The crystal structures of 4-methoxybenzoate bound CYP199A2 and CYP199A4: structural changes on substrate binding and the identification of an anion binding site

AbstractThe symporter melibiose permease MelB is the best-studied representative from MFS_2 family and the only protein in this large family with crystal structure determined. Previous thermodynamic studies show that MelB utilizes a cooperative binding as the core mechanism for its obligatory symport. Here we present two sugar-bound X-ray crystal structures of a Salmonella typhimurium MelB D59C uniport mutant that binds and catalyzes melibiose transport uncoupled to either cation, as determined by biochemical and biophysical characterizations. The two structures with bound nitrophenyl-α-D-galactoside or dodecyl-β-D-melibioside, which were refined to a resolution of 3.05 or 3.15 Å, respectively, are virtually identical at an outward-facing conformation; each one contains a α-galactoside molecule in the middle of protein. In the substrate-binding site, the galactosyl moiety on both ligands are at an essentially same configuration, so a galactoside specificity determinant pocket can be recognized, and hence the molecular recognition mechanism for the binding of sugar in MelB is deciphered. The data also allow to assign the conserved cation-binding pocket, which is directly connected to the sugar specificity determinant pocket. The intimate connection between the two selection sites lays the structural basis for the cooperative binding and coupled transport. This key structural finding answered the long-standing question on the substrate binding for the Na+-coupled MFS family of transporters.SignificanceMajor facilitator superfamily_2 transporters contain >10,000 members that are widely expressed from bacteria to mammalian cells, and catalyze uptake of varied nutrients from sugars to phospholipids. While several crystal structures with bound sugar for other MFS permeases have been determined, they are either uniporters or symporters coupled solely to H+. MelB catalyzes melibiose symport with either Na+, Li+, or H+, a prototype for Na+-coupled MFS transporters, but its sugar recognition has been a long-unsolved puzzle. Two high-resolution crystal structures presented here clearly reveal the molecular recognition mechanism for the binding of sugar in MelB. The substrate-binding site is characterized with a small specificity groove adjoining a large nonspecific cavity, which could offer a potential for future exploration of active transporters for drug delivery.

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Crystal structures of substrate-bound chitinase from the psychrophilic bacteriumMoritella marinaand its structure in solution

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004713032264 ◽

2014 ◽

Vol 70 (3) ◽

pp. 676-684 ◽

Cited By ~ 9

Author(s):

Piotr H. Malecki ◽

Constantinos E. Vorgias ◽

Maxim V. Petoukhov ◽

Dmitri I. Svergun ◽

Wojciech Rypniewski

Keyword(s):

Binding Site ◽

Crystal Structures ◽

Domain Structure ◽

Active Site ◽

Substrate Binding ◽

Glycoside Hydrolases ◽

Substrate Binding Site ◽

Domain Structures ◽

Structure In Solution

The four-domain structure of chitinase 60 fromMoritella marina(MmChi60) is outstanding in its complexity. Many glycoside hydrolases, such as chitinases and cellulases, have multi-domain structures, but only a few have been solved. The flexibility of the hinge regions between the domains apparently makes these proteins difficult to crystallize. The analysis of an active-site mutant ofMmChi60 in an unliganded form and in complex with the substrates NAG4and NAG5revealed significant differences in the substrate-binding site compared with the previously determined complexes of most studied chitinases. A SAXS experiment demonstrated that in addition to the elongated state found in the crystal, the protein can adapt other conformations in solution ranging from fully extended to compact.

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Crystal Structures of Substrate Binding Site Mutants of Manganese Peroxidase

Journal of Biological Chemistry ◽

10.1074/jbc.272.28.17574 ◽

1997 ◽

Vol 272 (28) ◽

pp. 17574-17580 ◽

Cited By ~ 61

Author(s):

Munirathinam Sundaramoorthy ◽

Katsuyuki Kishi ◽

Michael H. Gold ◽

Thomas L. Poulos

Keyword(s):

Binding Site ◽

Crystal Structures ◽

Manganese Peroxidase ◽

Substrate Binding ◽

Substrate Binding Site

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Chiral deaza-coelenterazine analogs for probing a substrate-binding site in the Ca2+-binding photoprotein aequorin

PLoS ONE ◽

10.1371/journal.pone.0251743 ◽

2021 ◽

Vol 16 (6) ◽

pp. e0251743

Author(s):

Satoshi Inouye ◽

Yuto Sumida ◽

Yuri Tomabechi ◽

Jumpei Taguchi ◽

Mikako Shirouzu ◽

...

Keyword(s):

Hydrogen Bonding ◽

Molecular Recognition ◽

Binding Site ◽

Crystal Structures ◽

Molecular Oxygen ◽

Substrate Binding ◽

Chemical Probes ◽

Substrate Binding Site ◽

Improvement Method ◽

Related Proteins

The Ca2+-binding photoprotein aequorin is a complex of apoAequorin (apoprotein) and (S)-2-peroxycoelenterazine. Aequorin can be regenerated by the incubation of apoAequorin with coelenterazine and molecular oxygen (O2). In this study, to investigate the molecular recognition of apoAequorin for coelenterazine using chemical probes, the chiral deaza-analogs of (S)- and (R)-deaza-CTZ (daCTZ) for coelenterazine and of (S)-2- and (R)-2-hydroxymethyl-deaza-CTZ (HM-daCTZ) for 2-peroxycoelenterazine were efficiently prepared by the improvement method. The chiral deaza-analogs of (S)-daCTZ and (S)-HM-daCTZ selectively inhibited the regeneration step to aequorin by binding the catalytic site of coelenterazine in the apoAequorin molecule. The crystal structures of the apoAequorin complexes with (S)-daCTZ and (S)-HM-daCTZ were determined, suggesting that the hydroxy moiety at the C6-hydroxyphenyl group and the carbonyl moiety of the imidazopyrazinone ring in coelenterazine are essential to bind the apoAequorin molecule through hydrogen bonding. Therefore, the chiral deaza-analogs of coelenterazine can be used as a probe to study the interaction between coelenterazine and the related proteins including photoprotein, luciferase, and coelenterazine-binding protein.

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Protease Domain Sodium, Calcium, and Zinc Sites in Factor VIIa: Crystal Structures and Kinetic Studies.

Blood ◽

10.1182/blood.v104.11.122.122 ◽

2004 ◽

Vol 104 (11) ◽

pp. 122-122 ◽

Cited By ~ 1

Author(s):

Amy E. Schmidt ◽

Pooja Shah ◽

Emily M. Gauthier ◽

S. Paul Bajaj

Keyword(s):

Binding Site ◽

Crystal Structures ◽

Protein C ◽

Factor Viia ◽

Substrate Binding ◽

Activated Protein C ◽

Kinetic Studies ◽

Protease Domain ◽

Substrate Binding Site ◽

Hydrolysis Of

Abstract During physiologic coagulation, the factor VIIa (FVIIa)/tissue factor (TF) complex activates FIX and FX. FVIIa consists of a N-terminal γ-carboxyglutamic acid (Gla) domain, two epidermal growth factor-like (EGF) domains, and a C-terminal serine protease domain. We obtained crystals of FVIIa/soluble TF in the presence of Na+, Rb+, or Choline+ (Ch+) under conditions containing micromolar concentrations of Zn2+. Rb+ is a large monovalent ion and has been used to identify Na+-sites in several proteins; whereas, Ch+ cannot substitute for Na+. The various crystals diffracted from 2.0 to 2.4 Å and belonged to the space group P212121. In the crystal structures, Na+ or Rb+ in FVIIa coordinates to the carbonyl groups of residues 185 (chymotrypsin numbering), 185A, 221, and 224 as well as to two water molecules. Thus, the Na+-site in FVIIa is similar to that of FXa and activated protein C but not to that of thrombin. Ca2+ in the protease domain of FVIIa is coordinated to the carboxylates of Glu70 and Glu80 as seen earlier by Banner and coworkers. Additionally, the crystal structures also showed two Zn2+-sites, one involving His71 and the other involving His117. The Zn2+-sites are unique to FVIIa since the His residues are not present in other proteases. To investigate the role of Na+, Ca2+, and Zn2+-sites in the protease domain of FVIIa, a series of biochemical and kinetic studies were performed. Na+ increased the kcat for hydrolysis of S-2288 (H-D-Ile-Pro-Arg-p-nitroanilide) ~22-fold by FVIIaWT whereas Ca2+ increased it ~by 230-fold. In the presence of Ca2+, Na+ had virtually no effect on the hydrolysis of S-2288; however, in the presence of Na+, Ca2+ increased the kcat ~12-fold. Thus, the increase in kcat by Ca2+ in the presence or absence of Na+ was similar (~250-fold). Further, Na+ had no effect on Km whereas Ca2+ increased it ~3.5-fold. However, the increase by in Km is biologically not pertinent since the Gla and EGF1 domains of FVIIa determine the Km for activation of FIX and FX. Moreover, FVIIaF225P (Na+-site mutant) showed little response to Na+ and FVIIaE80V (Ca2+-site mutant) showed no response to Ca2+ in hydrolyzing S-2288. These data indicate that the Na+ and Ca2+ effects observed are due to the occupancy of the protease domain Na+ and Ca2+ sites. Consistent with the Km data, Na+ had no effect on the binding of p-aminobenzamidine (pAB, S1 site probe) to FVIIaWT. Interestingly, Ca2+ decreased the Ki for pAB binding by ~5-fold indicating that the increase in Km for S-2288 caused by Ca2+ is not related to the S1 site but rather to the S2 and/or S3/S4 sites in FVIIa. In further studies, Zn2+ inhibited the potentiation of S-2288 hydrolysis by FVIIaWT with Ki ~1 of μM in the absence and ~30 μM in the presence of Ca2+. We conclude that the Na+-site in FVIIa is not linked to the synthetic substrate binding site(s), and that the Ca2+-site is linked to the substrate binding site(s). These observations are in contrast to what has been previously observed for FXa and activated protein C. Thus, in the absence of TF, Na+ and Ca2+ are positive regulators for catalysis by FVIIa; whereas, Zn2+ exerts a negative effect. Conceivably, occupancy of the Na+-site and the protease domain Ca2+-site may render FVIIa in a conformation suitable for TF binding and substrate hydrolysis. The local Zn2+ concentration following release by activated platelets at the site of hemostasis could dampen coagulation as a regulatory mechanism.

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Molecular mechanism for Rabex-5 GEF activation by Rabaptin-5

eLife ◽

10.7554/elife.02687 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 25

Author(s):

Zhe Zhang ◽

Tianlong Zhang ◽

Shanshan Wang ◽

Zhou Gong ◽

Chun Tang ◽

...

Keyword(s):

Binding Site ◽

Crystal Structures ◽

Molecular Mechanism ◽

Ternary Complex ◽

Substrate Binding ◽

Unknown Mechanism ◽

Substrate Binding Site ◽

Biochemical Analyses ◽

Α Helix

Rabex-5 and Rabaptin-5 function together to activate Rab5 and further promote early endosomal fusion in endocytosis. The Rabex-5 GEF activity is autoinhibited by the Rabex-5 CC domain (Rabex-5CC) and activated by the Rabaptin-5 C2-1 domain (Rabaptin-5C21) with yet unknown mechanism. We report here the crystal structures of Rabex-5 in complex with the dimeric Rabaptin-5C21 (Rabaptin-5C212) and in complex with Rabaptin-5C212 and Rab5, along with biophysical and biochemical analyses. We show that Rabex-5CC assumes an amphipathic α-helix which binds weakly to the substrate-binding site of the GEF domain, leading to weak autoinhibition of the GEF activity. Binding of Rabaptin-5C21 to Rabex-5 displaces Rabex-5CC to yield a largely exposed substrate-binding site, leading to release of the GEF activity. In the ternary complex the substrate-binding site of Rabex-5 is completely exposed to bind and activate Rab5. Our results reveal the molecular mechanism for the regulation of the Rabex-5 GEF activity.

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Crystal Structures of Creatininase Reveal the Substrate Binding Site and Provide an Insight into the Catalytic Mechanism

Journal of Molecular Biology ◽

10.1016/j.jmb.2004.01.022 ◽

2004 ◽

Vol 337 (2) ◽

pp. 399-416 ◽

Cited By ~ 13

Author(s):

Tadashi Yoshimoto ◽

Nobutada Tanaka ◽

Naota Kanada ◽

Takahiko Inoue ◽

Yoshitaka Nakajima ◽

...

Keyword(s):

Binding Site ◽

Crystal Structures ◽

Catalytic Mechanism ◽

Substrate Binding ◽

Substrate Binding Site ◽

Insight Into

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Catalysis by a rigid enzyme

10.1101/815415 ◽

2019 ◽

Author(s):

F. Ben Bdira ◽

C. A. Waudby ◽

A. N. Volkov ◽

S. P. Schröder ◽

E. AB ◽

...

Keyword(s):

Catalytic Activity ◽

Nmr Spectroscopy ◽

Complex Formation ◽

Crystal Structures ◽

Structural Changes ◽

Substrate Binding ◽

Protein Matrix ◽

Catalytic Intermediates ◽

Multiple Conformations ◽

Rigid Matrix

AbstractMany enzymes are dynamic entities, sampling conformational states that are relevant for catalytic activity. Crystal structures of catalytic intermediates suggest, however, that not all enzymes require structural changes for activity. The single-domain enzyme xylanase from Bacillus circulans (BCX) is involved in the degradation of hemicellulose. We demonstrate that BCX in solution undergoes minimal structural changes during catalysis. NMR spectroscopy results show that the rigid protein matrix provides a frame for fast substrate binding in multiple conformations, accompanied by slow, enzyme induced substrate distortion. Therefore, we propose a model in which the rigid enzyme takes advantage of substrate flexibility to induce a conformation that facilitates catalysis.One Sentence SummaryThe rigid matrix of BCX uses substrate flexibility in Michaelis complex formation.

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Crystal structures and kinetic studies of human Kappa class glutathione transferase provide insights into the catalytic mechanism

Biochemical Journal ◽

10.1042/bj20110753 ◽

2011 ◽

Vol 439 (2) ◽

pp. 215-225 ◽

Cited By ~ 10

Author(s):

Bing Wang ◽

Yingjie Peng ◽

Tianlong Zhang ◽

Jianping Ding

Keyword(s):

Binding Site ◽

Crystal Structures ◽

Cellular Localization ◽

Catalytic Mechanism ◽

Glutathione Transferase ◽

Substrate Binding ◽

Kinetic Studies ◽

Thiol Group ◽

Kinetic Mechanism ◽

Cellular Detoxification

GSTs (glutathione transferases) are a family of enzymes that primarily catalyse nucleophilic addition of the thiol of GSH (reduced glutathione) to a variety of hydrophobic electrophiles in the cellular detoxification of cytotoxic and genotoxic compounds. GSTks (Kappa class GSTs) are a distinct class because of their unique cellular localization, function and structure. In the present paper we report the crystal structures of hGSTk (human GSTk) in apo-form and in complex with GTX (S-hexylglutathione) and steady-state kinetic studies, revealing insights into the catalytic mechanism of hGSTk and other GSTks. Substrate binding induces a conformational change of the active site from an ‘open’ conformation in the apo-form to a ‘closed’ conformation in the GTX-bound complex, facilitating formations of the G site (GSH-binding site) and the H site (hydrophobic substrate-binding site). The conserved Ser16 at the G site functions as the catalytic residue in the deprotonation of the thiol group and the conserved Asp69, Ser200, Asp201 and Arg202 form a network of interactions with γ-glutamyl carboxylate to stabilize the thiolate anion. The H site is a large hydrophobic pocket with conformational flexibility to allow the binding of different hydrophobic substrates. The kinetic mechanism of hGSTk conforms to a rapid equilibrium random sequential Bi Bi model.

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