Allosteric inactivation of a trypsin-like serine protease by an antibody binding to the 37- and 70-loops

TY - JOUR

T1 - Allosteric inactivation of a trypsin-like serine protease by an antibody binding to the 37- and 70-loops

AU - Kromann-Hansen, Tobias

AU - Lund, Ida K

AU - Liu, Zhuo

AU - Andreasen, Peter A

AU - Høyer-Hansen, Gunilla

AU - Sørensen, Hans Peter

PY - 2013/10/8

Y1 - 2013/10/8

N2 - 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.

AB - 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.

KW - Allosteric Regulation

KW - Animals

KW - Antibodies, Monoclonal

KW - Catalytic Domain

KW - Enzyme Activation

KW - Epitopes

KW - Fibrinolysin

KW - Kinetics

KW - Mice

KW - Models, Molecular

KW - Protein Conformation

KW - Serine Endopeptidases

KW - Serine Proteinase Inhibitors

KW - Surface Plasmon Resonance

KW - Urokinase-Type Plasminogen Activator

U2 - 10.1021/bi400491k

DO - 10.1021/bi400491k

M3 - Journal article

C2 - 24079451

SN - 0006-2960

VL - 52

SP - 7114

EP - 7126

JO - Biochemistry

JF - Biochemistry

IS - 40

ER -