Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Aug 9;119(32):e2207459119.
doi: 10.1073/pnas.2207459119. Epub 2022 Aug 1.

Structural insight and characterization of human Twinkle helicase in mitochondrial disease

Amanda A Riccio  1 , Jonathan Bouvette  2 , Lalith Perera  3 , Matthew J Longley  1 , Juno M Krahn  4 , Jason G Williams  5 , Robert Dutcher  6 , Mario J Borgnia  2 , William C Copeland  1
Affiliations

Structural insight and characterization of human Twinkle helicase in mitochondrial disease

Amanda A Riccio et al. Proc Natl Acad Sci U S A. .

Abstract

Twinkle is the mammalian helicase vital for replication and integrity of mitochondrial DNA. Over 90 Twinkle helicase disease variants have been linked to progressive external ophthalmoplegia and ataxia neuropathies among other mitochondrial diseases. Despite the biological and clinical importance, Twinkle represents the only remaining component of the human minimal mitochondrial replisome that has yet to be structurally characterized. Here, we present 3-dimensional structures of human Twinkle W315L. Employing cryo-electron microscopy (cryo-EM), we characterize the oligomeric assemblies of human full-length Twinkle W315L, define its multimeric interface, and map clinical variants associated with Twinkle in inherited mitochondrial disease. Cryo-EM, crosslinking-mass spectrometry, and molecular dynamics simulations provide insight into the dynamic movement and molecular consequences of the W315L clinical variant. Collectively, this ensemble of structures outlines a framework for studying Twinkle function in mitochondrial DNA replication and associated disease states.

Keywords: Twinkle helicase; cryo-electron microscopy; mitochondrial DNA; mitochondrial DNA replication; progressive external ophthalmoplegia.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
Twinkle W315L analysis for structural studies. (A) Schematic of the Twinkle construct used in this study. Transport of Twinkle to the mitochondria removes the predicted mitochondrial transport sequence (MTS) producing Twinkle helicase (43-684), Twinkle FL ΔMTS). Twinkle domain structure consists of an N-terminal region (NTR) (light peach), a primase-like domain (light orange), and a C-terminal helicase domain (bright red). (B) Representative cryo-EM micrograph with scale bar (white). (C) Example cryo-EM 2D class averages of a heptamer and octamer Twinkle helicase. (D and E) Cryo-EM density maps of (D) heptamer (blue-gray) and (E) octamer (salmon) colored with size measurements indicated in angstroms of the lumen and width as measured in IMOD (49).
Fig. 2.
Fig. 2.
Overview of W315L Twinkle helicase structure.(A) Top view of DeepEMhancer cryo-EM map of Twinkle helicase heptamer (EMD ID: 25744) (blue) fit with PDB ID: 7T8C (cartoon representation). The oval (Top Left) indicates an asymmetric unit or a monomer. (B) Zoom-in on a monomer unit of the heptamer PDB ID: 7T8C (blue) and octamer PDB ID: 7T8B (salmon) in cartoon representation. The asterisk indicates the helix containing W315L, and the arrow points to a stick representation of W315L. (C) (Top) Schematic of a monomer unit colored according to domain: N-terminal region (NTR) (light peach), primase-like domain (light orange), and C-terminal helicase domain (bright red). (Bottom) Mixed cartoon representation of a Twinkle heptamer (transparent gray) and a surface representation of a monomer (subdomain colors) within the heptamer. (D) (Left) Schematic of Twinkle subdomains mapped with cartoon representations of cylindrical helices. The cylindrical representation is colored as in (C), and the asterisk indicates the location of the helix containing W315L. (Right) The cryo-EM map (mesh) (EMD ID: 25743) fit with the PDB ID: 7T8B cartoon representation of the helix containing W315L. Residues are shown in stick representation. The asterisk is maintained in the same position in (D) and (B).
Fig. 3.
Fig. 3.
XL-MS of Twinkle helicase W315L. (A) 4–12% SDS-PAGE with Coomassie staining of W315L Twinkle helicase in the presence and absence of Bs3d0 (0.5 mM). (B) 2D representation of Twinkle crosslinks displayed using XiNET (22). A schematic of the Twinkle residue numbers and domains are shown below the plot. The black tick marks indicate residues in increments of one hundred. (C) Histogram of linking distances derived from the map of Twinkle. Permissible crosslinks are defined as Cα-Cα ≤30 Å, distances were measured in Chimera. (D) (Left) A sample of Twinkle crosslinks modeled onto a Twinkle monomer (PDB ID: 7T8B). (Right) Crosslinked lysine side chains (sticks) near the multimerization interface are shown.
Fig. 4.
Fig. 4.
Twinkle W315L oligomeric interface. Throughout the figure chain A of Twinkle is light gray blue and chain B is dark gray blue, in schematic a when displaying the oligomer chains c–g are colored alternating these same colors. (A) Schematic of Twinkle heptamer head-to-tail assembly and simplified dimer system used for MD simulations. (B) (Top) Schematic of Twinkle domain structure and the black dashed box pertaining to the region of the Bottom Panel. (Bottom) Zoom-in of the average RMSF plots of the Cα atoms for chain A (Left) and chain B (Right) of W315L calculated over the entire MD trajectory. X-axis is a schematic of the relevant domains of Twinkle and residue numbering. Arrows indicate the region on the schematic of an asymmetric unit pertaining to residues ∼360–390 in the “tail region” of the Twinkle monomer for both chain A and chain B in a simplified dimer system. (C) A tilted view surface representation of Twinkle heptamer (PDB ID: 7T8C) and a black box highlights a cylindrical representation of two asymmetric units. The Twinkle heptamer is rotated to a side view of Twinkle, and a black box highlights the interface between asymmetric units. The asymmetric unit has a height of 60 Å as determined in Chimera. (Right) Zoom-in on the interface of chain A and chain B of PDB ID: 7T8C. Residues shown in stick representation are previously identified Twinkle clinical mutations (16). (D) A histogram of per-residue MM-GBSA binding energies of clinical variants at the interface of two asymmetric units of W315L. y axis represents the binding energies, and error bars are SE.
Fig. 5.
Fig. 5.
Proposed model of Twinkle WT and W315L octamer-heptamer monomer ejection. The loss of one (WT/W315L) or two monomer (W315L) units is required for DNA binding. Each attempt to bind DNA allows for ring opening and unrestrained movement exploiting the inherent flexibility of Twinkle. Twinkle top views are shown in cartoon surface representation for the heptamer (blue) and octamer (pink). Tilted views are shown using the Adaptive Poisson-Boltzmann Solver (APBS) electrostatics plugin of PyMOL onto the surface representation of both oligomeric assemblies. 3D surface cartoon representation color-coded electrostatic surface −5 (red) to +5 (blue) KbT/ec.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Korhonen J. A., Pham X. H., Pellegrini M., Falkenberg M., Reconstitution of a minimal mtDNA replisome in vitro. EMBO J. 23, 2423–2429 (2004). - PMC - PubMed
    1. Wanrooij S., et al. , Human mitochondrial RNA polymerase primes lagging-strand DNA synthesis in vitro. Proc. Natl. Acad. Sci. U.S.A. 105, 11122–11127 (2008). - PMC - PubMed
    1. Hakonen A. H., et al. , Recessive Twinkle mutations in early onset encephalopathy with mtDNA depletion. Brain 130, 3032–3040 (2007). - PubMed
    1. Peter B., Farge G., Pardo-Hernandez C., Tångefjord S., Falkenberg M., Structural basis for adPEO-causing mutations in the mitochondrial TWINKLE helicase. Hum. Mol. Genet. 28, 1090–1099 (2019). - PMC - PubMed
    1. Milenkovic D., et al. , TWINKLE is an essential mitochondrial helicase required for synthesis of nascent D-loop strands and complete mtDNA replication. Hum. Mol. Genet. 22, 1983–1993 (2013). - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources