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 A site-directed mutagenesis study of human placental aromatase.

1992 ◽  
Vol 267 (2) ◽  
pp. 762-768
Author(s):  
D J Zhou ◽  
K R Korzekwa ◽  
T Poulos ◽  
S A Chen
Keyword(s):  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
A Site ◽  
Mutagenesis Study

scholarly journals Crystal structure of the capsular polysaccharide synthesizing protein CapE of Staphylococcus aureus

2013 ◽  
Vol 33 (3) ◽  
Author(s):  
Takamitsu Miyafusa ◽  
Jose M. M. Caaveiro ◽  
Yoshikazu Tanaka ◽  
Martin E. Tanner ◽  
Kouhei Tsumoto
Keyword(s):  
Staphylococcus Aureus ◽  
Conformational Changes ◽  
Capsular Polysaccharide ◽  
Site Directed Mutagenesis ◽  
Structural Elements ◽  
Contact Interface ◽  
Protein Subunits ◽  
Polysaccharide Synthesis ◽  
Substrate Binding Domain ◽  
Primary Sequence Alignment

Enzymes synthesizing the bacterial CP (capsular polysaccharide) are attractive antimicrobial targets. However, we lack critical information about the structure and mechanism of many of them. In an effort to reduce that gap, we have determined three different crystal structures of the enzyme CapE of the human pathogen Staphylococcus aureus. The structure reveals that CapE is a member of the SDR (short-chain dehydrogenase/reductase) super-family of proteins. CapE assembles in a hexameric complex stabilized by three major contact surfaces between protein subunits. Turnover of substrate and/or coenzyme induces major conformational changes at the contact interface between protein subunits, and a displacement of the substrate-binding domain with respect to the Rossmann domain. A novel dynamic element that we called the latch is essential for remodelling of the protein–protein interface. Structural and primary sequence alignment identifies a group of SDR proteins involved in polysaccharide synthesis that share the two salient features of CapE: the mobile loop (latch) and a distinctive catalytic site (MxxxK). The relevance of these structural elements was evaluated by site-directed mutagenesis.

Role of Acidic and Aromatic Amino Acids inRhodobacter CapsulatusCytochromec1. A Site-Directed Mutagenesis Study†

Biochemistry ◽  
2003 ◽  
Vol 42 (29) ◽  
pp. 8818-8830 ◽  
Author(s):  
Jun Li ◽  
Artur Osyczka ◽  
Richard C. Conover ◽  
Michael K. Johnson ◽  
Hong Qin ◽  
...  
Keyword(s):  
Amino Acids ◽  
Aromatic Amino Acids ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
A Site ◽  
Mutagenesis Study

scholarly journals Structural insights into SETD3-mediated histidine methylation on β-actin

2018 ◽  
Author(s):  
Qiong Guo ◽  
Shanhui Liao ◽  
Sebastian Kwiatkowski ◽  
Weronika Tomaka ◽  
Huijuan Yu ◽  
...  
Keyword(s):  
Pulmonary Hypertension ◽  
Enzyme Activity ◽  
Small Molecule ◽  
Conformational Changes ◽  
Catalytic Mechanism ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Domain Protein ◽  
First Time ◽  
Structural Insights

SETD3 is a member of SET (Su(var)3-9, Enhancer of zeste, and Trithorax) domain protein superfamily and plays important roles in hypoxic pulmonary hypertension, muscle differentiation, and carcinogenesis. Recently, we have identified SETD3 as the actin-specific methyltransferase that methylates the N3 of His73 on β-actin. Here we present two structures of S-adenosyl-L-homocysteine-bound SETD3 in complex with either an unmodified β-actin peptide or its His-methylated variant. Structural analyses supported by the site-directed mutagenesis experiments and the enzyme activity assays indicated that the recognition and methylation of β-actin by SETD3 is highly sequence specific, and both SETD3 and β-actin adopt pronounce conformational changes upon binding to each other. In conclusion, the data show for the first time a catalytic mechanism of SETD3-mediated histidine methylation in β-actin, which not only throws light on protein histidine methylation phenomenon, but also facilitates the design of small molecule inhibitors of SETD3.

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