Molecular features of the UNC-45 chaperone critical for binding and folding muscle myosin

Hellerschmied, Doris; Lehner, Anita; Franicevic, Nina; Arnese, Renato; Johnson, Chloe A; Vogel, Antonia; Meinhart, Anton; Kurzbauer, R; Deszcz, Luiza; Gazda, Linn; Geeves, Michael A.; Clausen, Tim

doi:10.1038/s41467-019-12667-8

“…We further replicated these findings using Sf 9 cells engineered to stably express only mouse UNC45A, or UNC45B, demonstrating that endogenous insect HSP90 orthologs were sufficient to function with UNC45. Our findings confirm a recent report where overexpression of the C. elegans UNC45 ortholog was sufficient to fold muscle myosin (MHC-B) in Sf 9 cells without the overexpression of HSP90 ( 55 ). The Sf 9–UNC45 cell lines developed here facilitate the expression of recombinant MYO15 and may prove helpful for other proteins and myosin motors that require UNC45 paralogs to fold and mature correctly.…”

Section: Discussionsupporting

confidence: 92%

“…MYO3A, MYO6, and MYO7A have all been successfully purified from Sf 9 cells without the use of UNC45 coexpression, suggesting that this chaperone is not a prerequisite for folding ( 63 , 64 , 65 , 66 ). It is interesting to note that the UNC45 family chaperones also target the unfolded myosin motor domain in vitro ( 34 , 55 , 67 ) and potentially do so in response to tissue trauma in vivo ( 68 ). An exciting hypothesis is that UNC45 paralogs may maintain hair cell proteostasis and refold myosin motors in stereocilia as they are denatured and damaged.…”

Section: Discussionmentioning

confidence: 99%

The ATPase mechanism of myosin 15, the molecular motor mutated in DFNB3 human deafness

Jiang

¹

,

Takagi

²

,

Shams

³

et al. 2021

Journal of Biological Chemistry

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Cochlear hair cells each possess an exquisite bundle of actin-based stereocilia that detect sound. Unconventional myosin 15 (MYO15) traffics and delivers critical molecules required for stereocilia development and thus is essential for building the mechanosensory hair bundle. Mutations in the human MYO15A gene interfere with stereocilia trafficking and cause hereditary hearing loss, DFNB3, but the impact of these mutations is not known, as MYO15 itself is poorly characterized. To learn more, we performed a kinetic study of the ATPase motor domain to characterize its mechanochemical cycle. Using the baculovirus– Sf 9 system, we purified a recombinant minimal motor domain (S1) by coexpressing the mouse MYO15 ATPase, essential and regulatory light chains that bind its IQ domains, and UNC45 and HSP90A chaperones required for correct folding of the ATPase. MYO15 purified with either UNC45A or UNC45B coexpression had similar ATPase activities ( k cat = ∼ 6 s −1 at 20 °C). Using stopped-flow and quenched-flow transient kinetic analyses, we measured the major rate constants describing the ATPase cycle, including ATP, ADP, and actin binding; hydrolysis; and phosphate release. Actin-attached ADP release was the slowest measured transition (∼12 s −1 at 20 °C), although this did not rate-limit the ATPase cycle. The kinetic analysis shows the MYO15 motor domain has a moderate duty ratio (∼0.5) and weak thermodynamic coupling between ADP and actin binding. These findings are consistent with MYO15 being kinetically adapted for processive motility when oligomerized. Our kinetic characterization enables future studies into how deafness-causing mutations affect MYO15 and disrupt stereocilia trafficking necessary for hearing.

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“…Interestingly, one of the first identified C. elegans ts mutations in UNC45, which affects worm motility and sarcomere organization, is a point mutation in the corresponding codon resulting in E781K substitution (52). It has been recently found that that the worm UNC45 E781K mutant protein has different unfolding Tm as measured by CD, compared to other UCS ts mutants (20), suggesting that this mutation affects the structure of the UCS domain The Y750W (hs Y737W) mutation itself doesn't drastically affect chaperoning ability of UNC45 as shown in rescue experiments with worms (17) and myosin motor domain folding assay in insect cells (20). Our data show that the double mutant 2xW (Y737W, N745W) significantly affects the in vitro chaperoning function of UNC-45 ( Figs.…”

Section: Discussionmentioning

confidence: 99%

Mutational analysis of the structure and function of the chaperone domain of UNC-45B

Gaziova

¹

,

Moncrief

²

,

Christian

³

et al. 2020

Preprint

0

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UNC-45B is a multidomain molecular chaperone that is essential for the proper folding and assembly of myosin into muscle thick filaments in vivo. We have previously demonstrated that its UCS domain is responsible for the chaperone-like properties of UNC-45B. In order to better understand the chaperoning function of the UCS domain we engineered mutations designed to: i) disrupt chaperone-client interactions by removing and altering the structure of the putative client-interacting loop and ii) disrupt chaperone-client interactions by changing highly conserved residues in the putative client-binding groove. We tested the effect of these mutations by using a novel combination of complementary biophysical (circular dichroism, intrinsic tryptophan fluorescence, chaperone activity, and SAXS) and in vivo tools (C. elegans sarcomere structure). Removing the client-holding loop had a pronounced effect on the secondary structure, thermal stability, solution conformation and chaperone function of the UCS domain. These results are consistent with previous in vivo findings that this mutation neither rescue the defect in C. elegans sarcomere organization nor bind to myosin. We found that mutating several conserved residues in the client-binding groove do not affect UCS domain secondary structure or structural stability but reduced its chaperoning activity. We found that these groove mutations also significantly altered the structure and organization of the worm sarcomeres. We also tested the effect of R805W, a mutation distant from the client-binding region. Our in vivo data show that, to our surprise, the R805W mutation appeared to have the most drastic effect on the structure and organization of the worm sarcomeres. In humans, the R805W mutation segregates with human congenital/infantile cataract, indicating a crucial role of R805 in UCS domain stability and/or client interaction. Hence, our experimental approach combining biophysical and biological tools facilitates the study of myosin/chaperone interactions in mechanistic detail. Mutational analysis of a UCS protein3 Statement of SignificanceThe folding of myosin and the assembly of a functional sarcomere requires the chaperone UNC-45B. The molecular mechanism(s) for how UNC-45B assist in this assembly process or prevent stress-induced aggregation states are presently unknown.Answering this question is a problem at the core of muscle development and function.Here we developed a novel approach that combines biophysical and biological tools to study UNC-45B/myosin interactions in mechanistic detail. Our approach may provide critical insights into the molecular nature of the pathogenesis of many muscle disorders stemming from mutations in sarcomeric proteins including skeletal myopathies and cardiomyopathies, and possibly the age-associated decline in muscle mass and function found in the elderly known as sarcopenia.

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“…In vivo mutagenesis analysis revealed that in contrast to a Y750W mutant, which is still capable to restore muscle function, a N758Y mutant failed to rescue a temperature-sensitive unc-45 mutant (in worms carrying the ts allele unc-45(m94, mutation E781K) grown at the nonpermissive temperature. Further analysis of C. elegans mutations affecting myosin-binding groove by the same group confirmed that UNC-45 carrying Y750W mutation was able to support production of soluble myosin motor domain when coexpressed in insect cells, even though to less extent than wt UNC-45 (20). N758Y unfortunately failed to express in insect cells efficiently, suggesting structure destabilizing nature of this mutation.…”

Section: Introductionmentioning

confidence: 95%

“…Structurally, the myosin-interacting element shared by UCS proteins consists of nine copies of the armadillo domain (3,8,(16)(17)(18)(19)(20). Although no human UNC-45 structure is available, homology models have been created via in silico molecular modeling and simulations (21).…”

Section: Introductionmentioning

confidence: 99%

Mutational Analysis of the Structure and Function of the Chaperoning Domain of UNC-45B

Gaziova

¹

,

Moncrief

²

,

Christian

³

et al. 2020

Biophysical Journal

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UNC-45B is a multidomain molecular chaperone that is essential for the proper folding and assembly of myosin into muscle thick filaments in vivo. We have previously demonstrated that its UCS domain is responsible for the chaperone-like properties of UNC-45B. In order to better understand the chaperoning function of the UCS domain we engineered mutations designed to: i) disrupt chaperone-client interactions by removing and altering the structure of the putative client-interacting loop and ii) disrupt chaperone-client interactions by changing highly conserved residues in the putative client-binding groove. We tested the effect of these mutations by using a novel combination of complementary biophysical (circular dichroism, intrinsic tryptophan fluorescence, chaperone activity, and SAXS) and in vivo tools (C. elegans sarcomere structure). Removing the client-holding loop had a pronounced effect on the secondary structure, thermal stability, solution conformation and chaperone function of the UCS domain. These results are consistent with previous in vivo findings that this mutation neither rescue the defect in C. elegans sarcomere organization nor bind to myosin. We found that mutating several conserved residues in the client-binding groove do not affect UCS domain secondary structure or structural stability but reduced its chaperoning activity. We found that these groove mutations also significantly altered the structure and organization of the worm sarcomeres. We also tested the effect of R805W, a mutation distant from the client-binding region. Our in vivo data show that, to our surprise, the R805W mutation appeared to have the most drastic effect on the structure and organization of the worm sarcomeres. In humans, the R805W mutation segregates with human congenital/infantile cataract, indicating a crucial role of R805 in UCS domain stability and/or client interaction. Hence, our experimental approach combining biophysical and biological tools facilitates the study of myosin/chaperone interactions in mechanistic detail.

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Molecular features of the UNC-45 chaperone critical for binding and folding muscle myosin

Cited by 30 publications

References 72 publications

The ATPase mechanism of myosin 15, the molecular motor mutated in DFNB3 human deafness

The ATPase mechanism of myosin 15, the molecular motor mutated in DFNB3 human deafness

Mutational analysis of the structure and function of the chaperone domain of UNC-45B

Mutational Analysis of the Structure and Function of the Chaperoning Domain of UNC-45B

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