Yeast Protein Farnesyltransferase. Site-Directed Mutagenesis of Conserved Residues in the β-Subunit†

Biochemistry ◽  
1997 ◽  
Vol 36 (30) ◽  
pp. 9246-9252 ◽  
Author(s):  
Julia M. Dolence ◽  
David B. Rozema ◽  
C. Dale Poulter
Keyword(s):  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Β Subunit ◽  
Yeast Protein ◽  
Conserved Residues ◽  
Protein Farnesyltransferase

scholarly journals E. coli F1 -ATPase: Site-directed mutagenesis of the β-subunit

FEBS Letters ◽  
1988 ◽  
Vol 232 (1) ◽  
pp. 111-114 ◽  
Author(s):  
Derek Parsonage ◽  
Susan Wilke-Mounts ◽  
Alan E. Senior
Keyword(s):  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Β Subunit ◽  
E Coli ◽  
F1 Atpase

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.

scholarly journals Role of post-translational modifications at the β-subunit ectodomain in complex association with a promiscuous plant P4-ATPase

2016 ◽  
Vol 473 (11) ◽  
pp. 1605-1615 ◽  
Author(s):  
Sara R. Costa ◽  
Magdalena Marek ◽  
Kristian B. Axelsen ◽  
Lisa Theorin ◽  
Thomas G. Pomorski ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Β Subunit ◽  
Post Translational Modifications ◽  
Functional Roles ◽  
Evolutionarily Conserved ◽  
A Site ◽  
P Type ◽  
Cell Division Control

P-type ATPases of subfamily IV (P4-ATPases) constitute a major group of phospholipid flippases that form heteromeric complexes with members of the Cdc50 (cell division control 50) protein family. Some P4-ATPases interact specifically with only one β-subunit isoform, whereas others are promiscuous and can interact with several isoforms. In the present study, we used a site-directed mutagenesis approach to assess the role of post-translational modifications at the plant ALIS5 β-subunit ectodomain in the functionality of the promiscuous plant P4-ATPase ALA2. We identified two N-glycosylated residues, Asn181 and Asn231. Whereas mutation of Asn231 seems to have a small effect on P4-ATPase complex formation, mutation of evolutionarily conserved Asn181 disrupts interaction between the two subunits. Of the four cysteine residues located in the ALIS5 ectodomain, mutation of Cys86 and Cys107 compromises complex association, but the mutant β-subunits still promote complex trafficking and activity to some extent. In contrast, disruption of a conserved disulfide bond between Cys158 and Cys172 has no effect on the P4-ATPase complex. Our results demonstrate that post-translational modifications in the β-subunit have different functional roles in different organisms, which may be related to the promiscuity of the P4-ATPase.

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