Table 1 Mutational analysis of MDH-Pex5_eTPR interface

Pex5_eTPR	MDH	K_d [nM]	ΔH [kJ/mol]	−TΔS [kJ/mol]	ΔG [kJ/mol]	N^a	Statistical significance (p value)
Wild type	Wild type	435 ± 80	−26.5 ± 0.8	−9.8 ± 3.5	−36.3 ± 0.2	0.398 ± 0.007	-
Wild type	GSGS^b	148 ± 26	−24.0 ± 0.4	−15.0 ± 1.8	−39.0 ± 0.3	0.543 ± 0.005	<0.001
	Δ(62–70)^c,d	1001 ± 121	−25.4 ± 0.7	−8.8 ± 1.8	−34.3 ± 0.2	0.500 ± 0.009	<0.001
	PTS1^e	135 ± 19	−21.0 ± 0.2	−18.2 ± 0.7	−39.2 ± 0.3	0.933 ± 0.006	<0.001
R625A, D629A^f	Wild type	453 ± 64	−22.5 ± 0.5	−13.7 ± 1.0	−36.2 ± 0.2	0.500 ± 0.006	0.5
(Δ470–480)		102 ± 19	−28.8 ± 0.5	−11.1 ± 2.2	−40.0 ± 0.5	0.452 ± 0.004	<0.001
P490R		1389 ± 226	−26.9 ± 0.9	−6.0 ± 2.3	−32.9 ± 0.5	0.292 ± 0.006	<0.001
(Δ470–480)	PTS1^e	420 ± 93	−26.4 ± 0.6	−10.0 ± 2.8	−36.4 ± 0.3	1.040 ± 0.015	<0.001

Interactions of indicated mutants were characterized by Isothermal Titration Calorimetry (ITC). Welch’s t-test (two-tailed) p value is reported for comparing K_d values. Variants are compared to wild type; (Δ470–480)/PTS1 is compared to WT/PTS1.
^aN value is derived from data fitting and is related to binding stoichiometry with following formula: N = St*(AF_cell/AF_syr), where St—stoichiometry, AF_cell—active fraction of protein in the cell (MDH), AF_syr—active fraction of protein in the syringe (Pex5_eTPR), ^bdisrupted sites 1 and 2, ^cdisrupted site 1, ^d62-KLPPVPRDP-70 loop was substituted with GS linker, ^ePTS1 peptide derived from MDH (NH2-ARSKL-COOH), ^fdisrupted site 2.

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