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Phosphorylation by PINK1 releases the UBL domain and initializes the conformational opening of the E3 ubiquitin ligase Parkin.

Caulfield TR, Fiesel FC, Moussaud-Lamodière EL, Dourado DF, Flores SC, Springer W - PLoS Comput. Biol. (2014)

Bottom Line: At steady state, the Ub ligase is maintained inactive in a closed, auto-inhibited conformation that results from intra-molecular interactions.Phosphorylation of Ser65 results in widening of a newly defined cleft and dissociation of the regulatory N-terminal UBL domain.This may open up new avenues for the development of small molecule Parkin activators through targeted drug design.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Mayo Clinic Jacksonville, Florida, United States of America.

ABSTRACT
Loss-of-function mutations in PINK1 or PARKIN are the most common causes of autosomal recessive Parkinson's disease. Both gene products, the Ser/Thr kinase PINK1 and the E3 Ubiquitin ligase Parkin, functionally cooperate in a mitochondrial quality control pathway. Upon stress, PINK1 activates Parkin and enables its translocation to and ubiquitination of damaged mitochondria to facilitate their clearance from the cell. Though PINK1-dependent phosphorylation of Ser65 is an important initial step, the molecular mechanisms underlying the activation of Parkin's enzymatic functions remain unclear. Using molecular modeling, we generated a complete structural model of human Parkin at all atom resolution. At steady state, the Ub ligase is maintained inactive in a closed, auto-inhibited conformation that results from intra-molecular interactions. Evidently, Parkin has to undergo major structural rearrangements in order to unleash its catalytic activity. As a spark, we have modeled PINK1-dependent Ser65 phosphorylation in silico and provide the first molecular dynamics simulation of Parkin conformations along a sequential unfolding pathway that could release its intertwined domains and enable its catalytic activity. We combined free (unbiased) molecular dynamics simulation, Monte Carlo algorithms, and minimal-biasing methods with cell-based high content imaging and biochemical assays. Phosphorylation of Ser65 results in widening of a newly defined cleft and dissociation of the regulatory N-terminal UBL domain. This motion propagates through further opening conformations that allow binding of an Ub-loaded E2 co-enzyme. Subsequent spatial reorientation of the catalytic centers of both enzymes might facilitate the transfer of the Ub moiety to charge Parkin. Our structure-function study provides the basis to elucidate regulatory mechanisms and activity of the neuroprotective Parkin. This may open up new avenues for the development of small molecule Parkin activators through targeted drug design.

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Related in: MedlinePlus

Phosphorylation of Ser65 releases the safety belts of Parkin.A) Zoom into safety belt 1: The UBL blocks RING1 and IBR domains. Key cysteine residues of the E2 binding site in RING1 are indicated. The E2 binding site was defined as follows: Ile236, Thr237, Cys238, Ile239, Thr240, Cys241, Thr242, Asp243, Val244, Arg245, Ile259, Cys260, Leu261, Asp262, Cys263, Phe264, His265, Leu266, and Tyr267 B) The distance between UBL domain (Leu26) and RING1 (Cys238) significantly increased over time MDS. C) Similarly, the distance between UBL (Leu26) and IBR (Phe364) domains significantly increased over time MDS. D) Zoom into safety belt 2: The REP region blocks the E2 binding site in RING1 (as defined in A). E) Dynamic change in REP-RING1 interaction during Parkin opening motion. Graph shows the release of the REP region from the E2-binding site in RING1 as measured by RMSD. The RING1 is released from the REP region by MdMD time of 20 ns, exposing the E2 binding site. F) Loosened interaction between the center Tyr391 in REP region and Cys238 in RING1. The distance increases from 10 to 20 Å. During longer simulations, the distance eventually collapses as the UBL domain moves away and E2 binding has transiently occurred. Across many replicates, we find that the availability of adequate space for an E2 enzyme to approach the binding site in RING1 begins somewhere between 5–22 ns. G) Zoom into safety belt 3: Cys431 is buried by RING0. H) Release of the active site (Cys431) from RING0 (Arg163 C-alpha atom) as measured by RMSD for center-of-mass. RMSD increases moderately over time indicative of a less compacted area. I) SASA for Cys431 entire residue. During MDS, more water is available to Cys431, indicating its enhanced exposure.
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pcbi-1003935-g005: Phosphorylation of Ser65 releases the safety belts of Parkin.A) Zoom into safety belt 1: The UBL blocks RING1 and IBR domains. Key cysteine residues of the E2 binding site in RING1 are indicated. The E2 binding site was defined as follows: Ile236, Thr237, Cys238, Ile239, Thr240, Cys241, Thr242, Asp243, Val244, Arg245, Ile259, Cys260, Leu261, Asp262, Cys263, Phe264, His265, Leu266, and Tyr267 B) The distance between UBL domain (Leu26) and RING1 (Cys238) significantly increased over time MDS. C) Similarly, the distance between UBL (Leu26) and IBR (Phe364) domains significantly increased over time MDS. D) Zoom into safety belt 2: The REP region blocks the E2 binding site in RING1 (as defined in A). E) Dynamic change in REP-RING1 interaction during Parkin opening motion. Graph shows the release of the REP region from the E2-binding site in RING1 as measured by RMSD. The RING1 is released from the REP region by MdMD time of 20 ns, exposing the E2 binding site. F) Loosened interaction between the center Tyr391 in REP region and Cys238 in RING1. The distance increases from 10 to 20 Å. During longer simulations, the distance eventually collapses as the UBL domain moves away and E2 binding has transiently occurred. Across many replicates, we find that the availability of adequate space for an E2 enzyme to approach the binding site in RING1 begins somewhere between 5–22 ns. G) Zoom into safety belt 3: Cys431 is buried by RING0. H) Release of the active site (Cys431) from RING0 (Arg163 C-alpha atom) as measured by RMSD for center-of-mass. RMSD increases moderately over time indicative of a less compacted area. I) SASA for Cys431 entire residue. During MDS, more water is available to Cys431, indicating its enhanced exposure.

Mentions: First, the UBL-linker region must dissociate from RING1 and IBR domains in order to loosen the entire structure (Figure 5A). Based on our simulations, we measured the release of the inhibitory N-terminus that acts like a spring/clamp. The distance between the UBL and RING1 domains indeed increased from about 20 Å to more than 50 Å over time MDS (Figure 5B). Similarly, the distance between the UBL domain and the IBR region significantly increased from an initial 30 Å to almost 90 Å (Figure 5C).


Phosphorylation by PINK1 releases the UBL domain and initializes the conformational opening of the E3 ubiquitin ligase Parkin.

Caulfield TR, Fiesel FC, Moussaud-Lamodière EL, Dourado DF, Flores SC, Springer W - PLoS Comput. Biol. (2014)

Phosphorylation of Ser65 releases the safety belts of Parkin.A) Zoom into safety belt 1: The UBL blocks RING1 and IBR domains. Key cysteine residues of the E2 binding site in RING1 are indicated. The E2 binding site was defined as follows: Ile236, Thr237, Cys238, Ile239, Thr240, Cys241, Thr242, Asp243, Val244, Arg245, Ile259, Cys260, Leu261, Asp262, Cys263, Phe264, His265, Leu266, and Tyr267 B) The distance between UBL domain (Leu26) and RING1 (Cys238) significantly increased over time MDS. C) Similarly, the distance between UBL (Leu26) and IBR (Phe364) domains significantly increased over time MDS. D) Zoom into safety belt 2: The REP region blocks the E2 binding site in RING1 (as defined in A). E) Dynamic change in REP-RING1 interaction during Parkin opening motion. Graph shows the release of the REP region from the E2-binding site in RING1 as measured by RMSD. The RING1 is released from the REP region by MdMD time of 20 ns, exposing the E2 binding site. F) Loosened interaction between the center Tyr391 in REP region and Cys238 in RING1. The distance increases from 10 to 20 Å. During longer simulations, the distance eventually collapses as the UBL domain moves away and E2 binding has transiently occurred. Across many replicates, we find that the availability of adequate space for an E2 enzyme to approach the binding site in RING1 begins somewhere between 5–22 ns. G) Zoom into safety belt 3: Cys431 is buried by RING0. H) Release of the active site (Cys431) from RING0 (Arg163 C-alpha atom) as measured by RMSD for center-of-mass. RMSD increases moderately over time indicative of a less compacted area. I) SASA for Cys431 entire residue. During MDS, more water is available to Cys431, indicating its enhanced exposure.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222639&req=5

pcbi-1003935-g005: Phosphorylation of Ser65 releases the safety belts of Parkin.A) Zoom into safety belt 1: The UBL blocks RING1 and IBR domains. Key cysteine residues of the E2 binding site in RING1 are indicated. The E2 binding site was defined as follows: Ile236, Thr237, Cys238, Ile239, Thr240, Cys241, Thr242, Asp243, Val244, Arg245, Ile259, Cys260, Leu261, Asp262, Cys263, Phe264, His265, Leu266, and Tyr267 B) The distance between UBL domain (Leu26) and RING1 (Cys238) significantly increased over time MDS. C) Similarly, the distance between UBL (Leu26) and IBR (Phe364) domains significantly increased over time MDS. D) Zoom into safety belt 2: The REP region blocks the E2 binding site in RING1 (as defined in A). E) Dynamic change in REP-RING1 interaction during Parkin opening motion. Graph shows the release of the REP region from the E2-binding site in RING1 as measured by RMSD. The RING1 is released from the REP region by MdMD time of 20 ns, exposing the E2 binding site. F) Loosened interaction between the center Tyr391 in REP region and Cys238 in RING1. The distance increases from 10 to 20 Å. During longer simulations, the distance eventually collapses as the UBL domain moves away and E2 binding has transiently occurred. Across many replicates, we find that the availability of adequate space for an E2 enzyme to approach the binding site in RING1 begins somewhere between 5–22 ns. G) Zoom into safety belt 3: Cys431 is buried by RING0. H) Release of the active site (Cys431) from RING0 (Arg163 C-alpha atom) as measured by RMSD for center-of-mass. RMSD increases moderately over time indicative of a less compacted area. I) SASA for Cys431 entire residue. During MDS, more water is available to Cys431, indicating its enhanced exposure.
Mentions: First, the UBL-linker region must dissociate from RING1 and IBR domains in order to loosen the entire structure (Figure 5A). Based on our simulations, we measured the release of the inhibitory N-terminus that acts like a spring/clamp. The distance between the UBL and RING1 domains indeed increased from about 20 Å to more than 50 Å over time MDS (Figure 5B). Similarly, the distance between the UBL domain and the IBR region significantly increased from an initial 30 Å to almost 90 Å (Figure 5C).

Bottom Line: At steady state, the Ub ligase is maintained inactive in a closed, auto-inhibited conformation that results from intra-molecular interactions.Phosphorylation of Ser65 results in widening of a newly defined cleft and dissociation of the regulatory N-terminal UBL domain.This may open up new avenues for the development of small molecule Parkin activators through targeted drug design.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Mayo Clinic Jacksonville, Florida, United States of America.

ABSTRACT
Loss-of-function mutations in PINK1 or PARKIN are the most common causes of autosomal recessive Parkinson's disease. Both gene products, the Ser/Thr kinase PINK1 and the E3 Ubiquitin ligase Parkin, functionally cooperate in a mitochondrial quality control pathway. Upon stress, PINK1 activates Parkin and enables its translocation to and ubiquitination of damaged mitochondria to facilitate their clearance from the cell. Though PINK1-dependent phosphorylation of Ser65 is an important initial step, the molecular mechanisms underlying the activation of Parkin's enzymatic functions remain unclear. Using molecular modeling, we generated a complete structural model of human Parkin at all atom resolution. At steady state, the Ub ligase is maintained inactive in a closed, auto-inhibited conformation that results from intra-molecular interactions. Evidently, Parkin has to undergo major structural rearrangements in order to unleash its catalytic activity. As a spark, we have modeled PINK1-dependent Ser65 phosphorylation in silico and provide the first molecular dynamics simulation of Parkin conformations along a sequential unfolding pathway that could release its intertwined domains and enable its catalytic activity. We combined free (unbiased) molecular dynamics simulation, Monte Carlo algorithms, and minimal-biasing methods with cell-based high content imaging and biochemical assays. Phosphorylation of Ser65 results in widening of a newly defined cleft and dissociation of the regulatory N-terminal UBL domain. This motion propagates through further opening conformations that allow binding of an Ub-loaded E2 co-enzyme. Subsequent spatial reorientation of the catalytic centers of both enzymes might facilitate the transfer of the Ub moiety to charge Parkin. Our structure-function study provides the basis to elucidate regulatory mechanisms and activity of the neuroprotective Parkin. This may open up new avenues for the development of small molecule Parkin activators through targeted drug design.

Show MeSH
Related in: MedlinePlus