Abstract
Phage display technology has been exploited to study in detail the interaction between plasminogen activator inhibitor 1 (PAI-1) and either thrombin or an essential positively charged "loop" of tissue-type plasminogen activator (t-PA), denoted variable region 1 (VR1). For this purpose, a PAI-1 mutant phage library was used that served as a reservoir of PAI-1 proteins potentially deficient in the interaction with either VR1 or thrombin. A stringent two-step selection procedure was developed. (i) A negative selection was performed by incubating the pComb3/PAI-1 mutant library with an excess of a thrombin mutant with its VR1 domain substituted with that of t-PA (thrombin-VR1). (ii) The remaining phages were complexed with t-PA (positive selection) and selected by panning with an immobilized anti-t-PA monoclonal antibody. Four consecutive panning rounds yielded an enrichment of pComb3/PAI-1 mutant phages of approximately 50-fold. Sequence analysis of 16 different cDNAs, encoding PAI-1 mutants that are hampered in the binding to thrombin-VR1, revealed the following mutations. Four independent variants share a mutation of the P4' residue (Glu350 --> Lys). Nine independent PAI-1 variants share a substitution of P1' (Met347 --> Lys), whereas three others share a P2 substitution (Ala345 --> Asp). Kinetic analysis of representative PAI-1 mutants provides evidence that the P4' residue is essential for the interaction with the VR1 domain, consistent with the data of Madison et al. (Madison, E.L., Goldsmith, E.J., Gething, M.J., Sambrook, J.F., and Gerard, R.D. (1990) J. Biol. Chem. 265, 21423-21426), whereas the P1' and P2 residues confer thrombin specificity. Concordant with the design of the selection procedure, mutants were obtained that inhibit thrombin-VR1 at least 100-fold slower than wild-type PAI-1, identifying residues that are central to the interaction with either thrombin or VR1. This study demonstrates that phage technology can be used to analyze large numbers of mutants defective in their interaction with other (domains of) proteins, provided an adequate selection scheme is devised.
Highlights
Plasminogen activator inhibitor 1 (PAI-1)1 is the main inhibitor of the serine proteases tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator and a major physiological regulator of the fibrinolytic system
A number of studies have delineated regions or amino acids of PAI-1 that are involved in the interaction with t-PA: first, the reactive-site P1 residue (Arg346) of PAI-1 that interacts with the catalytic center of t-PA; second, the region between amino acids 110 and 145 of PAI-1 that binds to an unknown domain on t-PA [2]; and third, presumably negatively charged residues on PAI-1 that are involved in an interaction with the positively charged variable region 1 (VR1) on the protease domain of t-PA [3,4,5]
It is assumed that PAI-1 mutants with an altered VR1-binding site will be virtually unable to inhibit thrombinVR1, whereas such mutants would still be capable of inhibiting t-PA, albeit at a diminished rate
Summary
Materials—The construction of the phage-displayed pComb3/PAI-1 mutant library (containing 1.7 ϫ 106 independent colonies) has been previously described [14]. Elution of PAI-1 phages, and, amplification of phages and the determination of the number of colony-forming units were done as described [14] This selection procedure was repeated three times. An increasing amount of freshly grown PAI-1 phages (1013 colony-forming units/ml) were incubated for 90 min at 37 °C with either 0.5 nM t-PA or thrombin-VR1 in 25 l of HBS buffer (50 mM HEPES (pH 7.4), 100 mM NaCl, 1 mM EDTA, and 0.1% (w/v) polyethylene glycol 6000). Inhibition of t-PA or Thrombin-VR1 by Purified Individual PAI-1 Mutants Obtained after Four Rounds of Selection—Wells of microtiter plates (Nunc Maxisorp, Gibco BRL) were pretreated for 1 h at 37 °C with HO buffer (50 mM HEPES (pH 7.4), 100 mM NaCl, 0.1% (v/v) Tween 80, and 0.1% (w/v) ovalbumin) and washed with distilled water.
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