The role of MeH73 in actin polymerization and ATP hydrolysis
Nyman, Tomas ; Schüler, Herwig LU
; Korenbaum, Elena
; Schutt, Clarence E
; Karlsson, Roger
and Lindberg, Uno
(2002)
In Journal of Molecular Biology
317(4).
p.89-577
- Abstract
In actin from many species H73 is methylated, but the function of this rare post-translational modification is unknown. Although not within bonding distance, it is located close to the gamma-phosphate of the actin-bound ATP. In most crystal structures of actin, the delta1-nitrogen of the methylated H73 forms a hydrogen bond with the carbonyl of G158. This hydrogen bond spans the gap separating subdomains 2 and 4, thereby contributing to the forces that close the interdomain cleft around the ATP polyphosphate tail. A second hydrogen bond stabilizing interdomain closure exists between R183 and Y69. In the closed-to-open transition in beta-actin, both of these hydrogen bonds are broken as the phosphate tail is exposed to solvent. Here we... (More)
In actin from many species H73 is methylated, but the function of this rare post-translational modification is unknown. Although not within bonding distance, it is located close to the gamma-phosphate of the actin-bound ATP. In most crystal structures of actin, the delta1-nitrogen of the methylated H73 forms a hydrogen bond with the carbonyl of G158. This hydrogen bond spans the gap separating subdomains 2 and 4, thereby contributing to the forces that close the interdomain cleft around the ATP polyphosphate tail. A second hydrogen bond stabilizing interdomain closure exists between R183 and Y69. In the closed-to-open transition in beta-actin, both of these hydrogen bonds are broken as the phosphate tail is exposed to solvent. Here we describe the isolation and characterization of a mutant beta-actin (H73A) expressed in the yeast Saccharomyces cerevisiae. The properties of the mutant are compared to those of wild-type beta-actin, also expressed in yeast. Yeast does not have the methyl transferase necessary to methylate recombinant beta-actin. Thus, the polymerization properties of yeast-expressed wild-type beta-actin can be compared with normally methylated beta-actin isolated from calf thymus. Since earlier studies of the actin ATPase almost invariably employed rabbit skeletal alpha-actin, this isoform was included in these comparative studies on the polymerization, ATP hydrolysis, and phosphate release of actin. It was found that H73A-actin exchanged ATP at an increased rate, and was less stable than yeast-expressed wild-type actin, indicating that the mutation affects the spatial relationship between the two domains of actin which embrace the nucleotide. At physiological concentrations of Mg(2+), the kinetics of ATP hydrolysis of the mutant actin were unaffected, but polymer formation was delayed. The comparison of methylated and unmethylated beta-actin revealed that in the absence of a methyl group on H73, ATP hydrolysis and phosphate release occurred prior to, and seemingly independently of, filament formation. The comparison of beta and alpha-actin revealed differences in the timing and relative rates of ATP hydrolysis and P(i)-release.
(Less)
- author
- Nyman, Tomas
; Schüler, Herwig
LU
; Korenbaum, Elena
; Schutt, Clarence E
; Karlsson, Roger
and Lindberg, Uno
- publishing date
- 2002-04-05
- type
- Contribution to journal
- publication status
- published
- keywords
- Actins/chemistry, Adenosine Triphosphatases/chemistry, Adenosine Triphosphate/metabolism, Animals, Biopolymers/chemistry, Chickens/genetics, Deoxyribonuclease I/metabolism, Enzyme Stability, Histidine/metabolism, Hydrogen Bonding, Hydrolysis, Kinetics, Methylation, Myosin Subfragments/metabolism, Phosphates/metabolism, Protein Binding, Protein Structure, Tertiary, Saccharomyces cerevisiae/genetics, Temperature, Thermodynamics, Viscosity
- in
- Journal of Molecular Biology
- volume
- 317
- issue
- 4
- pages
- 13 pages
- publisher
- Elsevier
- external identifiers
-
- pmid:11955010
- scopus:0036290484
- ISSN
- 0022-2836
- DOI
- 10.1006/jmbi.2002.5436
- language
- English
- LU publication?
- no
- additional info
- Copyright 2002 Elsevier Science Ltd.
- id
- 659204e7-ea1f-4c0f-b687-c37efa691385
- date added to LUP
- 2024-11-21 18:07:06
- date last changed
- 2025-07-05 12:38:25
@article{659204e7-ea1f-4c0f-b687-c37efa691385,
abstract = {{<p>In actin from many species H73 is methylated, but the function of this rare post-translational modification is unknown. Although not within bonding distance, it is located close to the gamma-phosphate of the actin-bound ATP. In most crystal structures of actin, the delta1-nitrogen of the methylated H73 forms a hydrogen bond with the carbonyl of G158. This hydrogen bond spans the gap separating subdomains 2 and 4, thereby contributing to the forces that close the interdomain cleft around the ATP polyphosphate tail. A second hydrogen bond stabilizing interdomain closure exists between R183 and Y69. In the closed-to-open transition in beta-actin, both of these hydrogen bonds are broken as the phosphate tail is exposed to solvent. Here we describe the isolation and characterization of a mutant beta-actin (H73A) expressed in the yeast Saccharomyces cerevisiae. The properties of the mutant are compared to those of wild-type beta-actin, also expressed in yeast. Yeast does not have the methyl transferase necessary to methylate recombinant beta-actin. Thus, the polymerization properties of yeast-expressed wild-type beta-actin can be compared with normally methylated beta-actin isolated from calf thymus. Since earlier studies of the actin ATPase almost invariably employed rabbit skeletal alpha-actin, this isoform was included in these comparative studies on the polymerization, ATP hydrolysis, and phosphate release of actin. It was found that H73A-actin exchanged ATP at an increased rate, and was less stable than yeast-expressed wild-type actin, indicating that the mutation affects the spatial relationship between the two domains of actin which embrace the nucleotide. At physiological concentrations of Mg(2+), the kinetics of ATP hydrolysis of the mutant actin were unaffected, but polymer formation was delayed. The comparison of methylated and unmethylated beta-actin revealed that in the absence of a methyl group on H73, ATP hydrolysis and phosphate release occurred prior to, and seemingly independently of, filament formation. The comparison of beta and alpha-actin revealed differences in the timing and relative rates of ATP hydrolysis and P(i)-release.</p>}},
author = {{Nyman, Tomas and Schüler, Herwig and Korenbaum, Elena and Schutt, Clarence E and Karlsson, Roger and Lindberg, Uno}},
issn = {{0022-2836}},
keywords = {{Actins/chemistry; Adenosine Triphosphatases/chemistry; Adenosine Triphosphate/metabolism; Animals; Biopolymers/chemistry; Chickens/genetics; Deoxyribonuclease I/metabolism; Enzyme Stability; Histidine/metabolism; Hydrogen Bonding; Hydrolysis; Kinetics; Methylation; Myosin Subfragments/metabolism; Phosphates/metabolism; Protein Binding; Protein Structure, Tertiary; Saccharomyces cerevisiae/genetics; Temperature; Thermodynamics; Viscosity}},
language = {{eng}},
month = {{04}},
number = {{4}},
pages = {{89--577}},
publisher = {{Elsevier}},
series = {{Journal of Molecular Biology}},
title = {{The role of MeH73 in actin polymerization and ATP hydrolysis}},
url = {{http://dx.doi.org/10.1006/jmbi.2002.5436}},
doi = {{10.1006/jmbi.2002.5436}},
volume = {{317}},
year = {{2002}},
}