Characterizing Opa1: A Protein

Directly Linked to Blindness

Dr. Andrew Kehr
Structural Biologist/Biochemist
February 18, 2019
Loras College
Talk Itinerary
- Scientific Background
- Biochemistry of Opa1GG
- Structural Biology of S-Opa1
- Transitioning to a primarily undergraduate institution
- Expanding beyond Opa1
My Experience As a Researcher

K. Sinha, S. S. Sangani, A. D. Kehr, et. al. Biochemistry. (2016).

 My Experience As a Researcher

L. Kong, … A. D. Kehr, … J.E. Hinshaw. Nature. (2018).

Fusion and Fission

A. D. Kehr, J. E. Hinshaw. COCIS (2018),

 Fusion and Fission
Are Everywhere

Dynamin 1 GTPase Middle PH GED PRD

Vesicle fission

TM
Opa1 MTS
D
GTPase Middle GED
Mitochondrial
fusion (inner)
TM TM
Mitofusin GTPase Middle D D GED

Mitochondrial
fusion (outer)

A. D. Kehr, J. E. Hinshaw. COCIS (2018),

Mitochondrial Fission and Fusion

Modified from Figure 12-6a Molecular Cell Biology, Sixth Edition

R. McFarland, D. M. Turnbull, J. Int. Med. 265, 210–228 (2009).
https://vimeo.com/107211731
Opa1 Mutations Lead to Blindness

B. Y. Chun, J. F. Rizzo. Curr Opin Ophthalmol. 27, 475–480 (2016).

 Opa1 Knockout Causes Adverse
Mitochondrial Morphology
Control

Opa1-siRNA Control Opa1-siRNA

Adapted from: A. Olichon et al.. J. Biol. Chem. 278, 7743–7746 (2003).

 Proteolytic Processing: The Long and Short
of Opa1

Adapted from S. A. Muñoz-Gómez et al., Current Biology. 25, 1489–1495 (2015).

Adapted from G. Meglei, G. A. McQuibban. Biochemistry. 48, 1774–1784 (2009). Adapted from T. MacVicar, T. Langer. J. Cell. Sci. 129, 2297–2306 (2016).
Research Goals
• Does Opa1 behave like other dynamin-like GTPases?
• Does it dimerize in the presence of specific nucleotides?
• Does it have similar enzymatic activity?
• Does Opa1 look like other dynamin-like GTPases?
• Structural Biology techniques

Dyn1
DynGG as a Model Dimer Monomer

GTPase

GED
C-term

Dynamin 1 GTPase Middle PH GED PRD

TM
Opa1 MTS GTPase Middle GED
D

J. S. Chappie et al. Molecular Biology of the Cell. 20, 3561–3571 (2009).

Two Opa1GG Constructs

Opa1GG Opa1∆NGG
Predicted Structures

Opa1GG MTS TMD N-Term GTPase Middle GED

Opa1∆NGG MTS TMD N-Term GTPase Middle GED

 Opa1GG Purification
TEV Protease
Ni-NTA Ni-NTA Anion Exchange Amylose-NTA Size Exclusion
& Dialysis
Cleaves Removes Removes
His tag Removes Ensures
His/MBP tag minor remaining
enriches Opa1 His/MBP tag uniformity
off Opa1 contaminants MBP tag
Opa1∆NGG Migrates Differently In Multiple
Nucleotide States
Opa1GG Opa1∆NGG
158 66.5
Dimer Monomer

Dimer

?
N-term Dimer G Domain Dimer
Opa1 N-Terminal Domain Alone Sufficient
For Dimerization MBP-N Opa

NOpa

MBP

MTS TMD N-Term GTPase Middle GED

Opa1 Oligomerizes In Mitochondria

T. Varanita et al., Cell Metab. 21, 834–844 (2015).

A Potential Model for Mitochondrial Cristae
Maintenance
Outer
Membrane

Inner
Membrane

N-term Dimer

Inter-Membrane G Domain Dimer

Space
Research Goals
• Does Opa1 behave like other dynamin-like GTPases?
• Does it dimerize in the presence of specific nucleotides?
• Yes, but only the Opa1∆NGG.
• Does it have similar enzymatic activity?
• Does Opa1 look like other dynamin-like GTPases?
• Structural Biology techniques
Opa1GG: Similar, Yet Different To Dyn1

37°C Opa1GG - basal Dyn1 - basal

KM 30 uM 102 uM
Kcat 0.02 min-1 2.58 min-1

Dyn1: 1. B. D. Song, et al, J. Biol. Chem. 279, 40431–40436 (2004).

Opa1∆NGG is Active
Research Goals
• Does Opa1 behave like other dynamin-like GTPases?
• Does it dimerize in the presence of specific nucleotides?
• Yes, but only the Opa1∆NGG.
• Does it have similar enzymatic activity?
• Similar KM, slower catalysis (kcat).
• Does Opa1 look like other dynamin-like GTPases?
• Structural Biology techniques
 Techniques of Structural Biology
X-ray Crystallography Cryo Electron Microscopy Nuclear Magnetic Resonance

50 nm

K. Sinha, S. S. Sangani, A. D. Kehr, et. al. Biochemistry. (2016). L. Kong, … A. D. Kehr, … J.E. Hinshaw. Nature. (2018). L. Prade et al., Structure. (1997)
X-Ray Crystal Screening of Opa1∆NGG

Opa1∆NGG

Collaboration with Fred Dyda

E. Callaway. Nature. 525, 172–174 (2015).
 Research Goals
• Does Opa1 look like other dynamin-like GTPases?
• Previous evidence indicates tubulation using Opa1S

Predicted Model

GTPase Domain
Middle/Stalk
GTPase Effector
T. Ban, et al. Hum Mol Genet. 19, 2113–2122 (2010). Domain (GED)
 Cryo Electron Microscopy of Opa1-GDP/AlFx

3 mm

50 nm
Cryo Electron Microscopy of Opa1-GDP/AlFx

20181220_01612 20181220_01636

20181220_0162 20181220_01641
9
A Brief Diversion: Strands/Starts,
Handedness, and Pitch

5.4Å 33.2Å cm?

One Start: α-helix Two Start: DNA Three Start: Cord

Current 3D Model of Opa1-GDP/AlFx

34nm

90°

11nm
Research Goals
• Does Opa1 behave like other dynamin-like GTPases?
• Does it dimerize in the presence of specific nucleotides?
• Yes, but only the Opa1∆NGG.
• Does it have similar enzymatic activity?
• Similar KM, slower catalysis (kcat).
• Does Opa1 look like other dynamin-like GTPases?
• Yes – it helically oligomerizes on lipid in a right-handed helix
• No – it is 6 start!
Research Aims: Transitioning to Loras
College
• Determine the biochemical and structural mechanism of higher order
oligomerization of Opa1.

• Determine the structural and molecular mechanisms of Opa1

associated disease.
Biochemical Mechanism of Opa1
Oligomerization
Opa1∆NGG

MBP
? MBP
Structural Mechanism of Opa1
Oligomerization

? ?

? ?
 Molecular Mechanisms of Opa1 Associated
Disease

Ferré, M. et al.. Hum. Mutat. 36, 20–25 (2014).

What YOU Can Expect to Do in My Lab:
• Molecular Biology
• PCR, bacterial culture, DNA extraction and purification, agarose gel electrophoresis
• Protein expression
• Bacterial/yeast liquid culture, Western blots, expression tests
• Protein purification
• Column chromatography, dialysis, protease digestion, SDS-PAGE
• Enzyme assays
• Kinetics, Assays with pretty colors, (ultra)centrifugation
• Structural biology
• X-ray, Cryo-EM, NMR
Expanding Beyond Opa1
? ?
Dr. ???

?
YOU!

? ?
Acknowledgments
Hinshaw Lab EM and Computation David Chan Lab
Dr. Jenny Hinshaw Dr. Huaibin Wang Haase Lab
Dr. John Jimah
Dr. Haifeng He Hanover Lab
Dr. Melissa Mikolaj
Dr. Bertram Dyda Lab
Dr. Nidhi Kundu
Canagarajah
Abby Stanton
Lieza Chan FAES/OITE
Summer Students NIDDK Biotechnology Core
Past Matt Martin
Dr. Leo Kong Dawit Teshome All of you
Dr. Marisa Rubio
Shunming Fang
Questions?

Opah fish