Structural and Biochemical Studies to Assess Protein Interactions and (re)classify VUSs Aishwarya Prakash Assistant Professor of Oncologic Sciences Mitchell Cancer Institute
DNA Repair Video Conference Feb 19th, 2019 Outline
• PART I: Specialized functions of the NEIL1 DNA Glycosylase – BER & NEIL1 – The interaction between NEIL1 and mitochondrial SSB, in vitro – Concluding remarks • PART II: (Re)Classification of Variants of Uncertain Significance in Lynch syndrome patients – Identification of Variants of Uncertain Significance – Functional Characterization – Concluding remarks DNA Damage and Repair Pathways
Reactive Oxygen Species
ROS X-rays Ionizing Radiation Replication Errors Anti-tumor Drugs UV Light T T G T
Mismatch Repair Excision Recombinational Nucleotide Excision (MMR) Repair (BER, Repair (HR, NHEJ) Repair (NER) SSBR)
Adapted from Tubbs, Cell 2017; https://diethylstilbestrol.co.uk/dna-repair-system/ PART I: Base excision repair & NEIL1
5’ 3’ 3’ 5’ Monofunctional glycosylases Bifunctional glycosylases MYH NEIL1 MPG TDG OGG1 UNG MBD4 NEIL2 NTH1 NEIL3 SMUG1 5’ 3’ β-elimination βδ-elimination 3’ 5’
5’ P 3’ 5’ P P 3’ APE1 3’ 5’ 3’ 5’
OH APE1 5’ dRP 3’ PNK 3’ 5’ Polβ Polβ OH P Polδ 5’ 3’ 5’ 3’ Polε 3’ 5’ 3’ 5’
Polβ Polβ 5’ 3’ 3’ 5’ 5’ P 3’ FEN1 3’ 5’
5’ P 3’ Lig III / XRCC1 3’ 5’ 5’ 3’ Lig I 3’ 5’ Short-Patch BER 5’ 3’ 3’ 5’ Long-Patch BER Adapted from Hegde ML, Cell Research, 2008 and Duclos et al., 2011 Structural features of the Fpg/Nei DNA glycosylase family H2TH DNA binding motif EcoNei 263 aa EcoFpg 269 aa Zincless finger NEIL1 390 aa Zinc finger NEIL2 332 aa Ran-GTP Zinc finger
NEIL3 605 aa
H2TH
Blue – NEIL1 Pink - EcoFpg
Zinc finger PDB: 1R2Y 5ITX C-terminal Domain N-terminal Domain
Fromme, JC, Verdine, GL. (2003) J Biol Chem 278 51543-51548 Zhu, C. et. al. (2016) Proc.Natl.Acad.Sci.USA 113: 7792-7797 The C-terminal tail of NEIL1 is involved with protein interactions 2 334 2 NEIL1-△56 290 390 NEIL1
290 Polδ/Lig I 390
312 349 FEN-1/RPA/WRN/RF-C
290 PCNA 349 C-terminal disordered region
Prakash A. et al., NAR. 2017 Hegde M. et al., JMB. 2013 Sharma N. et al., DNA Repair. 2018 Doublié S. et al., PNAS. 2004 Doublié S. et al., PNAS. 2004 Adapted from Rangaswamy et al., Genes. 2017 Role of NEIL1 in mitochondrial DNA repair
Question: Does NEIL1 interact with proteins associated with mtDNA replication?
Nidhi Sharma Some proteins involved with mitochondrial DNA maintenance mtSSB, Homotetramer, PDB: 3ULL Polymerase Gamma, Heterotrimer, PDB: 5C51 Twinkle helicase, Nucleic Acids Res. 2015 Apr 30; 43(8): 4284–4295.
Transcription factor A (TFAM), PDB: 3TMM Complement Component 1, q subcomponent binding protein (C1QBP), homotrimer, PDB: 3RPX
Sharma N. et al. DNA Repair. 2018 Prakash Lab, unpublished. 2018 A combined approach to studying the interaction between NEIL1 and mtSSB
Structural Interactions between NEIL1 and mtSSB in solution • Protein Painting & Far-western analysis • Size-exclusion chromatography (SEC), Multi-Angle Light Scattering (MALS), & Small Angle X-ray Scattering (SAXS)
NEIL1-DNA, PDB: 5ITX mtSSB Tetramer, PDB: 3ULL
Yang C. et. al., Nat. Struct. Biol. 1997 https://fineartamerica.com/featured/p Zhu, C. et. al. Proc.Natl.Acad.Sci. 2016 rotein-structure-epstudio-design.html Protein painting to determine interface contacts
Coat surface Denature/ with molecular Dissociate paints Reduction/ Alkylation/ Trypsin Binding NEIL1/2 Digestion Partner Protease cleavage
Set A: Peptides from Set B: Peptides from individual proteins protein complex
Mass Spectrometry B A B – A = Interface contacts
Adapted from Luchini A., et al. Nature Communications 5, Article number: 4413 (2014) Examples of molecular protein paints
RBB, ANSA, Anthraquinone Naphthalene Derivative
CR (Congo red), AO50 (Acid Orange 50), Acryl azo Aryl azo compound compound
MV (Methyl violet), Triarylmethane DECI, compound Polymethine compound
Adapted from Luchini A., et al. Nature Communications 5, Article number: 4413 (2014) Protein Painting with NEIL1, mtSSB, and the N-terminal NEIL1-mtSSB complexC-terminal Tetramer interface
Dimer interface
mtSSB Structure (PDB ID: 3ULL) Protein painting with the NEIL1-mtSSB complex
Full-length NEIL1 Model: RaptorX NEIL1 residues 289 – 312 are required for an interaction with mtSSB 390 NEIL1-FL 349 NEIL1-Δ40 334 NEIL1-Δ56 312 NEIL1-Δ78 289 NEIL1-Δ100 289 390 GST-289-389 312 390 GST-312-389 289 349 GST-289-349 312 349 GST-312-349
GST
75 50 37 25 20
Coomassie stain Far Western Sharma N. et al. DNA Repair. 2018 NEIL1:mtSSB complex formation via SEC
Ø Size exclusion chromatography (SEC) ) separates molecules based on molecular weight and shape or Stokes radius. mAU Ø Molecules with bigger Stokes radii elute first
Absorbance ( Absorbance Volume (ml) ü Complex formation between NEIL1-mtSSB 24 26 28 30 32 34 36 38 40 Fraction (no.)
in absence of DNA was confirmed as the )
molecule is eluted as single peak. mAU 26 27 28 29 30 31 32 33
50 ü We noted a smaller Stokes radius for the 37 NEIL1-mtSSB complex. 25
Absorbance ( 15 Volume (ml)
24 26 28 30 32 34 36 38 40 Fraction (no.) ü Absence of the NEIL1 C-terminal 100 Theoretical molecular Protein/complexresidues abolishes the interaction with Elution Volume (ml) Stokes radius (Å) mtSSB weight (kDa) mtSSB 60.78 14.29 39.2 NEIL1 44.75 13.73 42.2 mtSSB-NEIL1 105 14.26 39.4
Sharma et. al. DNA Repair, 2018 Complex formation of NEIL1 and mtSSB in the presence of DNA )
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTAGACCTGGACG mAU CATCTGXACCTGC Absorbance ( Absorbance
Volume (ml)
Theoretical molecular Protein/complex Elution Volume (ml) Stokes radius (Å) weight (kDa) mtSSB 60.78 14.29 39.2 NEIL1 44.75 13.73 42.2 mtSSB-NEIL1-DNA 128 12.22 50.3
ü Size exclusion chromatography indicated the formation of a larger ternary complex (NEIL1-DNA- mtSSB) in presence of DNA
Sharma et. al. DNA Repair, 2018 Information obtained from SEC-MALS-SAXS
ØAbsolute molar mass and stoichiometry (MALS)
ØMaximum particle dimension (Dmax; SAXS)
ØSize and shape of molecule – P(r) function (SEC & SAXS)
ØFlexibility/disorder (Kratky plot; SAXS)
ØEstimation of molecular weight (MALS and SAXS)
ØOligomerization state and organization in solution (MALS & SAXS)
ØLow resolution molecular envelopes (Blob-o-logy; SAXS) SEC-SAXS/SEC-MALS-SAXS setup
Sample/Complex
SEC Detector at large distance Scattering Curves
UV detector 2θ < 5 degrees mtSSB NEIL1 mtSSB-DNA I
10 NEIL1-mtSSB NEIL1-DNA-mtSSB X-rays Buffer
subtraction RelativeLog
Momentum Transfer (Å)
Multi-angle light scattering • ATSAS Package/Scatter
• Data analysis- I0, Rg, Dmax , MW
• Ab-initio shape modeling
Differential refractive index (dRI) Srinivas Chakravarthy, PhD BioCAT, Argonne National Labs Shape Information from SAXS Analysis
2D Scattering Curves
Distribution of inter-atomic distances, p(r)
Svergun DI, Koch MHJ. Rep. Prog. Phys. 66 (2003) 1735–1782 SEC-SAXS analysis for multimeric complexes
mtSSB Ab ini&o shape NEIL1 mtSSB-DNA reconstruc0on NEIL1-mtSSB NEIL1-DNA-mtSSB
Relativep(r) Å 150 Distance r (Å) Pair-wise Distance Distribu0on GNOM analysis
mtSSB NEIL1 mtSSB-DNA NEIL1-mtSSB NEIL1-DNA-mtSSB I 2 q
Kratky Analysis Momentum Transfer (Å) Sharma et. al. DNA Repair, 2018 http://www.bioisis.net/tutorial/6 Summary of SAXS and MALS data for NEIL1, mtSSB, and complexes
mtSSB NEIL1 mtSSB-DNA NEIL1-mtSSB NEIL1-DNA- (tetramer) complex complex mtSSB complex Structural parameters
Rg (Å) from 27.52 37.15 28.18 28.12 46.86 P(r)
Rg from 27.44±1.21 33.04±2.46 28.40±0.86 28.48±0.80 44.83±1.80 Guinier Dmax (Å) 103.21 149.96 98.74 94.32 165.72 Molecular weight determination (kDa) Theoretical 60.78 44.72 78 105.5 123
MW(Vp) 61 46 79 60.3 146.6
MW(Vc) 59 36 71 55.4 134.3 MW (MALS) 59.8±1.67% 48.5±2.23% 69±1.46% 58±1.54% 129.6±1.54%
Sharma et. al. DNA Repair, 2018 Ab initio shape reconstruction of NEIL1, mtSSB, & complexes
NEIL1 NEIL1-mtSSB NEIL1-mtSSB-DNA
Oblate Oblate
X2 = 1.072 X2 = 1.311 X2 = 1.072
mtSSB Prolate Prolate X2 = 1.041 X2 = 1.311 X2 = 1.066
mtSSB-DNA Unknown Unknown
X2 = 0.945 X2 = 1.312 X2 = 1.069
Sharma et. al. DNA Repair, 2018 Conclusions and Significance
Ø NEIL1 interacts with mtSSB via its disordered C- terminal tail (protein painting, far western, SEC-MALS)
Ø Absolute molar mass values obtained via MALS indicate that NEIL1 interacts with a monomer of mtSSB
Ø The NEIL enzymes may have acquired specific functions during cellular processes such as replication and transcription Ø NEIL1-mediated disruption of replication proteins indicates a potential mechanistic switch between DNA replication and repair Outline
• PART I: Specialized functions of the NEIL1 DNA Glycosylase – BER & NEIL1 – The interaction between NEIL1 and mitochondrial SSB, in vitro – Concluding remarks • PART II: (Re)Classification of Variants of Uncertain Significance in Lynch syndrome patients – Identification of Variants of Uncertain Significance – Functional Characterization – Concluding remarks 5’ 3’ 3’ 5’ 5’ 3’ Mismatch Repair (MMR) Overview
5’ 3’ 5’ MutSa MutLa MutSb MutLa
Leading Strand Synthesis MSH6 PMS2 MSH3 PMS2 MSH2 MLH1 MSH2 MLH1 T G Mismatch recognition T 1 2 4 G MSH6T MSH2 G base-base Insertion – deletion mispair mismatch Incision
MSH6T PMS2 Polymerase d/e MSH2G MLH1 PCNA Removal MSH6 ExoI MSH6T MSH2 MutSa MSH2G G PMS2 MLH1 MutLa Resynthesis and Ligation ExoI C ExoI (5’ to 3’) G RPA Brandon D’Arcy, Adapted from Hsieh P et al., 2017, and http://erielab.web.unc.edu/research/ Lynch syndrome and cancer
Heterozygous mutations in 1 of the 4 main MMR genes (MSH2, MSH6, MLH1, and PMS2) and EPCAM causes Lynch syndrome (LS).
LS is an autosomal dominant hereditary syndrome where individuals have a very high lifetime risk of developing CRC, endometrial cancer, ovarian, gastric, and others.
LS is one of the most common cancer predispositions, representing 2-7% of colorectal cancer cases in the US.
MSH6 EPCAM EPCAM PMS2 10% 1% 1.2% 5% MSH2 MSH6 23.6% MSH2 29.4% 50% MLH1 MLH1 34% PMS2 21.6% 24.2%
LS incidence based on meeting LS incidence using multigene panel Amsterdam or Bethesda criteria testing regardless of family history
Blount and Prakash, Clinical Genetics, 2017 Variants of uncertain significance
• 20-30% of missense mutations identified in MMR genes are unclassified and are called VUSs
• A VUS result complicates the medical management of patients
Goal: Help with the assignment of a VUS as pathogenic or benign to assist with proper medical management.
Focus: PMS2. Of the 4 MMR genes, PMS2 has the highest incidence of VUSs. Brandon D’Arcy - For this talk, we will focus on 2 PMS2 variants (Post-doc)
Jessa Blount (Genetic Counselor) PMS2 Variant c.620G>A Family Pedigree
Breast cancer Colon (80) Cancer (70)
Lung Cancer Breast cancer (70) (Unknown Age)
Lymphoma Leukemia Melanoma Ovarian Cancer (66) (59) (60) (59) PMS2 VUS
Lymphoma Testicular Breast Uterine Cancer Renal Cancer (16) Cancer Cancer (27) (31) Sex Unknown (45) (32) Ovarian Cancer (39) Male
Female
Diagnosed with cancer (age at diagnosis)
- Variant causes G207 à E mutation Deceased
- Does not meet Amsterdam II Criteria for LS + Mutation present
Proband
D’Arcy et al., Human Mutation, 2019 PMS2 Variant c.123_131delGTTAGTAGA Family Pedigree
Lung cancer Liver Cancer (60s) (30s)
Melanoma Melanoma Breast Cancer (60s) Melanoma (60s) Stomach (Unknown Age) (82) Breast Cancer (70s) Colon Polyps Cancer Colon Polyps (70s)
Age 50 - This family does not meet PMS2 VUS Amsterdam II criteria for Sex Unknown Male clinical diagnosis of LS Glioblastoma (11) Colon Cancer (17) Constitutional Mismatch Repair Female - Variant causes deletion of Deficiency Syndrome Diagnosed with cancer amino acids 42-44 (Δ42-44) (age at diagnosis) - CMMRD – rare autosomal Deceased recessive condition + Mutation present Proband
D’Arcy et al., Human Mutation, 2019 Location of the PMS2 variants
1 365 506 862 ATPase Domain Endonuclease Domain MLH1 Interaction
G207 L42
N45 AGS 42-44 V43 Mg2+ AGS
E41
E44
PDB ID: 1H7U, Guarne, A. et al., 2001, Embo J The Δ42-44 variant is unstable in cells 1 365 506 862 ATPase Domain Endonuclease Domain MLH1 Interaction HCT116 Whole Cell Extracts HCT116 Nuclear Extracts
PMS2
MLH1
PCNA
150150 ns 150150 ns *** ** ns ns ** **** 100100 100100
5050 5050 Expression (%) Expression (%) PMS2 Expression (%) PMS2 Expression (%) 00 00 WT WT G207E G207E del42-44 del42-44 D’Arcy et al., Human Mutation, 2019
Non-Transfected Non-Transfected The Δ42-44 variant is deficient in MMR
2000 1500 HindIII AAGCTTCGAG Nt.BstNBI 3’ Nick Site 1000 GAGTCCGATATC 850 TTCGGAGCTC 650
XhoI
ns DNA MMR Substrate 1975 bp ** 150150 *
(%) * 100100 AseI
5050 Repair Activity (%) Repair Efficiency 00
WT G207E del42-44
Non-Transfected D’Arcy et al., Human Mutation, 2019 PMS2 variants: purification and activity
1 365 75 ATPase Domain ~40 kDa
50 - Δ42-44 was insoluble and 37 found in the pellet - Multiple attempts to purify this 25 variant included different tags Coomassie Stained SDS-PAGE gel and refolding procedures
60 His-PMS2 WT PMS2 WT 40 His-PMS2 G207E PMS2 G207E 20 His-PMS2 del42-44
ATPase Activity ATPase BSA
(nmol Pi/umol protein/min) 0
0 10 20 30 40 50 60 PMS2 ATPase domain construct, Wei Yang Time (min) D’Arcy et al., Human Mutation, 2019 The G207E variant is properly folded whereas the Δ42-44 lacks alpha helical elements
) 2 -1 mol
2 0
-2 (deg cm -4 WT -4 Δ42-44 ] x 10
θ G207E [ -6 200 220 240 260 Wavelength (nm)
Enzyme Helix 1 Helix 2 Strand 1 Strand 2 Turns Unordered WT 40% 8% 15% 11% 8% 18% G207E 43% 10% 12% 9% 7% 18% Δ42-44 0.2% 2.3% 11.3% 24.7% 17.6% 43.8%
Vijay Rangachari, USM D’Arcy et al., Human Mutation, 2019 Δ42-44 is likely an aggregated protein
1 365 ATPase Domain ~40 kDa
400 Column: Superdex 200 Exclusion limit: 10 – 600 kDa 300
200 Black = WT Blue = G207E Red = Δ42-44-GST 280 nm (mAU)
λ 100
0 0 20 40 60 80 Time (min)
D’Arcy et al., Human Mutation, 2019 SAXS data reveal that the Δ42Rearanged High Intensity Plot -44 variant Grouped Replotted del42-44: is aggregatedRearanged High Intensi 1000000 100000 10000 [I(q)] [I(q)] 1000 10 10 100 10 1 Black = WT 0.1 Blue = G207E Relative Log 0.01 Relative Log Relative Red = Δ42-44-GST
0.00 0.1 0.2 0.3 0.4 q (Å-1)
WT G207E WT G207E del42-44
D’Arcy et al., Human Mutation, 2019 SAXS data reveal that WT and G207E have similar properties in solution
Distance distribution function Kratky analysis
Black = WT Blue = G207E Red = Δ42-44
D’Arcy et al., Human Mutation, 2019 Crystal structure of G207E
WT Q208
β7 G207/ E207 G209 β8 L206 50 μm N-term K210 Q205 G207E
β8 β7
WT = Grey (1H7S) G207E = Blue C-term Mean r.m.s.d. = 1.8 Å G207E Structure Twinned (k, h, -l)
SG: P212121 (Orthorhombic) Resolution: 2.6 Å (R-work = 23.06 %, R-free = 24.24%) D’Arcy et al., Human Mutation, 2019 Summary and Checklist
Goal of the study: Reclassify variants of uncertain significance as pathogenic or benign based on impact on protein function
Δ42-44 variant is: - Unstable in cells and is MMR deficient - An inactive ATPase - An aggregated protein and lacks key secondary structure elements
The G207E is: - MMR proficient - An active ATPase - Well folded and functions like WT enzyme Summary and Checklist
Variant American College of Medical Genetics and Genomics (ACMG) Standards & Pathogenicity Guidelines Δ42-44 Criteria Implication Likely Pathogenic PS3 Well-established in vitro or in vivo studies to support damaging (1 strong and 2 moderate criteria) effect PM1 Located in a mutational hot spot and/or critical and well- established functional domain (active site) PM2 Absent from controls (or at extremely low frequency in recessive) PM3 For a recessive disorders, detected in trans with a pathogenic variant PM4 Protein length changes as a result of deletion PP3 Multiple lines of computational evidence support a deleterious effect on the gene or gene product G207E BS3 Well-established in vitro or in vivo studies show no damaging Likely Benign (1 effect on protein function strong and 1 supporting BP4 Multiple lines of computational evidence suggest no impact on criteria) gene or gene product (conservation, evolutionary, etc.)
In the future: • We hope to study and assist with the classification of variants to help with medical management • Develop high-throughput assays to assess pathogenicity D’Arcy et al., Human Mutation, 2019 Acknowledgements Prakash Laboratory Nidhi Sharma, Ph.D. Brandon M. D’Arcy, Ph.D. Monica Pasala Jennifer Arrington Mackenzie Terry Collaborators: Jessa Blount, MS, CGC (MCI) Lew Pannell, Ph.D. (MCI) Lindsay Schambeau (MCI) Bill Copeland, Ph.D. (NIEHS) Vijay Rangachari (USM) Synchrotron Access: Advanced Photon Source: BioCAT - Srinivas Chakravarthy, Ph.D. Advanced Light Source: SIBYLS Current & Pending Funding: - Susan Tsukatawa, Ph.D. NIEHS: R00-ES024417; ONES R01-ES030084 Mitchell Cancer Institute, Start up Funds
Past Funding: NIEHS: K99-ES024417 Thank you & Questions