causes listeriosis, a potentially fatal food-borne disease. The condition is especially harmful to pregnant women. outbreaks can originate from diverse foods, highlighting the need for novel strategies to improve food safety. The first step in invasion is internalization of the bacteria, which is mediated by the interaction of the internalin family of virulence factors with host cell receptors. A crucial interaction for invasion of the placenta, and thus a target for therapeutic intervention, is between internalin B (InlB) and the receptor c-Met. Single-domain antibodies (VH, also called nanobodies, or sdAbs) from camel heavy-chain antibodies are a novel solution for preventing infections. The VH R303, R330, and R326 all bind InlB with high affinity; however, the molecular mechanism behind their mode of action was unknown. We demonstrate that despite a high degree of sequence and structural diversity, the VH bind a single epitope on InlB. A combination of gentamicin protection assays and florescent microscopy establish that InlB-specific VH inhibit invasion of HeLa cells. A high-resolution X-ray structure of VH R303 in complex with InlB showed that the VH binds at the c-Met interaction site on InlB, thereby acting as a competitive inhibitor preventing bacterial invasion. These results point to the potential of VH as a novel class of therapeutics for the prevention of listeriosis.

The variable region of camelid heavy-chain antibodies produces the smallest known antibody fragment with antigen-binding capability (a V H H). The V H H R303 binds internalin B (InlB), a virulence factor expressed by the pathogen Listeria monocytogenes. InlB is critical for initiation of Listeria infection, as it binds a receptor (c-Met) on epithelial cells, triggering the entry of bacteria into host cells. InlB is surface-exposed and is required for virulence, hence a V H H targeting InlB has potential applications for pathogen detection or therapeutic intervention. Here, the expression, purification, crystallization and X-ray diffraction of R303 are reported. Crystals of R303 were obtained following in situ proteolysis with trypsin. Gel filtration and SDS-PAGE revealed that trypsin removed the C-terminal tag region of R303, facilitating crystal formation. Crystals of R303 diffracted to 1.3 Å resolution and belonged to the monoclinic space group P2 1 , with unit-cell parameters a = 46.4, b = 31.2, c = 74.8 Å , = 93.8 . The crystals exhibited a Matthews coefficient of 1.95 Å 3 Da À1 with two molecules in the asymmetric unit.

ObjectiveTo determine the X-ray structure and biophysical properties of a Camelid VHH isolated from a naïve phage display library.ResultsSingle domain antibodies (VHH) derived from the unique immune system of the Camelidae family have gained traction as useful tools for biotechnology as well as a source of potentially novel therapeutics. Here we report the structure and biophysical characterization of a VHH originally isolated from a naïve camelid phage display library. VHH R419 has a melting temperate of 66 °C and was found to be a monomer in solution. The protein crystallized in space group P6522 and the structure was solved by molecular replacement to a resolution of 1.5 Å. The structure revealed a flat paratope with CDR loops that could be classified into existing canonical loop structures. A combination of high expression yield, stability and rapid crystallization might make R419 into a candidate scaffold for CDR grafting and homology modeling.

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