scholarly journals Multiple interactions of the ‘transducer’ govern its function in calpain activation by Ca2+

2005 ◽  
Vol 388 (3) ◽  
pp. 741-744 ◽  
Author(s):  
Zoltán BOZÓKY ◽  
Anita ALEXA ◽  
Peter TOMPA ◽  
Peter FRIEDRICH
Keyword(s):  
Structural Changes ◽  
Catalytic Domain ◽  
Intramolecular Interactions ◽  
Site Directed Mutagenesis ◽  
Structural Element ◽  
X Ray ◽  
Calpain Activation ◽  
Ef Hand ◽  
Domain Iii ◽  
Multiple Interactions

Typical calpains in mammals become activated on binding of 8–12 Ca2+ ions per enzyme molecule, giving an example of integrated, manifold regulation by calcium. Besides two identified Ca2+ sites in catalytic domain II and several EF-hand motifs in domains IV and VI, an acidic loop in the centrally positioned domain III seems to harbour Ca2+. The mediator of distant Ca2+-induced structural transitions is an elongated structural element, the ‘transducer’. By site-directed mutagenesis along the transducer, we have generated various forms of rat m-calpain in which critical intramolecular interactions, as judged from the X-ray structure, would be abolished or modified. The kinetic parameters of these mutant enzymes support a model featuring shrinkage of transducer as a contributor to structural changes involved in calpain activation.

scholarly journals Electrostatic interactions of domain III stabilize the inactive conformation of μ-calpain

2004 ◽  
Vol 382 (2) ◽  
pp. 607-617 ◽  
Author(s):  
Amaury FERNÁNDEZ-MONTALVÁN ◽  
Irmgard ASSFALG-MACHLEIDT ◽  
Dietmar PFEILER ◽  
Hans FRITZ ◽  
Marianne JOCHUM ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Electrostatic Interactions ◽  
Catalytic Domain ◽  
Structural Information ◽  
Cysteine Proteases ◽  
Enzymic Activity ◽  
Site Directed Mutagenesis ◽  
Activation Process ◽  
Acidic Residues ◽  
Domain Iii

The ubiquitous μ- and m-calpains are Ca2+-dependent cysteine proteases. They are activated via rearrangement of the catalytic domain II induced by cooperative binding of Ca2+ to several sites of the molecule. Based on the crystallographic structures, a cluster of acidic residues in domain III, the acidic loop, has been proposed to function as part of an electrostatic switch in the activation process. Experimental support for this hypothesis was obtained by site-directed mutagenesis of recombinant human μ-calpain expressed with the baculovirus system in insect cells. Replacing the acidic residues of the loop individually with alanine resulted in an up to 7-fold reduction of the half-maximal Ca2+ concentration required for conformational changes (probed with 2-p-toluidinylnapthalene-6-sulphonate fluorescence) and for enzymic activity. Along with structural information, the contribution of individual acidic residues to the Ca2+ requirement for activation revealed that interactions of the acidic loop with basic residues in the catalytic subdomain IIb and in the pre-transducer region of domain III stabilize the structure of inactive μ-calpain. Disruption of these electrostatic interactions makes the molecule more flexible and increases its Ca2+ sensitivity. It is proposed that the acidic loop and the opposing basic loop of domain III constitute a double-headed electrostatic switch controlling the assembly of the catalytic domain.

scholarly journals Origins of the difference in Ca2+ requirement for activation of μ- and m-calpain

2002 ◽  
Vol 367 (1) ◽  
pp. 263-269 ◽  
Author(s):  
Previn DUTT ◽  
Cherie N. SPRIGGS ◽  
Peter L. DAVIES ◽  
Zongchao JIA ◽  
John S. ELCE
Keyword(s):  
Large Subunit ◽  
Cysteine Proteases ◽  
Intramolecular Interactions ◽  
Global Response ◽  
X Ray ◽  
Kd Values ◽  
Ef Hand ◽  
The Difference ◽  
Type Sequence

The μ- and m-calpains are closely related Ca2+-dependent cysteine proteases having different in vitro Ca2+ requirements (Kd), of approx. 25 and 325μM respectively. The two isoforms are heterodimers of slightly different large (80kDa) subunits and an identical small (28kDa) subunit, so that the difference in Kd values must reside in the large subunits. As assayed here, these Kd values relate to the Ca2+ required for the first phase of calpain activation and do not reflect the lower Ca2+ then required by fully activated calpain. On the basis of sequence comparison and the X-ray structure of m-calpain, many m-type residues in the C-terminal EF-hand-containing domain IV were converted into the corresponding μ-type residues, but these mutations did not produce the expected decrease in Kd. In a series of hybrid (μ/m) large-subunit calpains, the Kd values decreased progressively towards that of μ-calpain as the proportion of μ-type sequence increased from 0 to 90%. Kd values cannot therefore be ascribed to one or a few specific intramolecular interactions, but reflect the global response of the whole molecule to Ca2+ binding. Nonetheless, 25% of the difference in Kd values between μ- and m-calpain can be ascribed to the N-terminal peptide of the large subunit, whereas the C-terminal EF-hand-containing domain IV accounts for 65% of the difference.

Characterization of Recombinant Human Coagulation Factor XFriuli

1996 ◽  
Vol 75 (02) ◽  
pp. 313-317 ◽  
Author(s):  
D J Kim ◽  
A Girolami ◽  
H L James
Keyword(s):  
Catalytic Domain ◽  
Coagulation Factor ◽  
Molecular Size ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Bleeding Tendency ◽  
Factor X ◽  
Amino Terminal ◽  
Normal Plasma ◽  
Plasma Factor

SummaryNaturally occurring plasma factor XFriuli (pFXFr) is marginally activated by both the extrinsic and intrinsic coagulation pathways and has impaired catalytic potential. These studies were initiated to obtain confirmation that this molecule is multi-functionally defective due to the substitution of Ser for Pro at position 343 in the catalytic domain. By the Nelson-Long site-directed mutagenesis procedure a construct of cDNA in pRc/CMV was derived for recombinant factor XFriuli (rFXFr) produced in human embryonic (293) kidney cells. The rFXFr was purified and shown to have a molecular size identical to that of normal plasma factor X (pFX) by gel electrophoretic, and amino-terminal sequencing revealed normal processing cleavages. Using recombinant normal plasma factor X (rFXN) as a reference, the post-translational y-carboxy-glutamic acid (Gla) and (β-hydroxy aspartic acid (β-OH-Asp) content of rFXFr was over 85% and close to 100%, respectively, of expected levels. The specific activities of rFXFr in activation and catalytic assays were the same as those of pFXFr. Molecular modeling suggested the involvement of a new H-bond between the side-chains of Ser-343 and Thr-318 as they occur in anti-parallel (3-pleated sheets near the substrate-binding pocket of pFXFr. These results support the conclusion that the observed mutation in pFXFr is responsible for its dysfunctional activation and catalytic potentials, and that it accounts for the moderate bleeding tendency in the homozygous individuals who possess this variant procoagulant.

scholarly journals Structural basis of the stereoselective formation of the spirooxindole ring in the biosynthesis of citrinadins

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zhiwen Liu ◽  
Fanglong Zhao ◽  
Boyang Zhao ◽  
Jie Yang ◽  
Joseph Ferrara ◽  
...  
Keyword(s):  
High Resolution ◽  
Organic Synthesis ◽  
Indole Alkaloids ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Directed Mutagenesis ◽  
Computational Studies ◽  
X Ray ◽  
Evolutionary Branch ◽  
Catalytic Mechanisms

AbstractPrenylated indole alkaloids featuring spirooxindole rings possess a 3R or 3S carbon stereocenter, which determines the bioactivities of these compounds. Despite the stereoselective advantages of spirooxindole biosynthesis compared with those of organic synthesis, the biocatalytic mechanism for controlling the 3R or 3S-spirooxindole formation has been elusive. Here, we report an oxygenase/semipinacolase CtdE that specifies the 3S-spirooxindole construction in the biosynthesis of 21R-citrinadin A. High-resolution X-ray crystal structures of CtdE with the substrate and cofactor, together with site-directed mutagenesis and computational studies, illustrate the catalytic mechanisms for the possible β-face epoxidation followed by a regioselective collapse of the epoxide intermediate, which triggers semipinacol rearrangement to form the 3S-spirooxindole. Comparing CtdE with PhqK, which catalyzes the formation of the 3R-spirooxindole, we reveal an evolutionary branch of CtdE in specific 3S spirocyclization. Our study provides deeper insights into the stereoselective catalytic machinery, which is important for the biocatalysis design to synthesize spirooxindole pharmaceuticals.

scholarly journals Fluorescent Orthopalladated Complexes of 4-Aryliden-5(4H)-Oxazolones from the Kaede Protein: Synthesis and Characterization

Molecules ◽  
2021 ◽  
Vol 26 (5) ◽  
pp. 1238
Author(s):  
Eduardo Laga ◽  
David Dalmau ◽  
Sofía Arregui ◽  
Olga Crespo ◽  
Ana I. Jimenez ◽  
...  
Keyword(s):  
Intramolecular Interactions ◽  
Ortho Position ◽  
Fluorescent Properties ◽  
Ancillary Ligands ◽  
X Ray ◽  
Mononuclear Complexes ◽  
Fluorescence Measurements ◽  
Anionic Ligands ◽  
Order Of Magnitude

The goal of the work reported here was to amplify the fluorescent properties of 4-aryliden-5(4H)-oxazolones by suppression of the hula-twist non-radiative deactivation pathway. This aim was achieved by simultaneous bonding of a Pd center to the N atom of the heterocycle and the ortho carbon of the arylidene ring. Two different 4-((Z)-arylidene)-2-((E)-styryl)-5(4H)-oxazolones, the structures of which are closely related to the chromophore of the Kaede protein and substituted at the 2- and 4-positions of the arylidene ring (1a OMe; 1b F), were used as starting materials. Oxazolones 1a and 1b were reacted with Pd(OAc)2 to give the corresponding dinuclear orthometalated palladium derivates 2a and 2b by regioselective C–H activation of the ortho-position of the arylidene ring. Reaction of 2a (2b) with LiCl promoted the metathesis of the bridging carboxylate by chloride ligands to afford dinuclear 3a (3b). Mononuclear complexes containing the orthopalladated oxazolone and a variety of ancillary ligands (acetylacetonate (4a, 4b), hydroxyquinolinate (5a), aminoquinoline (6a), bipyridine (7a), phenanthroline (8a)) were prepared from 3a or 3b through metathesis of anionic ligands or substitution of neutral weakly bonded ligands. All species were fully characterized and the X-ray determination of the molecular structure of 7a was carried out. This structure has strongly distorted ligands due to intramolecular interactions. Fluorescence measurements showed an increase in the quantum yield (QY) by up to one order of magnitude on comparing the free oxazolone (QY < 1%) with the palladated oxazolone (QY = 12% for 6a). This fact shows that the coordination of the oxazolone to the palladium efficiently suppresses the hula-twist deactivation pathway.

scholarly journals Pore Modification and Phosphorus Doping Effect on Phosphoric Acid-Activated Fe-N-C for Alkaline Oxygen Reduction Reaction

Nanomaterials ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1519
Author(s):  
Jong Gyeong Kim ◽  
Sunghoon Han ◽  
Chanho Pak
Keyword(s):  
Phosphoric Acid ◽  
Photoelectron Spectroscopy ◽  
Structural Changes ◽  
Electronic States ◽  
Nitrogen Adsorption ◽  
Reduction Reaction ◽  
Phosphorus Doping ◽  
X Ray ◽  
Precious Metal Catalysts ◽  
Adsorption Desorption

The price and scarcity of platinum has driven up the demand for non-precious metal catalysts such as Fe-N-C. In this study, the effects of phosphoric acid (PA) activation and phosphorus doping were investigated using Fe-N-C catalysts prepared using SBA-15 as a sacrificial template. The physical and structural changes caused by the addition of PA were analyzed by nitrogen adsorption/desorption and X-ray diffraction. Analysis of the electronic states of Fe, N, and P were conducted by X-ray photoelectron spectroscopy. The amount and size of micropores varied depending on the PA content, with changes in pore structure observed using 0.066 g of PA. The electronic states of Fe and N did not change significantly after treatment with PA, and P was mainly found in states bonded to oxygen or carbon. When 0.135 g of PA was introduced per 1 g of silica, a catalytic activity which was increased slightly by 10 mV at −3 mA/cm2 was observed. A change in Fe-N-C stability was also observed through the introduction of PA.

scholarly journals Structural Changes in French VF Treatment Wetland Porous Media during the Rest Period: An Ex Situ Study Using X-ray Tomography

Water ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 389
Author(s):  
German Dario Martinez-Carvajal ◽  
Laurent Oxarango ◽  
Jérôme Adrien ◽  
Pascal Molle ◽  
Nicolas Forquet
Keyword(s):  
Porous Media ◽  
Structural Changes ◽  
Rest Period ◽  
Treatment Wetlands ◽  
Ex Situ ◽  
Initial Value ◽  
Treatment Wetland ◽  
X Ray ◽  
The Impact ◽  
Deposit Layer

Clogging constitutes a major operational issue for treatment wetlands. The rest period is a key feature of French Vertical Flow (VF) treatment wetlands and serves to mitigate clogging. An ex-situ drying experiment was performed to mimic the rest period and record structural changes in the porous media using X-ray Computed Tomography (CT). Samples containing the deposit and gravel layers of a first stage French VF treatment wetland were extracted and left to dry in a control environment. Based on CT scans, three phases were identified (voids, biosolids, and gravels). The impact of the rest period was assessed by means of different pore-scale variables. Ultimately, the volume of biosolids had reduced to 58% of its initial value, the deposit layer thickness dropped to 68% of its initial value, and the void/biosolid specific surface area ratio increased from a minimum value of 1.1 to a maximum of 4.2. Cracks greater than 3 mm developed at the uppermost part of the deposit layer, while, in the gravel layer, the rise in void volume corresponds to pores smaller than 2 mm in diameter. Lastly, the air-filled microporosity is estimated to have increased by 0.11 v/v.

Sign in / Sign up

Export Citation Format

Share Document