Allosteric inactivation of a trypsin-like serine protease by an antibody binding to the 37- and 70-loops.
Serine protease catalytic activity is in many cases regulated by conformational changes initiated by binding of physiological modulators to exosites located distantly from the active site. Inhibitory monoclonal antibodies binding to such exosites are potential therapeutics and offer opportunities for elucidating fundamental allosteric mechanisms. The monoclonal antibody mU1 has previously been shown to be able to inhibit the function of murine urokinase-type plasminogen activator in vivo. We have now mapped the epitope of mU1 to the catalytic domain's 37- and 70-loops, situated about 20 Å from the S1 specificity pocket of the active site. Our data suggest that binding of mU1 destabilizes the catalytic domain and results in conformational transition into a state, in which the N-terminal amino group of Ile16 is less efficiently stabilizing the oxyanion hole and in which the active site has a reduced affinity for substrates and inhibitors. Furthermore, we found evidence for functional interactions between residues in uPA's C-terminal catalytic domain and its N-terminal A-chain, as deletion of the A-chain facilitates the mU1-induced conformational distortion. The inactive, distorted state is by several criteria similar to the E* conformation described for other serine proteases. Hence, agents targeting serine protease conformation through binding to exosites in the 37- and 70-loops represent a new class of potential therapeutics.
Danish-Chinese Centre for Proteases and Cancer and ‡Department of Molecular Biology and Genetics, Aarhus University , DK-8000 Aarhus C, Denmark.
Tobias
T
Kromann-Hansen
Ida K
IK
Lund
Zhuo
Z
Liu
Peter A
PA
Andreasen
Gunilla
G
Høyer-Hansen
Hans Peter
HP
Sørensen
D000494:Allosteric Regulation; D000818:Animals; D000911:Antibodies, Monoclonal; D020134:Catalytic Domain; D004789:Enzyme Activation / Q000187:drug effects; D000939:Epitopes / Q000378:metabolism; D005341:Fibrinolysin / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D011487:Protein Conformation / Q000187:drug effects; D012697:Serine Endopeptidases / Q000276:immunology*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology*; D020349:Surface Plasmon Resonance; D014568:Urokinase-Type Plasminogen Activator / Q000187:drug effects / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D015842:Serine Proteinase Inhibitors; C114880:trypsin-like serine protease; D012697:Serine Endopeptidases; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator