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Yeast As a Chassis for Developing Functional Assays to Study Human P53
Published on: August 4, 2019
His(73), often methylated, is an important structural determinant for actin. A mutagenic analysis of HIS(73) of yeast
1Department of Biochemistry, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
Abstract:
His(73), has been proposed to regulate the release of P(i) from the interior of actin following polymerization-dependent hydrolysis of bound ATP. Although it is a 3-methylhistidine in the vast majority of actins, His(73) is unmethylated in S. cerevisiae actin. We mutated His(73) in yeast actin to Arg, Lys, Ala, Gln, and Glu and detected no altered phenotypes associated with the mutations in vivo. However, they significantly affect actin function in vitro. Substitution of the more basic residues resulted in enhanced thermal stability, decreased rate of nucleotide exchange, and decreased susceptibility to controlled proteolysis relative to wild-type actin. The opposite effects are observed with the neutral and anionic substitutions. All mutations reduced the rate of polymerization. Molecular dynamics simulations predict a new conformation for the His(73) imidazole in the absence of a methyl group. It also predicts that Arg(73) tightens and stabilizes the actin and that Glu(73) causes a rearrangement of the bottom of actin's interdomain cleft leading possibly to our observed destabilization of actin. Considering the exterior location of His(73), this work indicates a surprisingly important role for the residue as a major structural determinant of actin and provides a clue to the impact caused by methylation of His(73).
Insights
Histidine 73 (His73) in yeast actin plays a critical structural role. Mutations reveal its impact on actin stability and polymerization, offering insights into methylation effects.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Histidine 73 (His73) is implicated in regulating phosphate release from actin after ATP hydrolysis.
- While typically methylated (3-methylhistidine), His73 is unmethylated in Saccharomyces cerevisiae actin.
Purpose of the Study:
- To investigate the in vivo and in vitro functional consequences of mutating His73 in yeast actin.
- To elucidate the structural role of His73 and the impact of its methylation status on actin dynamics.
Main Methods:
- Site-directed mutagenesis of His73 in yeast actin to various residues (Arg, Lys, Ala, Gln, Glu).
- In vivo phenotypic analysis and in vitro functional assays (thermal stability, nucleotide exchange, proteolysis, polymerization rates).
- Molecular dynamics simulations to predict structural changes.
Main Results:
- Mutations showed no in vivo phenotypes but significantly altered in vitro actin function.
- Basic substitutions (Arg, Lys) increased stability and decreased nucleotide exchange and proteolysis susceptibility.
- Neutral and anionic substitutions had opposite effects; all mutations reduced polymerization rates.
Conclusions:
- His73 is a crucial structural determinant of actin, influencing its stability and dynamics.
- Methylation of His73 likely impacts actin structure and function, with unmethylated His73 having distinct conformational predictions.
- The study provides insights into the functional significance of post-translational modifications in actin.
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