A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior

Tobias Kromann-Hansen,Emil Oldenburg,Kristen Wing Yu Yung,Gholamreza H Ghassabeh,Serge Muyldermans,Paul J Declerck,Mingdong Huang,Peter A Andreasen,Jacky Chi Ki Ngo

doi:10.1074/jbc.m116.732503

Abstract

A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30-40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors.

Highlights

Serine proteases catalyze the hydrolysis of peptide bonds and are involved in numerous physiological processes, including digestion, blood clotting, fibrinolysis, complement activation, and turnover of the extracellular matrix [1]
We describe a new type of serine protease inhibitor by developing a single-domain Camelid-derived antibody fragment, a so-called nanobody, which targets the active site of the trypsin-like serine protease urokinase-type plasminogen activator
Screening the library with the active two-chain urokinase-type plasminogen activator (uPA) as bait, we identified a nanobody (Nb4) that potently inhibited the proteolytic activity of uPA toward the macromolecular substrate plasminogen as well as uPA’s amidolytic activity toward the small peptidic chromogenic substrate pyro-Glu-Gly-Arg-p-nitroanilide (CS61(44)) (Fig. 1, a and b)

Summary

Results

Inhibition of Human uPA by an Anti-uPA Nanobody—A nanobody library was generated by immunizing an Alpaca (Vicugna pacos) with zymogen single-chain uPA. UPA-Nb4 Complex Crystal Structure Reveals That Nb4 Binds to uPA in a Substrate-like Manner—The x-ray crystal structure of the catalytic domain of human uPA in complex with Nb4 was determined to a resolution of 1.97 Å (Rfree ϭ 0.207 and Rwork ϭ 0.177) with two complexes in the asymmetric unit (Table 1). The structure of the complex revealed that Nb4 inserts the CDR-H3 into the active site region of uPA utilizing the S4-S3Ј substrate-binding pockets (Fig. 3a). Arg-111 does not interact with uPA extensively, our crystal structure suggests that it may exert its effect on the inhibitory activity of Nb4 by stabilizing the conformation of the CDR-H3 loop through a salt bridge with Asp-99 and cation-␲ interaction with Tyr-113 (Fig. 4a). 1.80 Ϯ 0.15 2.1 Ϯ 0.1 Ͼ4000 61 Ϯ 4.0 3.6 Ϯ 0.1 11 Ϯ 1.0 Ͼ4000 Ͼ4000 2.6 Ϯ 0.1 61 Ϯ 4.0

Ͼ4000 33

Discussion

Experimental Procedures