scholarly journals Structures of plasmepsin II fromPlasmodium falciparumin complex with two hydroxyethylamine-based inhibitors

2015 ◽  
Vol 71 (12) ◽  
pp. 1531-1539 ◽  
Author(s):  
Rosario Recacha ◽  
Janis Leitans ◽  
Inara Akopjana ◽  
Lilija Aprupe ◽  
Peteris Trapencieris ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Complex Structure ◽  
Antimalarial Agents ◽  
Flexible Loop ◽  
Open Conformation ◽  
Active Site Cavity ◽  
Plasmepsin Ii ◽  
Complex Structural ◽  
New Compounds

Plasmepsin II (PMII) is one of the ten plasmepsins (PMs) identified in the genome ofPlasmodium falciparum, the causative agent of the most severe and deadliest form of malaria. Owing to the emergence ofP. falciparumstrains that are resistant to current antimalarial agents such as chloroquine and sulfadoxine/pyrimethamine, there is a constant pressure to find new and lasting chemotherapeutic drug therapies. Previously, the crystal structure of PMII in complex with NU655, a potent antimalarial hydroxyethylamine-based inhibitor, and the design of new compounds based on it have been reported. In the current study, two of these newly designed hydroxyethylamine-based inhibitors, PG418 and PG394, were cocrystallized with PMII and their structures were solved, analyzed and compared with that of the PMII–NU655 complex. Structural analysis of the PMII–PG418 complex revealed that the flap loop can adopt a fully closed conformation, stabilized by interactions with the inhibitor, and a fully open conformation, causing an overall expansion in the active-site cavity, which in turn causes unstable binding of the inhibitor. PG418 also stabilizes the flexible loop Gln275–Met286 of another monomer in the asymmetric unit of PMII, which is disordered in the PMII–NU655 complex structure. The crystal structure of PMII in complex with the inhibitor PG418 demonstrates the conformational flexibility of the active-site cavity of the plasmepsins. The interactions of the different moieties in the P1′ position of PG418 and PG394 with Thr217 have to be taken into account in the design of new potent plasmepsin inhibitors.

scholarly journals Crystal Structure of a ligand-bound LacY–Nanobody Complex

2018 ◽  
Vol 115 (35) ◽  
pp. 8769-8774 ◽  
Author(s):  
Hemant Kumar ◽  
Janet S. Finer-Moore ◽  
Xiaoxu Jiang ◽  
Irina Smirnova ◽  
Vladimir Kasho ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Lactose Permease ◽  
Transmembrane Helices ◽  
Helical Bundle ◽  
Flexible Loop ◽  
Sugar Binding ◽  
Open Conformation ◽  
Substrate Analog ◽  
Multiple Conformations ◽  
Alternating Access

The lactose permease of Escherichia coli (LacY), a dynamic polytopic membrane transport protein, catalyzes galactoside/H+ symport and operates by an alternating access mechanism that exhibits multiple conformations, the distribution of which is altered by sugar-binding. Camelid nanobodies were made against a double-mutant Gly46 → Trp/Gly262 → Trp (LacYWW) that produces an outward-open conformation, as opposed to the cytoplasmic open-state crystal structure of WT LacY. Nanobody 9047 (Nb9047) stabilizes WT LacY in a periplasmic-open conformation. Here, we describe the X-ray crystal structure of a complex between LacYWW, the high-affinity substrate analog 4-nitrophenyl-α-d-galactoside (NPG), and Nb9047 at 3-Å resolution. The present crystal structure demonstrates that Nb9047 binds to the periplasmic face of LacY, primarily to the C-terminal six-helical bundle, while a flexible loop of the Nb forms a bridge between the N- and C-terminal halves of LacY across the periplasmic vestibule. The bound Nb partially covers the vestibule, yet does not affect the on-rates or off-rates for the substrate binding to LacYWW, which implicates dynamic flexibility of the Nb–LacYWW complex. Nb9047-binding neither changes the overall structure of LacYWW with bound NPG, nor the positions of side chains comprising the galactoside-binding site. The current NPG-bound structure exhibits a more occluded periplasmic vestibule than seen in a previous structure of a (different Nb) apo-LacYWW/Nb9039 complex that we argue is caused by sugar-binding, with major differences located at the periplasmic ends of transmembrane helices in the N-terminal half of LacY.

scholarly journals Crystal structure of Prp8 reveals active site cavity of the spliceosome

Nature ◽  
2013 ◽  
Vol 493 (7434) ◽  
pp. 638-643 ◽  
Author(s):  
Wojciech P. Galej ◽  
Chris Oubridge ◽  
Andrew J. Newman ◽  
Kiyoshi Nagai
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Active Site Cavity

scholarly journals Crystal structure of the RNA 2′,3′-cyclic phosphodiesterase fromDeinococcus radiodurans

2017 ◽  
Vol 73 (5) ◽  
pp. 276-280 ◽  
Author(s):  
Wanchun Han ◽  
Jiahui Cheng ◽  
Congli Zhou ◽  
Yuejin Hua ◽  
Ye Zhao
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Deinococcus Radiodurans ◽  
Sulfate Ion ◽  
Open Conformation ◽  
Domains Of Life

2′,3′-Cyclic phosphodiesterase (CPDase) homologues have been found in all domains of life and are involved in diverse RNA and nucleotide metabolisms. The CPDase fromDeinococcus radioduranswas crystallized and the crystals diffracted to 1.6 Å resolution, which is the highest resolution currently known for a CPDase structure. Structural comparisons revealed that the enzyme is in an open conformation in the absence of substrate. Nevertheless, the active site is well formed, and the representative motifs interact with sulfate ion, which suggests a conserved catalytic mechanism.

Crystal structure of chikungunya virus nsP2 cysteine protease reveals a putative flexible loop blocking its active site

2018 ◽  
Vol 116 ◽  
pp. 451-462 ◽  
Author(s):  
Manju Narwal ◽  
Harvijay Singh ◽  
Shivendra Pratap ◽  
Anjali Malik ◽  
Richard J. Kuhn ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Cysteine Protease ◽  
Active Site ◽  
Chikungunya Virus ◽  
Flexible Loop

scholarly journals Crystal Structure ofN-Succinylarginine Dihydrolase AstB, Bound to Substrate and Product, an Enzyme from the Arginine Catabolic Pathway ofEscherichia coli

2005 ◽  
Vol 280 (16) ◽  
pp. 15800-15808 ◽  
Author(s):  
Ante Tocilj ◽  
Joseph D. Schrag ◽  
Yunge Li ◽  
Barbara L. Schneider ◽  
Larry Reitzer ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Catabolic Pathway ◽  
Major Pathway ◽  
Product Release ◽  
Flexible Loop ◽  
Ammonia Release ◽  
Arginine Catabolism ◽  
Insight Into

The ammonia-producing arginine succinyltransferase pathway is the major pathway inEscherichia coliand related bacteria for arginine catabolism as a sole nitrogen source. This pathway consists of five steps, each catalyzed by a distinct enzyme. Here we report the crystal structure ofN-succinylarginine dihydrolase AstB, the second enzyme of the arginine succinyltransferase pathway, providing the first structural insight into enzymes from this pathway. The enzyme exhibits a pseudo 5-fold symmetric α/β propeller fold of circularly arranged ββαβ modules enclosing the active site. The crystal structure indicates clearly that this enzyme belongs to the amidinotransferase (AT) superfamily and that the active site contains a Cys–His-Glu triad characteristic of the AT superfamily. Structures of the complexes of AstB with the reaction product and a C365S mutant with bound theN-succinylarginine substrate suggest a catalytic mechanism that consists of two cycles of hydrolysis and ammonia release, with each cycle utilizing a mechanism similar to that proposed for arginine deiminases. Like other members of the AT superfamily of enzymes, AstB possesses a flexible loop that is disordered in the absence of substrate and assumes an ordered conformation upon substrate binding, shielding the ligand from the bulk solvent, thereby controlling substrate access and product release.

scholarly journals Crystal Structure of Complete Rhinovirus RNA Polymerase Suggests Front Loading of Protein Primer

2005 ◽  
Vol 79 (1) ◽  
pp. 277-288 ◽  
Author(s):  
Todd C. Appleby ◽  
Hartmut Luecke ◽  
Jae Hoon Shim ◽  
Jim Z. Wu ◽  
I. Wayne Cheney ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Rna Polymerase ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Structural Information ◽  
Structural Features ◽  
Replication Initiation ◽  
Front Face ◽  
Large Cleft ◽  
Active Site Cavity

ABSTRACT Picornaviruses utilize virally encoded RNA polymerase and a uridylylated protein primer to ensure replication of the entire viral genome. The molecular details of this mechanism are not well understood due to the lack of structural information. We report the crystal structure of human rhinovirus 16 3D RNA-dependent RNA polymerase (HRV16 3Dpol) at a 2.4-Å resolution, representing the first complete polymerase structure from the Picornaviridae family. HRV16 3Dpol shares the canonical features of other known polymerase structures and contains an N-terminal region that tethers the fingers and thumb subdomains, forming a completely encircled active site cavity which is accessible through a small tunnel on the backside of the molecule. The small thumb subdomain contributes to the formation of a large cleft on the front face of the polymerase which also leads to the active site. The cleft appears large enough to accommodate a template:primer duplex during RNA elongation or a protein primer during the uridylylation stage of replication initiation. Based on the structural features of HRV16 3Dpo1 and the catalytic mechanism known for all polymerases, a front-loading model for uridylylation is proposed.

scholarly journals Crystal Structure of the Terminal Oxygenase Component of Cumene Dioxygenase from Pseudomonas fluorescens IP01

2005 ◽  
Vol 187 (7) ◽  
pp. 2483-2490 ◽  
Author(s):  
Xuesong Dong ◽  
Shinya Fushinobu ◽  
Eriko Fukuda ◽  
Tohru Terada ◽  
Shugo Nakamura ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Pseudomonas Fluorescens ◽  
Active Site ◽  
Complex Structure ◽  
Binding Pocket ◽  
Nonheme Iron ◽  
Single Amino Acid ◽  
Molecular Replacement ◽  
Catalytic Subunits ◽  
Single Amino Acid Residue

ABSTRACT The crystal structure of the terminal component of the cumene dioxygenase multicomponent enzyme system of Pseudomonas fluorescens IP01 (CumDO) was determined at a resolution of 2.2 Å by means of molecular replacement by using the crystal structure of the terminal oxygenase component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4 (NphDO). The ligation of the two catalytic centers of CumDO (i.e., the nonheme iron and Rieske [2Fe-2S] centers) and the bridging between them in neighboring catalytic subunits by hydrogen bonds through a single amino acid residue, Asp231, are similar to those of NphDO. An unidentified external ligand, possibly dioxygen, was bound at the active site nonheme iron. The entrance to the active site of CumDO is different from the entrance to the active site of NphDO, as the two loops forming the lid exhibit great deviation. On the basis of the complex structure of NphDO, a biphenyl substrate was modeled in the substrate-binding pocket of CumDO. The residues surrounding the modeled biphenyl molecule include residues that have already been shown to be important for its substrate specificity by a number of engineering studies of biphenyl dioxygenases.

Studies on a Tyr residue critical for the binding of coenzyme and substrate in mouse 3(17)α-hydroxysteroid dehydrogenase (AKR1C21): structure of the Y224D mutant enzyme

2010 ◽  
Vol 66 (2) ◽  
pp. 198-204
Author(s):  
Urmi Dhagat ◽  
Satoshi Endo ◽  
Hiroaki Mamiya ◽  
Akira Hara ◽  
Ossama El-Kabbani
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Unique Feature ◽  
Hydroxysteroid Dehydrogenase ◽  
Kinetic Constants ◽  
Mutant Enzyme ◽  
Side Chains ◽  
Wild Type ◽  
Stereospecific Reduction ◽  
Flexible Loop

Mouse 3(17)α-hydroxysteroid dehydrogenase (AKR1C21) is the only aldo–keto reductase that catalyzes the stereospecific reduction of 3- and 17-ketosteroids to the corresponding 3(17)α-hydroxysteroids. The Y224D mutation of AKR1C21 reduced theKmvalue for NADP(H) by up to 80-fold and completely reversed the 17α stereospecificity of the enzyme. The crystal structure of the Y224D mutant at 2.3 Å resolution revealed that the mutation resulted in a change in the conformation of the flexible loop B, including the V-shaped groove, which is a unique feature of the active-site architecture of wild-type AKR1C21 and is formed by the side chains of Tyr224 and Trp227. Furthermore, mutations (Y224F and Q222N) of residues involved in forming the safety belt for binding of the coenzyme showed similar alterations in kinetic constants for 3α-hydroxy/3-ketosteroids and 17-hydroxy/ketosteroids compared with the wild type.

scholarly journals Structure of a thermostable serralysin fromSerratiasp. FS14 at 1.1 Å resolution

2016 ◽  
Vol 72 (1) ◽  
pp. 10-15 ◽  
Author(s):  
Dongxia Wu ◽  
Tinting Ran ◽  
Weiwu Wang ◽  
Dongqing Xu
Keyword(s):  
Crystal Structure ◽  
Hydrogen Bonds ◽  
Active Site ◽  
Comparative Studies ◽  
Catalytic Domain ◽  
Molecular Surface ◽  
Terminal Domain ◽  
Catalytic Active Site ◽  
Active Site Cavity ◽  
Β Sheets

Serralysin is a well studied metalloprotease, and typical serralysins are not thermostable. The serralysin isolated fromSerratiasp. FS14 was found to be thermostable, and in order to reveal the mechanism responsible for its thermostability, the crystal structure of serralysin fromSerratiasp. FS14 was solved to a crystallographicRfactor of 0.1619 at 1.10 Å resolution. Similar to its homologues, it mainly consists of two domains: an N-terminal catalytic domain and a `parallel β-roll' C-terminal domain. Comparative studies show that the shape of the catalytic active-site cavity is more open owing to the 189–198 loop, with a short 310-helix protruding further from the molecular surface, and that the β-sheets comprising the `parallel β-roll' are longer than those in its homologues. The formation of hydrogen bonds from one of the nonconserved residues (Asn200) to Lys27 may contribute to the thermostability.

scholarly journals Crystal structure of papain-E64-c complex. Binding diversity of E64-c to papain S2 and S3 subsites

1992 ◽  
Vol 287 (3) ◽  
pp. 797-803 ◽  
Author(s):  
M J Kim ◽  
D Yamamoto ◽  
K Matsumoto ◽  
M Inoue ◽  
T Ishida ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Cysteine Proteinase ◽  
Complex Structure ◽  
Binding Mode ◽  
Accessible Surface Area ◽  
Cysteine Proteinase Inhibitor ◽  
Binding Modes ◽  
Replacement Method ◽  
Solvent Molecules

In order to investigate the binding mode of E64-c (a synthetic cysteine proteinase inhibitor) to papain at the atomic level, the crystal structure of the complex was analysed by X-ray diffraction at 1.9 A (1 A is expressed in SI units as 0.1 nm) resolution. The crystal has a space group P2(1)2(1)2(1) with a = 43.37, b = 102.34 and c = 49.95 A. A total of 21,135 observed reflections were collected from the same crystal, and 14811 unique reflections of up to 1.9 A resolution [Fo > 3 sigma(Fo)] were used for the structure solution and refinement. The papain structure was determined by means of the molecular replacement method, and then the inhibitor was observed on a (2 magnitude of Fo-magnitude of Fc) difference Fourier map. The complex structure was finally refined to R = 19.4% including 207 solvent molecules. Although this complex crystal (Form II) was polymorphous as compared with the previously analysed one (Form I), the binding modes of leucine and isoamylamide moieties of E64-c were significantly different from each other. By the calculation of accessible surface area for each complex atom, these two different binding modes were both shown to be tight enough to prevent the access of solvent molecules to the papain active site. With respect to the E64-c-papain binding mode, molecular-dynamics simulations proposed two kinds of stationary states which were derived from the crystal structures of Forms I and II. One of these, which corresponds to the binding mode simulated from Form I, was essentially the same as that observed in the crystal structure, and the other was somewhat different from the crystal structure of Form II, especially with respect to the binding of the isoamylamide moiety with the papain S subsites. The substrate specificity for the papain active site is discussed on the basis of the present results.

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