In silico and in vitro investigations on the protein–protein interactions of glutathione S-transferases with mitogen-activated protein kinase 8 and apoptosis signal-regulating kinase 1

Figures & data

Table 1. Details of the seven cytosolic GST isoforms.

Protein family	Protein	UniProt accession ID	UniProt sequence status
GSTs	GST-A1	P08263	Reviewed
	GST-A2	P09210	Reviewed
	GST-M1	P09488	Reviewed
	GST-M2	P28161	Reviewed
	GST-M5	P46439	Reviewed
	GST-P1	P09211	Reviewed
	GST-T1	P30711	Reviewed
MAPKs	MAPK1	P28482	Reviewed
	MAPK3	P27361	Reviewed
	MAPK4	P31151	Reviewed
	MAPK6	Q16659	Reviewed
	MAPK7	Q13164	Reviewed
	MAPK8	P45983	Reviewed
	MAPK9	P45984	Reviewed
	MAPK10	P53779	Reviewed
	MAPK11	Q15759	Reviewed
	MAPK12	P53778	Reviewed
	MAPK13	O15264	Reviewed
	MAPK14	Q16539	Reviewed
	MAPK15	Q8TD08	Reviewed
	NLK	Q9UBE8	Reviewed
	MAP3K5	Q99683	Reviewed

Figure 1. Study’s schematic workflow. Tool and database explanations and abbreviations are given in the main text.

Figure 2. Phylogenetic analysis of seven human glutathione S-transferase isoforms using four different methods: (a) Unweighted pair-group mean average (UPGMA), (b) Neighbour-joining (NJ), (c) Minimum evolution (ME) and (d) Maximum parsimony (MP). Each isoform’s UniProt accession number is also shown.

Figure 3. MD simulation results for GST-M1 with MAPK8: (a) Root Mean Square Deviation (RMSD), (b) Radius of Gyration (RoG), (c) Potential Energy and (d) Principal Component Analysis (PCA). (X-axis: time in graphs (a), (b) and (c). Y-axis: values for (a) RMSD, (b) RoG and (c) Potential energy. For graph (d), X-axis: principal component 1 and Y-axis: principal component 2.

Table 2. Docking score, estimated free energy of binding (ΔG) and estimated binding affinity (Kd) of GSTs with the MAPK8 and ASK1 complexes, obtained from the HADDOCK and PRODIGY web servers.

Protein–Protein complex	HADDOCK score	Binding affinity
		ΔG (kcal/mol)	Kd (M)
Seven GST isoforms with MAPK8
GST-A1:MAPK8	–83.7 +/6.2	–10.1	7.3e^–08
GST-A2:MAPK8	–76.6 +/10.4	–13.3	4.4e^–10
GST-M1:MAPK8	–105.7 +/20.1	–14.1	1.2e^–10
GST-M2:MAPK8	–159.0 +/20.2	–15.7	7.9e^–12
GST-M5:MAPK8	–151.7 +/6.8	–16.8	1.4e^–12
GST-P1:MAPK8	–86.6 +/8.3	–13.3	4.5e^–10
GST-T1:MAPK8	–91.8 +/7.7	–13.5	3.3e^–10
Seven GST isoforms with ASK1
GST-A1:ASK1	−86.5 +/9.1	−17.4	5.0e^–13
GST-A2:ASK1	−93.3 +/18.8	−15.1	2.4e^–11
GST-M1:ASK1	−68.3 +/11.0	−14.9	3.2e^–11
GST-M2:ASK1	−92.9 +/7.1	−14.4	7.2e^–11
GST-M5:ASK1	−130.1 +/19.6	−15.4	1.4e^–11
GST-P1:ASK1	−66.3 +/9.7	−14.4	6.9e^–11
GST-T1:ASK1	−44.3 +/9.0	−11.7	5.5e^–09

Table 3. Analysis of the intermolecular interactions of the protein–protein complexes formed by the seven GST isoforms with MAPK8 protein and with ASK1.

Intermolecular interactions of GSTs with MAPK8
	Salt bridges	H bonds	Nonbonded contacts
GST-A1:MAPK8	–	10	181
GST-A2:MAPK8	4	18	243
GST-M1:MAPK8	7	18	220
GST-M2:MAPK8	6	20	274
GST-M5:MAPK8	4	12	247
GST-P1:MAPK8	3	15	181
GST-T1:MAPK8	8	19	223
Intermolecular interactions of GSTs with ASK1
GST-A1:ASK1	8	21	267
GST-A2:ASK1	5	15	262
GST-M1:ASK1	5	19	255
GST-M2:ASK1	3	17	168
GST-M5:ASK1	8	26	266
GST-P1:ASK1	2	21	275
GST-T1:ASK1	7	22	240

Table 4. Time-averaged structural properties obtained from the MD simulations of seven GST isoforms with MAPK8 and with ASK1 protein.

Protein–Protein complexes	Average RMSD (nm)	Average RMSF (nm)	Average Rg (nm)	Potential energy (kj/mol)	Essential dynamics (nm^2)
Seven GST isoforms with MAPK8
GST-A1:MAPK8	0.184	0.126	2.49	–1,651,292.5	199.05
GST-A2:MAPK8	0.228	0.119	2.69	–1,998,776.75	188.292
GST-M1:MAPK8	0.167	0.110	2.46	–1,262,919.75	169.265
GST-M2:MAPK8	0.228	0.125	2.51	–1,625,000.375	192.007
GST-M5:MAPK8	0.225	0.116	2.46	–1,367,582.375	174.945
GST-P1:MAPK8	0.156	0.121	2.53	–1,585,577.125	183.181
GST-T1:MAPK8	0.231	0.148	2.65	–1,901,880.75	270.803
Seven GST isoforms with ASK1
GST-A1:ASK1	0.22	0.135	2.65	–1,908,568.875	244.06
GST-A2:ASK1	0.27	0.143	2.63	–1,587,740	278.932
GST-M1:ASK1	0.27	0.153	2.74	–1,954,723.375	301.066
GST-M2:ASK1	0.3	0.181	2.93	–2,066,349.25	442.799
GST-M5:ASK1	0.22	0.151	2.70	–1,895,464.75	292.549
GST-P1:ASK1	0.25	0.138	2.67	–1,783,174.5	246.417
GST-T1:ASK1	0.22	0.132	2.79	–1,922,931.375	235.815

Figure 4. MD simulation results for GST-T1 with ASK1, (a) RMSD, (b) Radius of Gyration, (c) Potential Energy and (d) Principal Component Analysis (X-axis: time in (a), (b) and (c). Y-axis: values for graphs (a) RMSD, (b) RoG and (c) Potential energy. For graph (d), X-axis: principal component 1 and Y-axis: principal component 2.

Figure 5. Predicted protein–protein complex obtained from HADDOCK’s easy interface. (a) GST-M1–MAPK8 complex and (b) GST-T1–ASK1 complex. Hydrophobic sites of GSTs interact with MAPK8’s kinase domain and with ASK1.

Data availability statement

The raw data files and docking complexes, MD simulation files and corresponding meta-data are available at: https://zenodo.org with doi: 10.5281/zenodo.3921901.