Case-study m.10191T>C

Leigh Disease is a very rare and severe mitochondrial disorder that arises in the first months of life; it has been associated with at least 75 nuclear and mitochondrially encoded genes with functional implications in mitochondrial biogenesis and metabolism (1). The disease is characterized by neurological regression with specific clinical lesions at basal ganglia and brainstem levels.

A mutation in the ND3 gene, m.10191T>C, has been associated with severe Complex I deficiency and neurological degeneration in adult patients (2) and children with Leigh disease (3,4). The mutation encodes for a Serine-Proline substitution in position 45. This missense variant is classified as Confirmed pathogenic mutation in MITOMAP, with Leigh Disease and Leigh Disease-like associated phenotypes.

We asked ourselves if theoretical compensatory variants exist for such mutation, within the same protein or in the interaction interface with another mitochondrial protein.

As a first step, we obtained 87 possibly inter-protein co-evolving pairs involving the site 45 of ND3. We considered only the pairs mapping on the physical interface between ND3 and ND1 and retrieved three PDB structures that resolved their interaction interface. Then, we calculated the ∆∆G of binding energy through the FoldX suite. ∆∆GA is the binding energy variation of the S45P mutant concerning the wild type ND3 protein; ∆∆GB is the partner mutant's binding energy variation for the wild type ND1 protein; ∆∆GAB is the binding energy variation when both proteins are mutated at their interaction interface.
Note that ∆∆GA is below the conventional threshold of ±0.61 Kcal/mol that identifies the disruptive variants for two PDB complexes, 5lc5 and 5ldx. We evidenced that three amino acid changes in the ND1 position 126 can restore the binding energy to, approximately, wild type levels (row in bold characters in the table below). Indeed, ∆∆GAB is proximal to 0, indicating that the interface with two co-occurring variations has similar binding energy to the wild-type protein complex.


Interactor

PDB ID

Chain 

(A protein)

Chain

(B protein)

Object variant

(A)

Partner variant (B)

∆∆G AB

∆∆G A

∆∆G B

MT-ND1

5lc5

A

H

S45P

A64P

2.340

-1.091

3.391

MT-ND1

5lc5

A

H

S45P

A64D

-1.071

-1.091

0.005

MT-ND1

5lc5

A

H

S45P

A64G

-1.410

-1.091

-0.339

MT-ND1

5lc5

A

H

S45P

A64V

-0.870

-1.091

0.204

MT-ND1

5lc5

A

H

S45P

A64S

-1.098

-1.091

-0.015

MT-ND1

5lc5

A

H

S45P

A64T

-0.923

-1.091

0.207

MT-ND1

5lc5

A

H

S45P

K62Q

-1.358

-1.091

-0.241

MT-ND1

5lc5

A

H

S45P

K62T

-0.341

-1.091

0.832

MT-ND1

5lc5

A

H

S45P

K62M

-1.309

-1.091

-0.217

MT-ND1

5lc5

A

H

S45P

K62E

-0.630

-1.091

0.412

MT-ND1

5lc5

A

H

S45P

K62N

-0.312

-1.091

0.846

MT-ND1

5lc5

A

H

S45P

N126K

-0.063

-1.091

0.553

MT-ND1

5lc5

A

H

S45P

N126D

1.607

-1.091

1.595

MT-ND1

5lc5

A

H

S45P

N126I

-1.125

-1.091

0.053

MT-ND1

5lc5

A

H

S45P

N126Y

0.031

-1.091

0.641

MT-ND1

5lc5

A

H

S45P

N126T

0.129

-1.091

0.124

MT-ND1

5lc5

A

H

S45P

N126S

0.097

-1.091

0.245

MT-ND1

5lc5

A

H

S45P

N126H

0.487

-1.091

0.694

MT-ND1

5ldw

A

H

S45P

A64P

1.517

-0.288

1.892

MT-ND1

5ldw

A

H

S45P

A64D

-0.286

-0.288

0.016

MT-ND1

5ldw

A

H

S45P

A64G

-0.586

-0.288

-0.290

MT-ND1

5ldw

A

H

S45P

A64V

-0.201

-0.288

0.074

MT-ND1

5ldw

A

H

S45P

A64S

-0.314

-0.288

-0.001

MT-ND1

5ldw

A

H

S45P

A64T

-0.184

-0.288

0.101

MT-ND1

5ldw

A

H

S45P

K62Q

-0.302

-0.288

-0.029

MT-ND1

5ldw

A

H

S45P

K62T

-0.157

-0.288

0.183

MT-ND1

5ldw

A

H

S45P

K62M

-0.296

-0.288

-0.012

MT-ND1

5ldw

A

H

S45P

K62E

0.035

-0.288

0.301

MT-ND1

5ldw

A

H

S45P

K62N

0.175

-0.288

0.465

MT-ND1

5ldw

A

H

S45P

N126K

-0.131

-0.288

-0.182

MT-ND1

5ldw

A

H

S45P

N126D

0.960

-0.288

0.906

MT-ND1

5ldw

A

H

S45P

N126I

-0.791

-0.288

0.457

MT-ND1

5ldw

A

H

S45P

N126Y

-0.186

-0.288

0.152

MT-ND1

5ldw

A

H

S45P

N126T

-0.684

-0.288

-0.612

MT-ND1

5ldw

A

H

S45P

N126S

-0.479

-0.288

-0.494

MT-ND1

5ldw

A

H

S45P

N126H

0.112

-0.288

0.233

MT-ND1

5ldx

A

H

S45P

A64P

4.036

0.854

3.812

MT-ND1

5ldx

A

H

S45P

A64D

0.685

0.854

-0.011

MT-ND1

5ldx

A

H

S45P

A64G

0.442

0.854

-0.325

MT-ND1

5ldx

A

H

S45P

A64V

0.603

0.854

-0.005

MT-ND1

5ldx

A

H

S45P

A64S

0.748

0.854

-0.004

MT-ND1

5ldx

A

H

S45P

A64T

0.884

0.854

0.107

MT-ND1

5ldx

A

H

S45P

K62Q

0.700

0.854

0.015

MT-ND1

5ldx

A

H

S45P

K62T

0.634

0.854

0.109

MT-ND1

5ldx

A

H

S45P

K62M

0.680

0.854

0.050

MT-ND1

5ldx

A

H

S45P

K62E

1.136

0.854

0.575

MT-ND1

5ldx

A

H

S45P

K62N

1.255

0.854

0.452

MT-ND1

5ldx

A

H

S45P

N126K

0.519

0.854

0.152

MT-ND1

5ldx

A

H

S45P

N126D

1.809

0.854

1.314

MT-ND1

5ldx

A

H

S45P

N126I

0.203

0.854

0.753

MT-ND1

5ldx

A

H

S45P

N126Y

0.866

0.854

0.512

MT-ND1

5ldx

A

H

S45P

N126T

0.133

0.854

-0.200

MT-ND1

5ldx

A

H

S45P

N126S

0.433

0.854

-0.153

MT-ND1

5ldx

A

H

S45P

N126H

1.079

0.854

0.557


The effect of ND3-ND1 interactions can also be observed in the Molecular Dynamics section of the MitImpact website. MD16, MD29, and MD31 collect results obtained by MD simulations of the amino acid pairs involving the positions 45 and 126 in ND3 and ND1, respectively.

Some interesting results about the S45P-N126S variant pairs regard:

  • The RMSD plot that shows how the S45P mutant subunit remains stably compact over the simulation time (i.e., lower RMSD) with respect to the wild-type protein complex (in red);
  • The RMSD of the double mutant that neatly overlaps that of the wild-type complex;
  • The RMSF plot, where we observe high residue fluctuations from the positions 20 to 50 in the ND3 protein (chain B). The S45P mutation causes lower mobility of that region, while the occurrence of both mutations on the proteins interface partially restores the wild-type condition.

Hands-on tutorial

From within the MitImpact home page, select Search by Gene or Protein position(1) and click on the Gene symbol(2) button. Select MT-ND3(3) from the drop-down menu, then type 45(4) as position and click Submit(5).

search form

The rsults page shows, in a multi-tab menu, all possible genomic variants that may occur in position 45 of the protein ND2. Select the variant 10191 (T>C)(6).

select the variant of interest

Data in the results page will be updated for the selected variant, which willbe shown to encode for the S45P amino acid change. Looking at the pathogenicity predictors (7), we can visualize pre-computed results from many computational algorithms. Clicking on the ? symbol (8), we obtain details for score and score interpretation.
Note that score thresholds are not dogmatic, i.e, they do not definitely separate neutral from pathogenic variants. Given the heterogeneity of predictions, we suggest to consider recent meta-predictors (i.e., APOGEE, Deogen2, MtoolBox, and Meta-SNP) for the classification of target variants.


pathogenicity predictors

MitImpact 3 moves from single variant prediction to inferred binary interactions, as collected in the Residue interaction(9) section. There, you can visualize the ∆∆G for single and wild-type protein mutants through the drop-down menus labeled ∆∆G intra, ∆∆G intra interface, and ∆∆G inter sections. The first variant pair in the ∆∆G inter menu is ND3 S45P vs. ND1 A64P (10), which was studied in the 5lc5 PDB structure.
∆∆G 1st and ∆∆G 2nd are both > |0.61| (11), then significantly altering the binding energy of the complex. ∆∆G both for the double mutant is 2.34 Kcal/mol(11), very far from 0, meaning that native ∆∆G is not restored by the co-occurrence of the two mutations (S45P and A64P).


DDG energy

Selecting the partner N126S (12), instead, we can see that the S45P variant (∆∆G 1st) considerably alters the binding energy (-1.09 Kcal/mol)(13), while N126S on the ND1 subunit has a lower effect (∆∆G 2nd = 0.25)(13). The global effect of the two mutations in the ND3-ND1 interface is ∆∆G both = 0.1(13).
We recall that if -0.1 ≤ ∆∆Gboth ≤ 0.1, then the two variants can be considered structurally compensated.


DDG energy

The potential compensatory effects on interesting missense variants can thus be retrieved by exploring the Residue interaction section. The users can download the flat file version of out MitImpact 3 database and evaluate the fields ΔΔG intraP, ΔΔG intraP interface, ΔΔG interP.
Further eetails on can be found in the Output legend page.

Refrences

  1. Gorman, G.S., Chinnery, P.F., DiMauro, S., Hirano, M., Koga, Y., McFarland, R., Suomalainen, A., Thorburn, D.R., Zeviani, M. and Turnbull, D.M. (2016) Mitochondrial diseases. Nature reviews. Disease primers, 2, 16080.
  2. Taylor, R.W., Singh-Kler, R., Hayes, C.M., Smith, P.E. and Turnbull, D.M. (2001) Progressive mitochondrial disease resulting from a novel missense mutation in the mitochondrial DNA ND3 gene. Annals of neurology, 50, 104-107.
  3. McFarland, R., Kirby, D.M., Fowler, K.J., Ohtake, A., Ryan, M.T., Amor, D.J., Fletcher, J.M., Dixon, J.W., Collins, F.A., Turnbull, D.M. et al. (2004) De novo mutations in the mitochondrial ND3 gene as a cause of infantile mitochondrial encephalopathy and complex I deficiency. Annals of neurology, 55, 58-64.
  4. Bugiani, M., Invernizzi, F., Alberio, S., Briem, E., Lamantea, E., Carrara, F., Moroni, I., Farina, L., Spada, M., Donati, M.A. et al. (2004) Clinical and molecular findings in children with complex I deficiency. Biochimica et biophysica acta, 1659, 136-147.