Human Miro Proteins Act as NTP Hydrolases through a Novel, Non-Canonical Catalytic Mechanism
Abstract
:1. Introduction
2. Results and Discussion
2.1. Miro1 and Miro2 N-Terminal GTPase Domains Are Catalytically Active
2.2. C-Terminal GTPase Domains of Miro1 and Miro2 Exhibit Hydrolytic Activity against GTP
2.3. EF Hands do not Influence C-Terminal Domain Catalytic Activity
2.4. The Miro1 and Miro2 C-Terminal GTPase Domains Function as an NTPase, with Propensities towards GTP and ATP as Substrates
2.5. Rationale for Miro NTP Binding through Structural Modelling
2.6. Structural Comparison with Ras GTPases Suggests that Miro Proteins Utilise a Different Catalytic Mechanism
2.7. The Miro Proteins’ Catalytic Mechanism May Involve an “Internal Arginine Finger”
3. Materials and Methods
3.1. Cloning of Miro1 and Miro2 Mutants
3.2. Expression and Purification of Miro Constructs
3.3. PiColourLock™ Assessment of Phosphate Generation
3.4. Structural Modelling and Analysis
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Clone | Forward Primers | Reverse Primers |
---|---|---|
Miro1 NT-GTPase (K3-P169) | 5′-TGCCATAGCATATGAAGAAAGACGTGCGGAT-3′ | 5′-TGCCATAGCTCGAGTCAAGGATGAAGAACAGCTTTCTGTG-3′ |
Miro1 CT-GTPase (Q415-P580) | 5′-TGCCATAGCATATGCAAAGAAATGTGTTCAGATGTAATG-3′ | 5′-TGCCATAGCTCGAGTCACGGATACATGGCCATTGTT-3′ |
Miro1 EF-CT-GTPase (E177-R590) | 5′-TGCCATAGCATATGGAGGAGAAGGAGATGAAACCA-3′ | 5′-TGCCATAGCTCGAGTCAAGCTCTTGGGGTCAGCTTGT-3′ |
Miro1 NT-GTPase (R3-P169) | 5′-TGCCATAGCATATGCGGGACGTGCGCATCCTGTTA-3′ | 5′-TGCCATAGCTCGAGTCATGGGATGCAGGACGGCCTT-3′ |
Miro1 CT-GTPase (Q413-F589) | 5′-TGCCATAGCATATGCAGCGGAGCGTCCTCCTGT-3′ | 5′-TGCCATAGCTCGAGTCAGAAGGAAGAGGGATGCAGCTCT-3′ |
Miro2 EF-CT-GTPase (E177-P586) | 5′-TGCCATAGCATATGGAGGCCAAGCAGTTGAGG-3′ | 5′-TGCCATAGCTCGAGTCAGGGATGCAGCTCTGCGTG-3′ |
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Peters, D.T.; Kay, L.; Eswaran, J.; Lakey, J.H.; Soundararajan, M. Human Miro Proteins Act as NTP Hydrolases through a Novel, Non-Canonical Catalytic Mechanism. Int. J. Mol. Sci. 2018, 19, 3839. https://doi.org/10.3390/ijms19123839
Peters DT, Kay L, Eswaran J, Lakey JH, Soundararajan M. Human Miro Proteins Act as NTP Hydrolases through a Novel, Non-Canonical Catalytic Mechanism. International Journal of Molecular Sciences. 2018; 19(12):3839. https://doi.org/10.3390/ijms19123839
Chicago/Turabian StylePeters, Daniel T., Laura Kay, Jeyanthy Eswaran, Jeremy H. Lakey, and Meera Soundararajan. 2018. "Human Miro Proteins Act as NTP Hydrolases through a Novel, Non-Canonical Catalytic Mechanism" International Journal of Molecular Sciences 19, no. 12: 3839. https://doi.org/10.3390/ijms19123839