Sterol Methyltransferase:  Functional Analysis of Highly Conserved Residues by Site-Directed Mutagenesis†

Biochemistry ◽  
2004 ◽  
Vol 43 (2) ◽  
pp. 569-576 ◽  
Author(s):  
W. David Nes ◽  
Pruthvi Jayasimha ◽  
Wenxu Zhou ◽  
Ragu Kanagasabai ◽  
Changxiao Jin ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Conserved Residues ◽  
Sterol Methyltransferase

scholarly journals Conserved Residues Control the T1R3-Specific Allosteric Signaling Pathway of the Mammalian Sweet-Taste Receptor

2019 ◽  
Vol 44 (5) ◽  
pp. 303-310 ◽  
Author(s):  
Jean-Baptiste Chéron ◽  
Amanda Soohoo ◽  
Yi Wang ◽  
Jérôme Golebiowski ◽  
Serge Antonczak ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Taste Receptor ◽  
Receptor Activation ◽  
Sweet Taste ◽  
Site Directed Mutagenesis ◽  
Activation Mechanism ◽  
Structural Basis ◽  
Directed Mutagenesis ◽  
Conserved Residues ◽  
Sweet Taste Receptor

Abstract Mammalian sensory systems detect sweet taste through the activation of a single heteromeric T1R2/T1R3 receptor belonging to class C G-protein-coupled receptors. Allosteric ligands are known to interact within the transmembrane domain, yet a complete view of receptor activation remains elusive. By combining site-directed mutagenesis with computational modeling, we investigate the structure and dynamics of the allosteric binding pocket of the T1R3 sweet-taste receptor in its apo form, and in the presence of an allosteric ligand, cyclamate. A novel positively charged residue at the extracellular loop 2 is shown to interact with the ligand. Molecular dynamics simulations capture significant differences in the behavior of a network of conserved residues with and without cyclamate, although they do not directly interact with the allosteric ligand. Structural models show that they adopt alternate conformations, associated with a conformational change in the transmembrane region. Site-directed mutagenesis confirms that these residues are unequivocally involved in the receptor function and the allosteric signaling mechanism of the sweet-taste receptor. Similar to a large portion of the transmembrane domain, they are highly conserved among mammals, suggesting an activation mechanism that is evolutionarily conserved. This work provides a structural basis for describing the dynamics of the receptor, and for the rational design of new sweet-taste modulators.

scholarly journals The PDZ Domain of the SpoIVB Serine Peptidase Facilitates Multiple Functions

2001 ◽  
Vol 183 (14) ◽  
pp. 4364-4373 ◽  
Author(s):  
Ngo T. Hoa ◽  
James A. Brannigan ◽  
Simon M. Cutting
Keyword(s):  
Gene Expression ◽  
Pdz Domain ◽  
Proteolytic Processing ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Critical Component ◽  
Conserved Residues ◽  
Multiple Functions ◽  
Serine Peptidase ◽  
Distinct Process

ABSTRACT During spore formation in Bacillus subtilis, the SpoIVB protein is a critical component of the ςKregulatory checkpoint. SpoIVB has been shown to be a serine peptidase that is synthesized in the spore chamber and which self-cleaves, releasing active forms. These forms can signal proteolytic processing of the transcription factor ςK in the outer mother cell chamber of the sporulating cell. This forms the basis of the ςK checkpoint and ensures accurate ςK-controlled gene expression. SpoIVB has also been shown to activate a second distinct process, termed the second function, which is essential for the formation of heat-resistant spores. In addition to the serine peptidase domain, SpoIVB contains a PDZ domain. We have altered a number of conserved residues in the PDZ domain by site-directed mutagenesis and assayed the sporulation phenotype and signaling properties of mutant SpoIVB proteins. Our work has revealed that the SpoIVB PDZ domain could be used for up to four distinct processes, (i) targeting of itself for transproteolysis, (ii) binding to the protease inhibitor BofC, (iii) signaling of pro-ςK processing, and (iv) signaling of the second function of SpoIVB.

Site-directed mutagenesis and functional analysis of an active site tryptophan of insect chitinase

2002 ◽  
Vol 32 (11) ◽  
pp. 1477-1488 ◽  
Author(s):  
Hong Zhang ◽  
Xin Huang ◽  
Tamo Fukamizo ◽  
Subbaratnam Muthukrishnan ◽  
Karl J Kramer
Keyword(s):  
Functional Analysis ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis
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