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 Modulation of erbB kinase activity and oncogenic potential by single point mutations in the glycine loop of the catalytic domain.

1994 ◽  
Vol 14 (10) ◽  
pp. 6868-6878 ◽  
Author(s):  
H K Shu ◽  
C M Chang ◽  
L Ravi ◽  
L Ling ◽  
C M Castellano ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Conformational Changes ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Single Point ◽  
Point Mutations ◽  
Full Length ◽  
Site Directed Mutagenesis ◽  
Tyrosine Kinase Domain ◽  
Amino Terminal

Avian c-erbB is activated to a leukemia oncogene following truncation of its amino-terminal ligand-binding domain by retroviral insertion. The insertionally activated transcripts encode protein products which have constitutive tyrosine kinase activity and can induce erythroleukemia but not sarcomas. We have previously found that a valine-to-isoleucine point mutation at position 157 (V157I mutant) within the tyrosine kinase domain of this truncated erbB can dramatically activate the sarcomagenic potential of the oncogene and increase the kinase activity of this oncoprotein. This mutation lies at position 157 of the insertionally activated c-erbB product, affecting a highly conserved valine residue of the glycine loop involved in ATP binding and phosphate transfer. To investigate the functional importance of this residue in the catalytic activity of kinases, we have introduced at this position, by site-directed mutagenesis, codons representing the remaining 18 amino acid residues. Most of the mutants have diminished activity, with six of them completely devoid of kinase activity, indicating the sensitivity of this region to conformational changes. Some of these mutants displayed increased kinase activity and greater transforming potential in comparison with IA c-erbB, but none had levels as high as those of the V157I mutant. In general, the sarcomagenic potential of the various erbB mutants correlated with their autophosphorylation state and their ability to cause phosphorylation of MAP kinase. However, there are important exceptions such as the V157G mutant, which lacks enhanced autophosphorylation but is highly sarcomagenic. Studies of this and other autophosphorylation site mutants point to the existence of an autophosphorylation-independent pathway in sarcomagenesis. The requirement for leukemogenic potential is much less stringent and correlates with positivity of kinase activity. When the valine-to-isoleucine substitution was put in context of the full-length erbB protein, the mutation relaxed the ligand dependence and had a positive effect on the transforming potential of the full-length c-erbB.

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 Contribution of Distinct Structural Elements to Activation of Calpain by Ca2+Ions

2004 ◽  
Vol 279 (19) ◽  
pp. 20118-20126 ◽  
Author(s):  
Anita Alexa ◽  
Zoltán Bozóky ◽  
Attila Farkas ◽  
Peter Tompa ◽  
Peter Friedrich
Keyword(s):  
Enzyme Activity ◽  
Conformational Changes ◽  
Site Directed Mutagenesis ◽  
Schematic Model ◽  
Structural Elements ◽  
Long Distance ◽  
Relay System ◽  
Domain Iii ◽  
A Site ◽  
Mutagenesis Study

The effect of Ca2+in calpain activation is mediated via several binding sites in the enzyme molecule. To test the contribution of structural elements suspected to be part of this Ca2+relay system, we made a site-directed mutagenesis study on calpains, measuring consequential changes in Ca2+binding and Ca2+sensitivity of enzyme activity. Evidence is provided for earlier suggestions that an acidic loop in domain III and the transducer region connecting domains III and IV are part of the Ca2+relay system. Wild-typeDrosophilaCalpain B domain III binds two to three Ca2+ions with aKdof 3400 μm. Phospholipids lower this value to 220 μm. Ca2+binding decreases in parallel with the number of mutated loop residues. Deletion of the entire loop abolishes binding of the ion. The Ca2+dependence of enzyme activity of various acidic-loop mutants of Calpain B and rat m-calpain suggests the importance of the loop in regulating activity. Most conspicuously, the replacement of two adjacent acidic residues in the N-terminal half of the loop evokes a dramatic decrease in the Ca2+need of both enzymes, lowering half-maximal Ca2+concentration from 8.6 to 1.3 mmfor Calpain B and from 250 to 7 μmfor m-calpain. Transducer-region mutations in m-calpain also facilitate Ca2+activation with the most profound effect seen upon shortening the region by deletion mutagenesis. All of these data along with structural considerations suggest that the acidic loop and the transducer region form an interconnected, extended structural unit that has the capacity to integrate and transduce Ca2+-evoked conformational changes over a long distance. A schematic model of this “extended transducer” mechanism is presented.

scholarly journals Agonist Binding and G Protein Coupling in Histamine H2 Receptor: A Molecular Dynamics Study

2020 ◽  
Vol 21 (18) ◽  
pp. 6693
Author(s):  
Marcus Conrad ◽  
Christian A. Söldner ◽  
Yinglong Miao ◽  
Heinrich Sticht
Keyword(s):  
Molecular Dynamics ◽  
G Protein ◽  
Conformational Changes ◽  
Structural Information ◽  
Conformational Stability ◽  
Site Directed Mutagenesis ◽  
Active Conformation ◽  
Histamine H2 Receptor ◽  
H2 Receptor ◽  
Ulcer Disease

The histamine H2 receptor (H2R) plays an important role in the regulation of gastric acid secretion. Therefore, it is a main drug target for the treatment of gastroesophageal reflux or peptic ulcer disease. However, there is as of yet no 3D-structural information available hampering a mechanistic understanding of H2R. Therefore, we created a model of the histamine-H2R-Gs complex based on the structure of the ternary complex of the β2-adrenoceptor and investigated the conformational stability of this active GPCR conformation. Since the physiologically relevant motions with respect to ligand binding and conformational changes of GPCRs can only partly be assessed on the timescale of conventional MD (cMD) simulations, we also applied metadynamics and Gaussian accelerated molecular dynamics (GaMD) simulations. A multiple walker metadynamics simulation in combination with cMD was applied for the determination of the histamine binding mode. The preferential binding pose detected is in good agreement with previous data from site directed mutagenesis and provides a basis for rational ligand design. Inspection of the H2R-Gs interface reveals a network of polar interactions that may contribute to H2R coupling selectivity. The cMD and GaMD simulations demonstrate that the active conformation is retained on a μs-timescale in the ternary histamine-H2R-Gs complex and in a truncated complex that contains only Gs helix α5 instead of the entire G protein. In contrast, histamine alone is unable to stabilize the active conformation, which is in line with previous studies of other GPCRs.

scholarly journals The family 21 carbohydrate-binding module of glucoamylase from Rhizopus oryzae consists of two sites playing distinct roles in ligand binding

2006 ◽  
Vol 396 (3) ◽  
pp. 469-477 ◽  
Author(s):  
Wei-I Chou ◽  
Tun-Wen Pai ◽  
Shi-Hwei Liu ◽  
Bor-Kai Hsiung ◽  
Margaret D.-T. Chang
Keyword(s):  
Ligand Binding ◽  
Binding Sites ◽  
Catalytic Domain ◽  
Rhizopus Oryzae ◽  
Structural Information ◽  
Molecular Model ◽  
Site Directed Mutagenesis ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Ligand Binding Sites

The starch-hydrolysing enzyme GA (glucoamylase) from Rhizopus oryzae is a commonly used glycoside hydrolase in industry. It consists of a C-terminal catalytic domain and an N-terminal starch-binding domain, which belong to the CBM21 (carbohydrate-binding module, family 21). In the present study, a molecular model of CBM21 from R. oryzae GA (RoGACBM21) was constructed according to PSSC (progressive secondary structure correlation), modified structure-based sequence alignment, and site-directed mutagenesis was used to identify and characterize potential ligand-binding sites. Our model suggests that RoGACBM21 contains two ligand-binding sites, with Tyr32 and Tyr67 grouped into site I, and Trp47, Tyr83 and Tyr93 grouped into site II. The involvement of these aromatic residues has been validated using chemical modification, UV difference spectroscopy studies, and both qualitative and quantitative binding assays on a series of RoGACBM21 mutants. Our results further reveal that binding sites I and II play distinct roles in ligand binding, the former not only is involved in binding insoluble starch, but also facilitates the binding of RoGACBM21 to long-chain soluble polysaccharides, whereas the latter serves as the major binding site mediating the binding of both soluble polysaccharide and insoluble ligands. In the present study we have for the first time demonstrated that the key ligand-binding residues of RoGACBM21 can be identified and characterized by a combination of novel bioinformatics methodologies in the absence of resolved three-dimensional structural information.

scholarly journals Evidence that pyruvate dehydrogenase kinase belongs to the ATPase/kinase superfamily

1999 ◽  
Vol 344 (1) ◽  
pp. 47-53 ◽  
Author(s):  
Melissa BOWKER-KINLEY ◽  
Kirill M. POPOV
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Catalytic Domain ◽  
Heat Shock Protein 90 ◽  
Enzymic Activity ◽  
Pyruvate Dehydrogenase Kinase ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
C Terminus ◽  
Km Value

In this study the roles of invariant Asn-247, Asp-282, Gly-284, Gly-286 and Gly-319 of pyruvate dehydrogenase kinase were investigated by site-directed mutagenesis. Recombinant kinases, wild-type, Asn-247Ala, Asp-282Ala, Gly-284Ala, Gly-286Ala and Gly-319Ala, were expressed in bacteria, purified, and characterized. Three mutant kinases, Asn-247Ala, Asp-282Ala and Gly-286Ala, lacked any appreciable activity. Two other mutants, Gly-284Ala and Gly-319Ala, were catalytically active, with apparent Vmax values close to that of the wild-type kinase (67 and 85 versus 70 nmol/min per mg, respectively). The apparent Km value of Gly-319Ala for nucleotide substrate increased significantly (1500 versus 16 μM). In contrast, Gly-284Ala had only a slightly higher Km value than the wild-type enzyme (28 versus 16 μM). ATP-binding analysis showed that Asn-247Ala, Asp-282Ala and Gly-286Ala could not bind nucleotide. The Kd value of Gly-284Ala was slightly higher than that of the wild-type enzyme (7 versus 4 μM, respectively). In agreement with kinetic analysis, the Gly-319Ala mutant bound ATP so poorly that it was difficult to determine the binding constant. Despite the fact that Asn-247Ala, Asp-282Ala and Gly-286Ala lacked enzymic activity, they were still capable of binding the protein substrate, as shown by their negative-dominant effect in the competition assay with the wild-type kinase. The results of CD spectropolarimetry indicated that there were no major changes in the secondary structures of Asp-282Ala and Gly-286Ala. These results suggest strongly that the catalytic domain of pyruvate dehydrogenase kinase is located at the C-terminus. Furthermore, the catalytic domain is likely to be folded similarly to the catalytic domains of the members of ATPase/kinase superfamily [molecular chaperone heat-shock protein 90 (Hsp90), DNA gyrase B and histidine protein kinases].

scholarly journals An allosteric ligand stabilizes distinct conformations in the M2 muscarinic acetylcholine receptor

2021 ◽  
Author(s):  
Jun Xu ◽  
Harald Hübner ◽  
Yunfei Hu ◽  
Xiaogang Niu ◽  
Peter Gmeiner ◽  
...  
Keyword(s):  
Muscarinic Receptors ◽  
Conformational Changes ◽  
Rational Design ◽  
Molecular Mechanisms ◽  
Structural Information ◽  
Muscarinic Acetylcholine Receptor ◽  
Activation Process ◽  
Allosteric Modulators ◽  
Positive Allosteric Modulator ◽  
M2 Muscarinic Receptor

AbstractAllosteric modulators provide therapeutic advantages over orthosteric drugs. A plethora of allosteric modulators have been identified for several GPCRs, particularly for muscarinic receptors (mAChRs)1,2. To study the molecular mechanisms governing allosteric modulation, we utilized a recently developed NMR system to investigate the conformational changes in the M2 muscarinic receptor (M2R) in response to the positive allosteric modulator (PAM) LY2119620. Our studies provide the first biophysical data showing that LY2119620 can substantially change the structure and dynamics of M2R in both the extracellular and G-protein coupling domains during the activation process. These NMR data suggest that LY2119620 may function by stabilizing distinct sets of conformations not observed in the presence of orthosteric agonists alone, which may account for the different signaling behaviors of the M2R when bound to LY2119620. Our studies provide new structural information for understanding the mechanism of GPCR allostery, and may facilitate the rational design of allosteric therapeutics targeting muscarinic receptors.

scholarly journals Modulation of erbB kinase activity and oncogenic potential by single point mutations in the glycine loop of the catalytic domain

1994 ◽  
Vol 14 (10) ◽  
pp. 6868-6878
Author(s):  
H K Shu ◽  
C M Chang ◽  
L Ravi ◽  
L Ling ◽  
C M Castellano ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Conformational Changes ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Single Point ◽  
Point Mutations ◽  
Full Length ◽  
Site Directed Mutagenesis ◽  
Tyrosine Kinase Domain ◽  
Amino Terminal

Avian c-erbB is activated to a leukemia oncogene following truncation of its amino-terminal ligand-binding domain by retroviral insertion. The insertionally activated transcripts encode protein products which have constitutive tyrosine kinase activity and can induce erythroleukemia but not sarcomas. We have previously found that a valine-to-isoleucine point mutation at position 157 (V157I mutant) within the tyrosine kinase domain of this truncated erbB can dramatically activate the sarcomagenic potential of the oncogene and increase the kinase activity of this oncoprotein. This mutation lies at position 157 of the insertionally activated c-erbB product, affecting a highly conserved valine residue of the glycine loop involved in ATP binding and phosphate transfer. To investigate the functional importance of this residue in the catalytic activity of kinases, we have introduced at this position, by site-directed mutagenesis, codons representing the remaining 18 amino acid residues. Most of the mutants have diminished activity, with six of them completely devoid of kinase activity, indicating the sensitivity of this region to conformational changes. Some of these mutants displayed increased kinase activity and greater transforming potential in comparison with IA c-erbB, but none had levels as high as those of the V157I mutant. In general, the sarcomagenic potential of the various erbB mutants correlated with their autophosphorylation state and their ability to cause phosphorylation of MAP kinase. However, there are important exceptions such as the V157G mutant, which lacks enhanced autophosphorylation but is highly sarcomagenic. Studies of this and other autophosphorylation site mutants point to the existence of an autophosphorylation-independent pathway in sarcomagenesis. The requirement for leukemogenic potential is much less stringent and correlates with positivity of kinase activity. When the valine-to-isoleucine substitution was put in context of the full-length erbB protein, the mutation relaxed the ligand dependence and had a positive effect on the transforming potential of the full-length c-erbB.

scholarly journals Interaction between residues in the Mg2+-binding site regulates BK channel activation

2013 ◽  
Vol 141 (2) ◽  
pp. 217-228 ◽  
Author(s):  
Junqiu Yang ◽  
Huanghe Yang ◽  
Xiaohui Sun ◽  
Kelli Delaloye ◽  
Xiao Yang ◽  
...  
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Electrostatic Interactions ◽  
Structural Information ◽  
Bk Channels ◽  
Bk Channel ◽  
Channel Activation ◽  
Cytosolic Domain ◽  
Membrane Spanning ◽  
Interdomain Interactions

As a unique member of the voltage-gated potassium channel family, a large conductance, voltage- and Ca2+-activated K+ (BK) channel has a large cytosolic domain that serves as the Ca2+ sensor, in addition to a membrane-spanning domain that contains the voltage-sensing (VSD) and pore-gate domains. The conformational changes of the cytosolic domain induced by Ca2+ binding and the conformational changes of the VSD induced by membrane voltage changes trigger the opening of the pore-gate domain. Although some structural information of these individual functional domains is available, how the interactions among these domains, especially the noncovalent interactions, control the dynamic gating process of BK channels is still not clear. Previous studies discovered that intracellular Mg2+ binds to an interdomain binding site consisting of D99 and N172 from the membrane-spanning domain and E374 and E399 from the cytosolic domain. The bound Mg2+ at this narrow interdomain interface activates the BK channel through an electrostatic interaction with a positively charged residue in the VSD. In this study, we investigated the potential interdomain interactions between the Mg2+-coordination residues and their effects on channel gating. By introducing different charges to these residues, we discovered a native interdomain interaction between D99 and E374 that can affect BK channel activation. To understand the underlying mechanism of the interdomain interactions between the Mg2+-coordination residues, we introduced artificial electrostatic interactions between residues 172 and 399 from two different domains. We found that the interdomain interactions between these two positions not only alter the local conformations near the Mg2+-binding site but also change distant conformations including the pore-gate domain, thereby affecting the voltage- and Ca2+-dependent activation of the BK channel. These results illustrate the importance of interdomain interactions to the allosteric gating mechanisms of BK channels.

scholarly journals Lys-197 and Asp-414 are critical residues for binding of ATP/Mg2+ by rat brain inositol 1,4,5-trisphosphate 3-kinase

1993 ◽  
Vol 291 (3) ◽  
pp. 811-816 ◽  
Author(s):  
D Communi ◽  
K Takazawa ◽  
C Erneux
Keyword(s):  
Enzyme Activity ◽  
Amino Acid ◽  
Rat Brain ◽  
Catalytic Domain ◽  
Enzymic Activity ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Calmodulin Binding ◽  
Inositol 1,4,5 Trisphosphate

Rat brain inositol 1,4,5-trisphosphate (InsP3) 3-kinase A was expressed in Escherichia coli in order to identify the amino acid residues involved in substrate ATP/Mg2+ binding. Two amino acid regions that are conserved in the catalytic domain of InsP3 3-kinase isoenzymes A and B had characteristics consistent with two ATP/Mg(2+)-binding motives. Site-directed mutagenesis was performed on residues Lys-197, Lys-207 and Asp-414 to generate three mutant enzymes, referred to as C5 K197I, C5 K207I and C5 D414N. Comparison of the wild-type and mutant proteins with regard to enzymic activity revealed that C5 K197I exhibited 10% of control enzyme activity, C5 D414N was totally inactive and C5 K207I was fully active. The reduced levels of enzyme activity for C5 K197I and C5 D414N were correlated with an altered ability of the mutant enzymes to bind ATP/Mg2+, as determined by ATP-agarose affinity chromatography. Neither Ca2+/calmodulin binding nor InsP3 binding appeared to be affected. Mutant C5 K207I showed the same characteristics as the wild-type enzyme. Taken together, these results strongly indicated (i) that amino acid residues Lys-197 and Asp-414 are necessary for InsP3 3-kinase activity and form part of the ATP/Mg(2+)-binding domain, and (ii) that amino acid residues Lys-197, Lys-207 and Asp-414 are not involved in either InsP3 binding or enzyme stimulation by Ca2+/calmodulin.

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