattering (SAXS) model of the complete Tle5–Tli5 complex. We identified two important motifs in Tli5 through sequence and structural analysis. One is a conserved loop‐β‐hairpin motif that exists in the Tle5 immunity homologs, the other is a long and sharp α‐α motif that directly interacts with Tle5 according to SAXS data. We also distinguished the structural features of Tle5 and Tle5B family immunity proteins. Together, our work provided insights into a novel inhibition mechanism that may enhance our understanding of phospholipase D family proteins.     

Structural and functional characterization of a novel α/β hydrolase from cariogenic pathogen Streptococcus mutans

The protein Smu.1393c from Streptococcus mutans is annotated as a putative α/β hydrolase, but it has low sequence identity to the structure‐known α/β hydrolases. Here we present the crystal structure of Smu.1393c at 2.0 Å resolution. Smu.1393c has a fully open alkaline substrate pocket, whose conformation is unique among other similar hydrolase structures. Three residues, Ser101, His251, and Glu125, were identified as the active center of Smu.1393c. By screening a series of artificial hydrolase substrates, we demonstrated Smu.1393c had low carboxylesterase activity towards short‐chain carboxyl esters, which provided a clue for exploring the in vivo function of Smu.1393c. Proteins 2014; 82:695–700. © 2013 Wiley Periodicals, Inc.     

Structural basis for Arf6-MKLP1 complex formation on the Flemming body responsible for cytokinesis

A small GTPase, Arf6, is involved in cytokinesis by localizing to the Flemming body (the midbody). However, it remains unknown how Arf6 contributes to cytokinesis. Here, we demonstrate that Arf6 directly interacts with mitotic kinesin-like protein 1 (MKLP1), a Flemming body-localizing protein essential for cytokinesis. The crystal structure of the Arf6-MKLP1 complex reveals that MKLP1 forms a homodimer flanked by two Arf6 molecules, forming a 2:2 heterotetramer containing an extended β-sheet composed of 22 β-strands that spans the entire heterotetramer, suitable for interaction with a concave membrane surface at the cleavage furrow. We show that, during cytokinesis, Arf6 is first accumulated around the cleavage furrow and, prior to abscission, recruited onto the Flemming body via interaction with MKLP1. We also show by structure-based mutagenesis and siRNA-mediated knockdowns that the complex formation is required for completion of cytokinesis. A model based on these results suggests that the Arf6-MKLP1 complex plays a crucial role in cytokinesis by connecting the microtubule bundle and membranes at the cleavage plane.     

Two alternative modes for optimizing nylon‐6 byproduct hydrolytic activity from a carboxylesterase with a β‐lactamase fold: X‐ray crystallographic analysis of directly evolved 6‐aminohexanoate‐dimer hydrolase

Promiscuous 6-aminohexanoate-linear dimer (Ald)-hydrolytic activity originally obtained in a carboxylesterase with a β-lactamase fold was enhanced about 80-fold by directed evolution using error-prone PCR and DNA shuffling. Kinetic studies of the mutant enzyme (Hyb-S4M94) demonstrated that the enzyme had acquired an increased affinity (K_m = 15 mM) and turnover (k_cat = 3.1 s⁻¹) for Ald, and that a catalytic center suitable for nylon-6 byproduct hydrolysis had been generated. Construction of various mutant enzymes revealed that the enhanced activity in the newly evolved enzyme is due to the substitutions R187S/F264C/D370Y. Crystal structures of Hyb-S4M94 with bound substrate suggested that catalytic function for Ald was improved by hydrogen-bonding/hydrophobic interactions between the Ald—COOH and Tyr370, a hydrogen-bonding network from Ser187 to , and interaction between and Gln27-O_ɛ derived from another subunit in the homo-dimeric structure. In wild-type Ald-hydrolase (NylB), Ald-hydrolytic activity is thought to be optimized by the substitutions G181D/H266N, which improve an electrostatic interaction with (Kawashima et al., FEBS J 2009; 276:2547–2556). We propose here that there exist at least two alternative modes for optimizing the Ald-hydrolytic activity of a carboxylesterase with a β-lactamase fold.     

Structural basis of selectivity and neutralizing activity of a TGFα/epiregulin specific antibody

Recent studies have implicated a role of the epidermal growth factor receptor (EGFR) pathway in kidney disease. Skin toxicity associated with therapeutics which completely block the EGFR pathway precludes their use in chronic dosing. Therefore, we developed antibodies which specifically neutralize the EGFR ligands TGFα (transforming growth factor‐alpha) and epiregulin but not EGF (epidermal growth factor), amphiregulin, betacellulin, HB‐EGF (heparin‐binding epidermal growth factor), or epigen. The epitope of one such neutralizing antibody, LY3016859, was characterized in detail to elucidate the structural basis for ligand specificity. Here we report a crystal structure of the LY3016859 Fab fragment in complex with soluble human TGFα. Our data demonstrate a conformational epitope located primarily within the C‐terminal subdomain of the ligand. In addition, point mutagenesis experiments were used to highlight specific amino acids which are critical for both antigen binding and neutralization, most notably Ala⁴¹, Glu⁴⁴, and His⁴⁵. These results illustrate the structural basis for the ligand specificity/selectivity of LY3016859 and could also provide insight into further engineering to alter specificity and/or affinity of LY3016859.     

Vibrational Circular Dichroism (VCD), VCD Exciton Coupling,and X‐ray Determination of the Absolute Configuration of an α,β‐Unsaturated Germacranolide

The absolute configuration of 1 was deduced by vibrational circular dichroism together with the evaluation of the Flack and Hooft X‐ray parameters. Vibrational circular dichroism exciton coupling, using the carbonyl group signals, confirmed the absolute configuration of 2 . In addition, sodium borohydride reduction of the 11,13‐double bond of 6‐epi‐desacetyllaurenobiolide ( 1 ) yields an almost equimolecular mixture of C11 epimers, while reduction of the same double bond of 6‐epi‐laurenobiolide ( 2 ) provided almost exclusively the (11S) diastereoisomer 4 . Chirality 27:247–252, 2015. © 2015 Wiley Periodicals, Inc.     

Mg2+ ions bind at the C‐terminal region of skeletal muscle α‐tropomyosin

Tropomyosin (Tm) is a dimeric coiled‐coil protein that polymerizes through head‐to‐tail interactions. These polymers bind along actin filaments and play an important role in the regulation of muscle contraction. Analysis of its primary structure shows that Tm is rich in acidic residues, which are clustered along the molecule and may form sites for divalent cation binding. In a previous study, we showed that the Mg²⁺‐induced increase in stability of the C‐terminal half of Tm is sensitive to mutations near the C‐terminus. In the present report, we study the interaction between Mg²⁺ and full‐length Tm and smaller fragments corresponding to the last 65 and 26 Tm residues. Although the smaller Tm peptide (Tm_{259‐284(W269)}) is flexible and to large extent unstructured, the larger Tm_{220‐284(W269)} fragment forms a coiled coil in solution whose stability increases significantly in the presence of Mg²⁺. NMR analysis shows that Mg²⁺ induces chemical shift perturbations in both Tm_{220‐284(W269)} and Tm_{259‐284(W269)} in the vicinity of His276, in which are located several negatively charged residues. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 583–590, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Structural determinants in bacterial 2‐keto‐3‐deoxy‐D‐gluconate dehydrogenase KduD for dual‐coenzyme specificity

Short‐chain dehydrogenase/reductase (SDR) is distributed in many organisms, from bacteria to humans, and has significant roles in metabolism of carbohydrates, lipids, amino acids, and other biomolecules. An important intermediate in acidic polysaccharide metabolism is 2‐keto‐3‐deoxy‐d ‐gluconate (KDG). Recently, two short and long loops in Sphingomonas KDG‐producing SDR enzymes (NADPH‐dependent A1‐R and NADH‐dependent A1‐R′) involved in alginate metabolism were shown to be crucial for NADPH or NADH coenzyme specificity. Two SDR family enzymes—KduD from Pectobacterium carotovorum (PcaKduD) and DhuD from Streptococcus pyogenes (SpyDhuD)—prefer NADH as coenzyme, although only PcaKduD can utilize both NADPH and NADH. Both enzymes reduce 2,5‐diketo‐3‐deoxy‐d ‐gluconate to produce KDG. Tertiary and quaternary structures of SpyDhuD and PcaKduD and its complex with NADH were determined at high resolution (approximately 1.6 Å) by X‐ray crystallography. Both PcaKduD and SpyDhuD consist of a three‐layered structure, α/β/α, with a coenzyme‐binding site in the Rossmann fold; similar to enzymes A1‐R and A1‐R′, both arrange the two short and long loops close to the coenzyme‐binding site. The primary structures of the two loops in PcaKduD and SpyDhuD were similar to those in A1‐R′ but not A1‐R. Charge neutrality and moderate space at the binding site of the nucleoside ribose 2′ coenzyme region were determined to be structurally crucial for dual‐coenzyme specificity in PcaKduD by structural comparison of the NADH‐ and NADPH‐specific SDR enzymes. The corresponding site in SpyDhuD was negatively charged and spatially shallow. This is the first reported study on structural determinants in SDR family KduD related to dual‐coenzyme specificity. Proteins 2016; 84:934–947. © 2016 Wiley Periodicals, Inc.     

Insight into a strategy for attenuating AmpC‐mediated β‐lactam resistance: Structural basis for selective inhibition of the glycoside hydrolase NagZ

NagZ is an exo‐N‐acetyl‐β‐glucosaminidase, found within Gram‐negative bacteria, that acts in the peptidoglycan recycling pathway to cleave N‐acetylglucosamine residues off peptidoglycan fragments. This activity is required for resistance to cephalosporins mediated by inducible AmpC β‐lactamase. NagZ uses a catalytic mechanism involving a covalent glycosyl enzyme intermediate, unlike that of the human exo‐N‐acetyl‐β‐glucosaminidases: O‐GlcNAcase and the β‐hexosaminidase isoenzymes. These latter enzymes, which remove GlcNAc from glycoconjugates, use a neighboring‐group catalytic mechanism that proceeds through an oxazoline intermediate. Exploiting these mechanistic differences we previously developed 2‐N‐acyl derivatives of O‐(2‐acetamido‐2‐deoxy‐D ‐glucopyranosylidene)amino‐N‐phenylcarbamate (PUGNAc), which selectively inhibits NagZ over the functionally related human enzymes and attenuate antibiotic resistance in Gram‐negatives that harbor inducible AmpC. To understand the structural basis for the selectivity of these inhibitors for NagZ, we have determined its crystallographic structure in complex with N‐valeryl‐PUGNAc, the most selective known inhibitor of NagZ over both the human β‐hexosaminidases and O‐GlcNAcase. The selectivity stems from the five‐carbon acyl chain of N‐valeryl‐PUGNAc, which we found ordered within the enzyme active site. In contrast, a structure determination of a human O‐GlcNAcase homologue bound to a related inhibitor N‐butyryl‐PUGNAc, which bears a four‐carbon chain and is selective for both NagZ and O‐GlcNAcase over the human β‐hexosamnidases, reveals that this inhibitor induces several conformational changes in the active site of this O‐GlcNAcase homologue. A comparison of these complexes, and with the human β‐hexosaminidases, reveals how selectivity for NagZ can be engineered by altering the 2‐N‐acyl substituent of PUGNAc to develop inhibitors that repress AmpC mediated β‐lactam resistance.     

Crystal structures of α‐dioxygenase from Oryza sativa: Insights into substrate binding and activation by hydrogen peroxide

α‐Dioxygenases (α‐DOX) are heme‐containing enzymes found predominantly in plants and fungi, where they generate oxylipins in response to pathogen attack. α‐DOX oxygenate a variety of 14–20 carbon fatty acids containing up to three unsaturated bonds through stereoselective removal of the pro‐R hydrogen from the α‐carbon by a tyrosyl radical generated via the oxidation of the heme moiety by hydrogen peroxide (H₂O₂). We determined the X‐ray crystal structures of wild type α‐DOX from Oryza sativa, the wild type enzyme in complex with H₂O₂, and the catalytically inactive Y379F mutant in complex with the fatty acid palmitic acid (PA). PA binds within the active site cleft of α‐DOX such that the carboxylate forms ionic interactions with His‐311 and Arg‐559. Thr‐316 aids in the positioning of carbon‐2 for hydrogen abstraction. Twenty‐five of the twenty eight contacts made between PA and residues lining the active site occur within the carboxylate and first eight carbons, indicating that interactions within this region of the substrate are responsible for governing selectivity. Comparison of the wild type and H₂O₂ structures provides insight into enzyme activation. The binding of H₂O₂ at the distal face of the heme displaces residues His‐157, Asp‐158, and Trp‐159 ～2.5 Å from their positions in the wild type structure. As a result, the Oδ2 atom of Asp‐158 interacts with the Ca atom in the calcium binding loop, the side chains of Trp‐159 and Trp‐213 reorient, and the guanidinium group of Arg‐559 is repositioned near Tyr‐379, poised to interact with the carboxylate group of the substrate.     

Structure of an early native‐like intermediate of β2‐microglobulin amyloidogenesis

To investigate early intermediates of β2‐microglobulin (β2m) amyloidogenesis, we solved the structure of β2m containing the amyloidogenic Pro32Gly mutation by X‐ray crystallography. One nanobody (Nb24) that efficiently blocks fibril elongation was used as a chaperone to co‐crystallize the Pro32Gly β2m monomer under physiological conditions. The complex of P32G β2m with Nb24 reveals a trans peptide bond at position 32 of this amyloidogenic variant, whereas Pro32 adopts the cis conformation in the wild‐type monomer, indicating that the cis to trans isomerization at Pro32 plays a critical role in the early onset of β2m amyloid formation.     

Structural basis for the nuclease activity of a bacteriophage large terminase

The DNA‐packaging motor in tailed bacteriophages requires nuclease activity to ensure that the genome is packaged correctly. This nuclease activity is tightly regulated as the enzyme is inactive for the duration of DNA translocation. Here, we report the X‐ray structure of the large terminase nuclease domain from bacteriophage SPP1. Similarity with the RNase H family endonucleases allowed interactions with the DNA to be predicted. A structure‐based alignment with the distantly related T4 gp17 terminase shows the conservation of an extended β‐sheet and an auxiliary β‐hairpin that are not found in other RNase H family proteins. The model with DNA suggests that the β‐hairpin partly blocks the active site, and in vivo activity assays show that the nuclease domain is not functional in the absence of the ATPase domain. Here, we propose that the nuclease activity is regulated by movement of the β‐hairpin, altering active site access and the orientation of catalytically essential residues.     

Crystal structure of the N‐terminal domains of the surface cell antigen 4 of Rickettsia

The obligate intracellular, gram‐negative bacterium Rickettsia is the causative agent of spotted fevers and typhus in humans. Surface cell antigen (sca) proteins surround these bacteria. We recently reported the co‐localization of one of these proteins, sca4, with vinculin in cells at sites of focal adhesions and demonstrated that two vinculin binding sites directed the sca4/vinculin interaction. Here we report the 2.2 Å crystal structure of the conserved N‐terminal 38 kDa domain of sca4 from Rickettsia rickettsii. The structure reveals two subdomains. The first is an all‐helical domain that is folded in a fashion similar to the dimeric assembly chaperone for rubisco, namely RbcX. The following and highly conserved β‐strand domain lacks significant structural similarity with other known structures and to the best of our knowledge represents a new protein fold.     

Molecular insights into the maturation of phosphodiesterase 6 by the specialized chaperone complex of HSP90 with AIPL1

Phosphodiesterase 6 (PDE6) is a key effector enzyme in vertebrate phototransduction, and its maturation and function are known to critically depend on a specialized chaperone, aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1). Defects in PDE6 and AIPL1 underlie several severe retinal diseases, including retinitis pigmentosa and Leber congenital amaurosis. Here, we characterize the complex of AIPL1 with HSP90 and demonstrate its essential role in promoting the functional conformation of nascent PDE6. Our analysis suggests that AIPL1 preferentially binds to HSP90 in the closed state with a stoichiometry of 1:2, with the tetratricopeptide repeat domain and the tetratricopeptide repeat helix 7 extension of AIPL1 being the main contributors to the AIPL1/HSP90 interface. We demonstrate that mutations of these determinants markedly diminished both the affinity of AIPL1 for HSP90 and the ability of AIPL1 to cochaperone the maturation of PDE6 in a heterologous expression system. In addition, the FK506-binding protein (FKBP) domain of AIPL1 encloses a unique prenyl-binding site that anchors AIPL1 to posttranslational lipid modifications of PDE6. A mouse model with rod PDE6 lacking farnesylation of its PDE6A subunit revealed normal expression, trafficking, and signaling of the enzyme. Furthermore, AIPL1 was unexpectedly capable of inducing the maturation of unprenylated cone PDE6C, whereas mutant AIPL1 deficient in prenyl binding competently cochaperoned prenylated PDE6C. Thus, we conclude neither sequestration of the prenyl modifications is required for PDE6 maturation to proceed, nor is the FKBP-lipid interaction involved in the conformational switch of the enzyme into the functional state.     

Structural basis for the function of DEAH helicases

DEAH helicases participate in pre‐messenger RNA splicing and ribosome biogenesis. The structure of yeast Prp43p‐ADP reveals the homology of DEAH helicases to DNA helicases and the presence of an oligonucleotide‐binding motif. A β‐hairpin from the second RecA domain is wedged between two carboxy‐terminal domains and blocks access to the occluded RNA binding site formed by the RecA domains and a C‐terminal domain. ATP binding and hydrolysis are likely to induce conformational changes in the hairpin that are important for RNA unwinding or ribonucleoprotein remodelling. The structure of Prp43p provides the framework for functional and genetic analysis of all DEAH helicases.