Table 2.

Binding of Wild-Type and Mutant PDHK2 Proteins to the Unaltered L2 Domain

	GST-L2 pulldown	ITC with L2
PDHK2	PDHK2 binding (%)a	KD (μM)	ΔH (kcal/mol)
wild-type	100 ± 9b	8.3 ± 0.3c	−46 ± 1
K17A	110 ± 20	26 ± 2	−12 ± 1
P22A	45 ± 14	NMd	–
L23A	10 ± 6	NDe	–
F28A	10 ± 5	NDe	–
F31A	17 ± 6	NMd	–
F44A	7 ± 1	NMd	–
L45A	47 ± 11	NMd	–
Q47A	130 ± 30	3.0 ± 0.1	−34 ± 1
L160A	48 ± 4	NMd	–
I167A	110 ± 5	13 ± 1	−24 ± 1
F168A	65 ± 8	NMd	–
K368A	52 ± 14	64 ± 6f	−12 ± 1
R372A	9 ± 3	NMd	–
E389A	100 ± 7	7.8 ± 0.3	−24 ± 1
K391A	62 ± 3	78 ± 3f	−4.2 ± 0.1

^aAs discussed in Experimental Procedures, fractions containing free and GST-L2-bound PDHK2 variants were separated via SDS–PAGE. Gels were stained with Coomassie R250. Stained gels were analyzed by scanning densitometry. Scans were quantified using an UN-SCAN-IT automated digitizing system (Silk Scientific, Inc.). Data represent the ratio between GST-L2 protein and corresponding PDHK2 protein. The ratio of wild-type PDHK2 to GST-L2 was taken to be 100%.

^bResults are expressed as means ± the standard deviation of at least three experiments.

^cResults are expressed as means ± the standard deviation of three experiments conducted with different preparations of proteins. ITC measurements were performed at 30 °C in a VP-ITC microcalorimeter (MicroCal). Unaltered L2 domain (250 μM) in the syringe was injected into the reaction cell containing 10 μM wild-type or mutant PDHK2. Dissociation constants and enthalpy changes were obtained using Origin, version 7.0, supplied by manufacturer.

^dNot measurable due to the insufficient amount of differential heat produced in the binding reaction.

^eNot done due to the limited yields of PDHK2-L23A and PDHK2-F28A proteins.

^fTo achieve an accurate determination of binding parameters, measurements were conducted using 20 μM PDHK2-K368A or PDHK2-K391A and 400 μM L2 in the syringe.