TABLE 3

Alanine scanning analysis of PAI-1 residues involved in binding of paionin-4

Initially, 45 PAI-1 mutants in the latent state were tested for paionin-4 binding by the use of phage ELISA. The level of phage binding was expressed relative to that of the wt. The following mutants did not differ measurably (i.e., less than 2-fold) from wt with respect to binding to paionin-4 in phage ELISA: K82E, M85A, E92A, S94A, T96A, F100A, Q102A, P113A, F116A, F119A, R120A, S121A, T122A, K124A, Q125A, V126A, K143A, T144A, H145A, T146A, K147A, Y172A, Y174A, W177F, K178A, E214A, Y222A, D224A, H231A, D233A, Y243A, E244A, K245A, K325A, N331A, S333A. The mutants that demonstrated decreased binding in phage ELISA were then characterized further by the use of surface plasmon resonance, using a BIACORE T100 instrument equipped with a CM5 chip coupled with paionin-4-D1D2, and, as a control for nonspecific binding, D1D2. The mutants were passed over the chip in concentrations of 1 μM. The levels of steady state binding were expressed relative to that of the wt. The table shows means and S.D. for at least three independent determinations. Also shown is the binding of PAI-1 wt to a chip with D1D2 (i.e., without paionin-4).

PAI-1 variant	Secondary Structural Element	Binding in Phage ELISA	Binding in BIACORE
		fraction of control
wt		1.00	1.00
Y81A	hD	0.14 ± 0.05^a	0.25 ± 0.03^a
P87A	hD-s2Aloop	0.23 ± 0.01^a	0.11 ± 0.01^a
W88A	hD-s2Aloop	0.18 ± 0.07^a	0.01 ± 0.02^a
K90A	s2A	0.36 ± 0.06^a	0.27 ± 0.00^a
D91A	s2A	0.17 ± 0.06^a	0.11 ± 0.01^a
E92A	s2A	0.17 ± 0.06^a	0.40 ± 0.14^a
Murine wt		N.D.	0.15 ± 0.01^a
Murine-hF-human chimera		N.D.	0.39 ± 0.06^a
Human-hF-murine chimera		N.D.	0.06 ± 0.00^a
wt, D1D2 chip			−0.01 ± 0.04^a

N.D., not determined, because the murine and murine-human chimeras react differently from the human protein with the antibodies employed in the ELISA.
↵ a Significantly different from wt binding to the paionin-4-D1D2 according to Student's t-test (P < 0.01).