Engagement of an extended beta-hairpin is a common substrate/inhibitor interaction at the active site of serine proteases and is a key feature of Laskowski mechanism inhibitors that present a substrate-like loop to a target protease. This loop is cleaved but subsequently religated forming a stable protease/inhibitor complex. Laskowski inhibitors are ubiquitous in nature and are used extensively in serine protease inhibitor design. However, most studies concentrate on engineering sidechain interactions rather than the direct contributions of the substrate-like beta-sheet to enzyme inhibition. Here we report the structure of an optimised beta-sheet inhibitory motif within the Sunflower Trypsin Inhibitor (SFTI) and describe its interactions in a bovine trypsin/SFTI crystal structure. For the first time we demonstrate the ability of a crystalised protease to cleave and religate a Laskowski inhibitor and show that an optimised SFTI variant, SFTI-TCTR-N12N14 has an internal hydrogen bond network that engages the inhibitor sidechains that would normally interact with a target protease, making main-chain interactions the dominant feature driving complex formation. Despite having reduced sidechain interactions, this SFTI variant is remarkably potent, inhibiting trypsin with a Ki of 0.7 nM. Furthermore, it inhibits a chymotrypsin-like enzyme, kallikrein-related peptidase 7 with a Ki of 16.4 nM compared to wild type SFTI with a Ki of over 10000 nM. Molecular modelling of the SFTI/KLK7 complex again indicates an interface dominated by bata-sheet interactions, highlighting the importance of this motif and the adaptability of SFTI as a scaffold for inhibitor design.