1. How Antibodies Communicate with DNA
Dr. John J. Tanner
University of Missouri-Columbia
Department of Chemistry

2. Molecular Recognition: How molecules meet and greet each other
Enzyme - Substrate
Antibody - Antigen
Protein - DNA
Protein - Solvent
Protein - Membrane
Protein - Protein

3. ssDNA
antibody

4. Outline 1. Importance of anti-DNA antibodies 2. X-ray Crystallography
3. Describe the antibody fold
4. How antibodies communicate with DNA

5. Anti-DNA Antibodies
Implicated in the autoimmune disease systemic lupus erythematosus (SLE).
Model system for studying protein-DNA interactions.
Few crystal structures of this type of antibody and only one Fab/DNA structure.
Homology modeling is difficult (impossible)

6. X-ray Crystallography
crystals
diffraction
phases

7. X-ray Crystallography
crystals
diffraction
phases

8. Nobel History of X-ray Crystallography
Röntgen
Discovery of X-rays
1901
von Laue
Diffraction of X-rays by crystals
1914
Bragg and Bragg, 1915
Determined first crystal structures
nl=2dsinq
1915

9. Nobel History of X-ray Crystallography
Sumner, Northrop, Stanley
Preparation and crystallization
of proteins (urease)
1946
Perutz and Kendrew
First crystal structures of proteins
(myoglobin and hemoglobin)
1962
Crick, Watson, Wilkins
Double-helix structure of DNA
from fiber X-ray diffraction
1962

10. Nobel History of X-ray Crystallography
Hodgkin
Crystal structures of penicillin and vitamin B12
1964
1976
Lipscomb
Crystal structures of boranes;
Hauptman & Karle
“Direct methods” determination of crystal structures
1985

11. Nobel History of X-ray Crystallography
Deisenhofer, Huber, & Michel
First crystal structure an integral membrane protein
1988
Crystal structures of the ribosome
200?

12. Fab DNA-1 Recombinant anti-ss DNA Fab
Selected from a bacteriophage display library generated from an autoimmune MRL/lpr mouse (Deutscher group).
Prefers oligodeoxythymidine, dTN, N = 3-15
Binding is enthalpy driven, which differs from that of anti-dsDNA Fabs
Mutation studies demonstrate enthalpy-entropy compensation

13. Schematic diagram of immunoglobulin G, IgG

14. MU Research Board NSLS at Brookhaven NL
Season Prewitt Jon Schuermann Prof. Susan Deutscher Dr. Andrey Komissarov
MU Research Board NSLS at Brookhaven NL

15. X-ray Crystallography
crystals
diffraction
phases

16. Purification of DNA-1 with 6xHis tag at the C-terminus of the heavy chain
sonication
Ni-NTA
cation exchange 1
cation exchange 2

17. Crystals of a DNA-1/dT5 complex (scale = 0.1 mm)
2 M AS
pH 5
His tag
2 M AS
pH 5
His tag
2 M AS
2 % PEG
pH 7.5
His tag
25 % PEG
0.2 M AS
pH 5
His tag
PEG
2-propanol
pH 6
No His tag

18. DNA-1/dT5 complex, Form 1 6xHis tagged Fab 1.8 M Ammonium sulfate
0.1 M Na acetate, pH 5
space group P6(5)22
a = b = 172 Å, c = 145 Å
2 Fab per asymmetric unit
cryoprotect in 30 % glycerol
2.5 Å resolution at home
2.1 Å resolution at synchrotron

19. X-ray Crystallography
crystals
diffraction
phases

20. National Synchrotron Light Source Brookhaven National Lab

22. Beamline X8C, Sept. 1999

23. 1.65 Å Data for GAPDH Collected at X8C
> 2.3 million observations

24. Coming in
A Division of E.O. Lawrence Berkeley National Lab

25. Superbend
beamline
4.2.2 will be
operational
in 2002

26. Data Collection Statistics
Software HKL
Resolution Å
Outer shell Å
No. observations 489,562
Unique reflections 72,520
Average multiplicity 6.8
Completeness 99% (96%)
Mean I /sI (3.6)
Refl. with I /sI > % (62 %)
R-merge (I) (0.294)

27. X-ray Crystallography
crystals
diffraction
phases

28. Refinement Statistics
Software CNS and O
Resolution Å
Outer shell Å
F/s cutoff none
R-cryst (0.280)
R-free - 10 % set (0.306)
No. protein atoms 6503
No. DNA atoms 82
No. water molecules 341
coordinate error - sA Å
RMSD bonds Å
RMSD bond angles 1.4 °

29. Schematic diagram of immunoglobulin G, IgG

30. Immunoglobulin Fold

31. Immunoglobulin Fold

32. variable
domain 6 3 4 5 7 2 1
constant
domain

33. Antibody-DNA Interactions

34. Two Fab molecules bind the same DNA fragment

35. M1
Fab 1 T3 T2
Fab 2 T1

36. Tyr L32
His A91 T1
Tyr H100

37. Tyr L32
His
A91 T1
Tyr
H100

38. Asn L50
Tyr L49 T2
Tyr H100A

39. Asn L50
Tyr L49 T2
Tyr H100A

40. Conformational Changes Induced by DNA Binding

41. DNA binding causes an elbow angle decrease 14°

42. 6 Å movement of HCDR3

43. 99
100A
100
100
100A 99
Salt
link
Salt
link
HCDR3 Without DNA
HCDR3 With DNA bound

48. T3 T2

50. Summary of DNA-1/dT5 Structure
ssDNA binds in narrow cleft
Base recognition dominates protein-DNA interface
Tyr-thymine stacking is a major theme
Interesting Glu-phosphate interaction
No protein-DNA ion pairs observed
Crystal structure of protein-DNA nanotube suggests strategies for designing nanomaterials
Possible induced fit mechanism mediated by L3 and H3 involves backbone and side chain motion, disorder to order transition

52. Ongoing and Future Studies
Crystal structures of uncomplexed Fab
Other DNA-1/dTN structures, N=1-5
Structure of mutants with and without DNA
ab initio electronic structure calculations using models of Tyrosine-Thymine-Tyrosine sandwich
Molecular dynamics simulations
Engineer anti-dsDNA Fab via random mutagenesis phage display approach directed at HCDR3
Inhibitor design

57. Working model Induced fit mechanism H3 & L3 are flexible
Other CDRs static
H3 & L3 direct ligand to various regions of the combining site

59. Protein-DNA Recognition
Protein-dsDNA recognition well documented (polymerases, nucleases, transcription factors…).
Fewer structures of protein-ssDNA complexes (rep protein A, rep helicase, telomere end binding protein...).
Different recognition motifs for dsDNA and ssDNA?
Do Abs possess unique DNA recognition motifs?

60. A Protein-DNA Nanotube

61. Adjacent nanotubes are connected through b-strand 7 of CH1 domains
Fab LH
Fab AB

62. Interactions between NCS-related CH1 domains
Lys H215
Val H213
Thr H211
Thr B211
Val B213
Lys B215
A possible design strategy for constructing nanomaterials?

63. Protein-DNA interactions stabilize the nanotube in the direction parallel to the 6(5) axis
Fab LH
Fab AB