The heme-containing enzyme myeloperoxidase (MPO) is critical for optimal antimicrobial activity of human neutrophils. We recently discovered that the bacterium expresses a novel immune evasion protein， called SPIN， that binds tightly to MPO， inhibits MPO activity， and contributes to bacterial survival following phagocytosis. A co-crystal structure of SPIN bound to MPO suggested that SPIN blocks substrate access to the catalytic heme by inserting an N-terminal β-hairpin into the MPO active-site channel. Here， we describe a series of experiments that more completely define the structure/function relationships of SPIN. Whereas the SPIN N terminus adopts a β-hairpin confirmation upon binding to MPO， ... More
The heme-containing enzyme myeloperoxidase (MPO) is critical for optimal antimicrobial activity of human neutrophils. We recently discovered that the bacterium expresses a novel immune evasion protein， called SPIN， that binds tightly to MPO， inhibits MPO activity， and contributes to bacterial survival following phagocytosis. A co-crystal structure of SPIN bound to MPO suggested that SPIN blocks substrate access to the catalytic heme by inserting an N-terminal β-hairpin into the MPO active-site channel. Here， we describe a series of experiments that more completely define the structure/function relationships of SPIN. Whereas the SPIN N terminus adopts a β-hairpin confirmation upon binding to MPO， the solution NMR studies presented here are consistent with this region of SPIN being dynamically structured in the unbound state. Curiously， whereas the N-terminal β-hairpin of SPIN accounts for ∼55% of the buried surface area in the SPIN-MPO complex， its deletion did not significantly change the affinity of SPIN for MPO but did eliminate the ability of SPIN to inhibit MPO. The flexible nature of the SPIN N terminus rendered it susceptible to proteolytic degradation by a series of chymotrypsin-like proteases found within neutrophil granules， thereby abrogating SPIN activity. Degradation of SPIN was prevented by the immune evasion protein Eap， which acts as a selective inhibitor of neutrophil serine proteases. Together， these studies provide insight into MPO inhibition by SPIN and suggest possible functional synergy between two distinct classes of immune evasion proteins.
