I presented two fascinating stories where Molecular Dynamics simulations contributed to enhancing our understanding of immunodeficiencies. In one of the projects, the treatment of patients could be improved. These slides were presented at the Basler Modeller Stammtisch, 26.02.2021

1. Translational Applications
of Biomolecular Modelling
• Bignucolo Olivier, Jan 26. 2021

2. Translational Applications
of Biomolecular Modelling
• Bignucolo Olivier, Jan 26. 2021
Project 1- When binding is too weak
Project 2- When binding is too strong

3. Inborn errors of immunity
Affecting the development and/or
function of the immune system
Genetic Determined
Not age restricted
Predisposition
to Malignancies
Opportunists
Infection Susceptibility
Can be associated with extra-
immunological symptoms
Syndromic
Autoimmunity/Autoinflammation

4. When the binding is too weak
A. Jauch M. Recher

5. When the binding is too weak
A. Jauch M. Recher
 Young female and her father with life-threatening
autoimmune manifestations
Various immunological aberrant parameters
One was unexpected!

6. The patient –P1- and her father display
- increased phosphorylation of DNA damage associated proteins
γH2Ax
+
p53BP1
+
γH2Ax
+ p53BP1
+
0
2
4
6
8
10
(%
of
CD3
+
)
*
*
*
*
*
*
Mother
P1
Father
HD
HD ≡ healthy donors
A. Jauch et al. Paper in preparation

7. γH2Ax
+
p53BP1
+
γH2Ax
+ p53BP1
+
0
2
4
6
8
10
(%
of
CD3
+
)
*
*
*
*
*
*
Mother
P1
Father
HD
The patient and her father display
- increased phosphorylation of DNA damage associated proteins
- augmented susceptibility to DNA damage
HD ≡ healthy donors
A. Jauch et al. Paper in preparation

8. Whole exome sequencing
 Novel missense mutation in ligase 4  LIG4
Cartoon of Lig4

9. LIG4 final DNA nick-sealing step of classical
nonhomologous end-joining

10. LIG4 final DNA nick-sealing step of classical
nonhomologous end-joining

11. LIG4 final DNA nick-sealing step of classical
nonhomologous end-joining

12. Novel missense mutation: R580Q
Arg580 highly conserved
Conserved region
R580 highly conserved among vertebrae

13. Missense mutation: R580Q
Arg580 highly conserved
R580Q: reduced ligation capacity

14. Missense mutation: R580Q
Arg580 highly conserved
R580Q: reduced ligation capacity
Question:
Which functional step is hindered ?
• Nuclear localisation/recruitment
• DNA binding
• ATP/AMP binding
• Mg2+ binding
• Binding of interaction partner protein

15. Missense mutation: R580Q
Arg580 highly conserved
R580Q: reduced ligation capacity
Question:
Which functional step is hindered ?
• Nuclear localisation/recruitment
• DNA binding
• ATP/AMP binding
• Mg2+ binding
• Binding of interaction partner protein
These questions can be treated with Molecular Modelling
and further explained with Molecular Dynamics

16. Where is Arg580 in the sequence?
DNA binding
Domain
Nucleotidyltransferase Oligonucleotide/
oligosaccharide-fold
Binding Domain
BRCA1 C Termini
>616  IDPs
580
271, 278, 293
Mg2+
331, 427 432, 443, 449
ATP ATP

17. Where is Arg580 in the structure?
DNA binding
Domain
Nucleotidyltransferase Oligonucleotide/
oligosaccharide-fold
Binding Domain
BRCA1 C Termini
>616  IDPs
580
R580
271, 278, 293
Mg2+
331, 427 432, 443, 449
ATP ATP
Arg580 does not bind to any relevant partner
in the LIG4 open structure
OPEN
Model generated on the basis of the X-ray structure code 6BKF

18. Where is Arg580 in the structure?
DNA binding
Domain
Nucleotidyltransferase Oligonucleotide/
oligosaccharide-fold
Binding Domain
BRCA1 C Termini
>616  IDPs
580
R580
271, 278, 293
Mg2+
331, 427 432, 443, 449
ATP ATP
LIG4 closed structure
Arg580 binds to the AMP-carrying DNA strand
R580
OPEN
CLOSED
Models generated on the basis of the X-ray structures codes 6BKF & 6BKG

19. Arg580 binds to a DNA backbone
Hypothesis R580Q  Probably weaker binding
Questions:
1) However, this is not always the case
 Here three ARG/LYS candidates in the surrounding
2) Quantification of the energy
of binding WT versus R580Q
K560
K451
R577
These questions can be treated
with Molecular Dynamics
But what is Molecular Dynamics?
Let’s have a rapid overview in images

20. Arg580 binds to a DNA backbone
Hypothesis R580Q  Probably weaker binding
Questions:
1) However, this is not always the case
 Here three ARG/LYS candidates in the surrounding
2) Quantification of the energy
of binding WT versus R580Q
K560
K451
R577
These questions can be treated
with Molecular Dynamics
But what is Molecular Dynamics?
Let’s have a rapid overview in images

21. Construct the model of the protein in silico

22. Add the nucleic acids:
Two strands of nicked DNA + intact strand
Magnesium

23. K+ and Cl- at 150 mmol/l

24. Water is often the largest component in terms of particle numbers
Here ~200’000 atoms

25. Now: how do we get the dynamics of the system?
 Solve the Newton’s equations of motion
for each of the ~ 200’000 particles
Timescale:
~ 17 s  ~ 45 ns
protonation at ~ 8 s

26. Let’s imagine that we
can transpose two or
three of the 200’000
particles from a 3D-
space to a 2D-space
To better understand:

27. Let’s imagine that we
can transpose two or
three of the 200’000
particles from a 3D-
space to a 2D-space
To better understand:
Originally a funny movie (pool)
allowing me to explain the principles
of MD to non experts
Removed here for the sake of file size

28. Estimate the LIG4/DNA binding energy
1) Construct two systems of nicked DNA bound to LIG4: WT versus R580Q
2) Run MD simulations: 6*500 ms WT and 6*500 ms R580Q
3) Calculate the DNA-protein interaction energy for each set of data

29. 8
9
10
11
12
f
WT
R580Q
**
0 200 400 600
time (ns)
Binding
energy
(-10
4,
kJ/mol)
Estimate the LIG4/DNA binding energy
1) Construct two systems of nicked DNA bound to LIG4: WT versus R580Q
2) Run MD simulations: 6*500 ms WT and 6*500 ms R580Q
3) Calculate the DNA-protein interaction energy for each set of data
A. Jauch et al. manuscript in preparation

30. 8
9
10
11
12
f
WT
R580Q
**
0 200 400 600
time (ns)
Binding
energy
(-10
4,
kJ/mol)
Estimate the LIG4/DNA binding energy
1) Construct two systems of nicked DNA bound to LIG4: WT versus R580Q
2) Run MD simulations: 6*500 ms WT and 6*500 ms R580Q
3) Calculate the DNA-protein interaction energy for each set of data
A. Jauch et al. manuscript in preparation

31. R at position 580 interacts more often with the DNA Backbone
Q at position 580 reorients in an unfavorable position
WT R580Q
Red and brown spheres in the movie  DNA backbone atoms are highlighted if and only when
they are interacting with R580 or Q580
 The number of flashes corresponds to the frequency of interaction

32. The surrounding residues do not compensate for the R580Q loss of charge

33. LIG4 project:
Loss-of-function, decreased binding energy
Little effects on the surrounding residues
8
9
10
11
12
f
WT
R580Q
**
0 200 400 600
time (ns)
Binding
energy
(-10
4,
kJ/mol)

34. When the binding is too strong
A. Burgener et al. 2019 Nature Immunology
C. Hess
A. V. Burgener
 A cohort of patients
affected by Persistent Polyclonal
B cell Lymphocytosis

35. Primary Immunodeficiencies (PIDs)
Rare inherited or somatic defects of the immune system:
Increased susceptibility to infections
Autoimmune diseases
Hematological malignancies
Mahlaoui (2014) Rare Diseases and Orphan Drugs
Prospective study of
PAD patients

36. Oxygen consumption rate in B cells:
HC < PAD < PPBL
A. Burgener et al. 2019 Nature Immunology
ECAR: extracellular acidification rate  Glycolysis
OCR:  oxidative phosphorylation
Figure: adapted from A. Jauch
Pyruvate
Persistent Polyclonal B cell Lymphocytosis
Primary Antibody Disorder
Healthy controls

37. A. Burgener et al. 2019 Nature Immunology
ECAR: extracellular acidification rate  Glycolysis
OCR:  oxidative phosphorylation
Figure: adapted from A. Jauch
Pyruvate
Oxygen consumption rate in B cells:
HC < PAD < PPBL
Identical Glycolysis

38. Higher-order organization of the mitochondrial
electron transport chain
J. Letts & L. Sazanov (2017) nature structural and molecular biology

39. Complex II is hyper-functional in PPBL
A. Burgener et al.

40. Accumulation of fumarate, the direct product of complex II
Fumarate/Succinate:
Fold change difference ~ 6

41. Fumarate is the direct product of complex II
A. Burgener et al. (2019) Nature Immunology

42. Structure of Complex II: SDHA, SDHB, SDHC & SDHD
A. Geleta et al. (2017) trends in biomedical sciences
SDHA
SDHB
SDHC
SDHD
e-

43. Whole genome sequencing  mutation in SDHA
A. Geleta et al. (2017) trends in biomedical sciences
SDHA
SDHB
SDHC
SDHD
Structural investigations
AIM: explain the complex II gain-of-function
on the basis of the A45T mutation in SDHA

44. SDHA
SDHB
C-ter
N-ter
Ala45
SDHA 1-42 = transit peptide
 A45 is the third residue of the mature protein
Molecular representation of the SDHA/SDHB complex
Location of the A45T mutation at the short N-ter loop of SDHA
Methods
- homology modelling SDHA & SDHB
- add missing loops
- add FAD in SDHB
- ReMDs of WT and p.A45T (~750 ns each)

45. Working hypothesis:
Mutation A45T
 Stronger SDHA-SDHB interactions
 facilitated e- transport
 increased succinate oxidation rate
 complex II gain-of-function phenotype
Strategy:
Compare the interactions between the N-ter loop of SDHA and SDHB
in WT versus A45T

46. Working hypothesis:
Mutation A45T
 Stronger SDHA-SDHB interactions
 facilitated e- transport
 increased succinate oxidation rate
 complex II gain-of-function phenotype
Strategy:
Compare the interactions between the N-ter loop of SDHA and SDHB
in WT versus A45T

47. Strategy:
Compare in silico the interactions between the N-ter loop of SDHA and SDHB
in WT versus A45T
Testing the working hypothesis:
Mutation A45T
 Stronger SDHA-SDHB interactions

48. SDHAA45T
 Enhanced SDHA-SDHB H-bond interactions
A45T
WT
A. Burgener et al.

49. SDHAA45T
 Enhanced SDHA-SDHB H-bond interactions
A45T
WT
A. Burgener et al.

50. Snapshot from a A45T trajectory: hydrophilic interactions
Interacting residues shown as sticks
A. Burgener et al.
Lys46
Ser44
Ala43
Lys23
Asp22
Asp20
SDHA
SDHB
Asp20 and Asp22 of SDHB: highly conserved

51. WT versus A45T trajectory
Interacting residues between SDHA N-ter and SDHB are shown as spheres
Video in A. Burgener et al.

52. A. Burgener et al.
Biochemical verification
Spectrophotometrically measurement of SDHA-SDHB activity
in B-cell lines carrying the A45T mutation confirms the prediction

53. Mechanism
What may enhance the interactions between the SDHA N-ter and SDHB?
Working hypothesis:
A45T mutation:
hydrophobic Ala  hydrophilic Thr
 Thr45 would engage in electrostatic interactions with
the conserved Asp20 and Asp22 of SDHB

54. Testing the hypothesis:
Count the contribution of each individual residue to the interaction
43 44 45 46 20 21 22 23 24 26 29 31 58 108
SDHA
Residue number
43 44 45 46 47 48
wt A S A K V S
Mut A S T K V S
Residue number
20 21 22 23 24 25 58 109
D P D K A G T K
SDHB

55. Negligible contribution of Thr45 to the interaction:
Working hypothesis was naïve, and is rejected
43 44 45 46 20 21 22 23 24 26 29 31 58 108
SDHA
Residue 45 does not contribute at all to the interactions !!
Residue number
43 44 45 46 47 48
wt A S A K V S
Mut A S T K V S
Residue number
20 21 22 23 24 25 58 109
D P D K A G T K
SDHB

56. New hypothesis:
A45T stabilizes the N-ter loop of SDHA in a favourable position for interacting
with SDHB
N-ter ”free”:
no interaction
with SDHB
N-ter “fastened along the core protein”:
 interaction with SDHB facilitated

57. Hypothetical mechanism:
Thr45, but not Ala45, would form tight interactions with adjacent residues of SDHA
 the short N-ter maintained in a position favourable for interacting with SDHB
SDHA
SDHB
Thr45
Arg458
Asp22
Ala43
H-bonds: Thr45 --- Arg458
Ala43 --- Asp22

58. Compare the fraction of interaction time between
residue 45 (A/T) and adjacent SDHA residues
WT: Ala45 interacts with
adjacent Arg458 during ~ 20 %
of the total SDHA Nter-SDHB
interaction time

59. Compare the fraction of interaction time between
residue 45 (A/T) and adjacent SDHA residues
WT: Ala45 interacts with
adjacent Arg458 during ~ 20 %
of the total SDHA Nter-SDHB
interaction time
A45T: Thr45 interacts with
Arg458 during ~ 65% of the
total SDHA Nter-SDHB
interaction time
Thr45
Arg458

60. Compare the fraction of interaction time between
residue 45 (A/T) and adjacent SDHA residues
A. Burgener et al.

61. Conclusions
LIG4: when the binding is too weak
- Experimental investigations  mutation  decreased ligation capability
- Molecular dynamics  describes mechanism at the molecular interface
- The first LIG4 mutation involving DNA binding
SDHA: when the binding is too strong
- MD predicts increased SDHA/SDHB interaction
- Experiment confirms prediction
- MD provides an amazing mechanism augmenting the binding stability

62. Conclusions
LIG4: when the binding is too weak
- Experimental investigations  mutation  decreased ligation capability
- Molecular dynamics  describes mechanism at the molecular interface
- The first LIG4 mutation involving DNA binding
SDHA: when the binding is too strong
- MD predicts increased SDHA/SDHB interaction
- Experiment confirms prediction
- MD provides an amazing mechanism augmenting the binding stability

63. Conclusions
LIG4: when the binding is too weak
- Experimental investigations  mutation  decreased ligation capability
- Molecular dynamics  describes mechanism at the molecular interface
SDHA: when the binding is too strong
- MD predicts increased SDHA/SDHB interaction
- Experiment confirms prediction
- MD provides an amazing mechanism augmenting the binding stability

64. Conclusions
LIG4: when the binding is too weak
- Experimental investigations  mutation  decreased ligation capability
- Molecular dynamics  describes mechanism at the molecular interface
SDHA: when the binding is too strong
- MD predicts increased SDHA/SDHB interaction
- Experiment confirms prediction
- MD provides an amazing mechanism of the augmented binding strength

65. Thank you for your time!
Immunobiology
Christoph Hess
and Team
Immunodeficiency
Mike Recher
and Team
Annaïse Jauch
Friends
Basel Modeller Stammtisch
Christian Kramer
And participants