Crystal Structure and Function of a DARPin Neutralizing Inhibitor of Lactococcal Phage TP901-1: COMPARISON OF DARPin AND CAMELID VHH BINDING MODE*

Combinatorial libraries of designed ankyrin repeat proteins (DARPins) have been proven to be a valuable source of specific binding proteins, as they can be expressed at very high levels and are very stable. We report here the selection of DARPins directed against a macromolecular multiprotein complex, the baseplate BppU·BppL complex of the lactococcal phage TP901-1. Using ribosome display, we selected several DARPins that bound specifically to the tip of the receptor-binding protein (RBP, the BppL trimer). The three selected DARPins display high specificity and affinity in the low nanomolar range and bind with a stoichiometry of one DARPin per BppL trimer. The crystal structure of a DARPin complexed with the RBP was solved at 2.1 Å resolution. The DARPin·RBP interface is of the concave (DARPin)-convex (RBP) type, typical of other DARPin protein complexes and different from what is observed with a camelid VHH domain, which penetrates the phage p2 RBP inter-monomer interface. Finally, phage infection assays demonstrated that TP901-1 infection of Lactococcus lactis cells was inhibited by each of the three selected DARPins. This study provides proof of concept for the possible use of DARPins to circumvent viral infection. It also provides support for the use of DARPins in co-crystallization, due to their rigidity and their ability to provide multiple crystal contacts.Lactococcus lactis is a Gram-positive bacterium widely used by the dairy industry for the production of an array of fermented milk products. Several industrial strains are sensitive to various distinct bacteriophages, mostly belonging to the Siphoviridae family. The lactococcal phage population is divided in at least 10 genetically distinct groups, of which the 936, c2, and P335 groups are prominent (1, 2). These L. lactis-infecting phages are considerably problematic in causing milk fermentation failures and resulting in decreased yields as well as low quality products (3). Preventing these infections has proven to be difficult because of lactococcal phage ubiquity, biodiversity, and genomic plasticity (4).Phage infection is initiated by binding of the phage receptor-binding protein (RBP),⁵ located within the baseplate at the distal part of the tail, to its receptor on the host cell surface (5). We have previously solved the crystal structures of the three RBPs of the lactococcal phages p2 (936) (6), bIL170 (936) (7), TP901-1 (P335) (8), and their chimera (9) as well as characterized their saccharide binding sites (10). The RBPs of these phages have a similar homotrimeric architecture related by a 3-fold axis. They comprise three domains: the N terminus shoulder domain, the interlaced β-prism neck domain, and the jellyroll head domain at the C terminus. The head domain has a saccharide binding site likely involved in host recognition. The lactococcal phage TP901-1 contains a double-disk-shaped baseplate at the tip of its tail which is made of a lower baseplate protein (BppL) and an upper baseplate protein (BppU) (11).One strategy to minimize bacteriophage infections is to competitively block phage adsorption by adding a protein that specifically binds to the phage RBP. A neutralizing llama VHH domain recognizing the head domain of the phage p2 RBP has been used to block L. lactis phage infection in milk fermentation (12). Lactococcal phages could readily escape neutralization by generating mutations interfering with VHH binding over the large interaction surface while keeping the central polysaccharide receptor binding pocket intact (10). Designed ankyrin repeat proteins (DARPins) may be another tool to neutralize viral infection, as they display distinct characteristics from VHHs and contain the required properties in terms of stability and facility of expression (13).Ankyrin repeat proteins are found in virtually all phyla and mediate specific protein-protein interactions in all cell compartments (14). The ankyrin elementary module is composed of 33 amino acids structured as a β-turn followed by two antiparallel α-helices and a loop connected to the β-turn of the next repeat. The repeats are stacked in a rigid manner. In creating a DARPin library, residues in each repeat were subdivided in two groups; (i) randomized residues constituting potential target interaction points and (ii) framework residues, important for maintaining the ankyrin fold (13). Libraries with varying repeat numbers were assembled and named according to the constituent repeat number; N2C and N3C libraries were used in this study, with two and three internal repeats inserted between the N and C capping repeats, respectively. DARPins are a powerful alternative to the use of antibodies, notably because of their very high expression rates in Escherichia coli, their high stability paired with high affinity, and successful reports of their use in co-crystallization (15–19). Their architecture results in a very rigid structure that facilitates multiple crystal contacts and may promote crystal formation of the protein of interest by providing additional surfaces for such crystal contacts.We report here the selection and analysis of DARPin binders directed against a macromolecular multiprotein ensemble, the TP901-1 baseplate BppU·BppL protein complex. Ribosome display selection, ELISA screening, and surface plasmon resonance (SPR) measurements allowed us to isolate and characterize three N2C DARPins that recognized the RBP (BppL of the BppU·BppL complex) with high specificity and affinity. Further studies showed that the three DARPins bound to a unique area of the RBP at the tip of the head domain. QELS, MALS, UV, and refractometry coupled online with a size exclusion chromatography (SEC) column allowed us to monitor complex formation in solution as well as to estimate DARPin binding stoichiometry. Crystals of one of these selected DARPins in complex with the RBP were obtained, and the x-ray structure was solved at 2.1 Å resolution. This constitutes the first structure of a DARPin complex originating from the N2C library and the highest resolution for a DARPin complex structure reported to date. Finally, phage adsorption inhibition experiments demonstrated that the three N2C DARPins strongly inhibited L. lactis infection by TP901-1. We describe the DARPin·RBP interface and compare it to other DARPin interfaces. We also compare it to the p2 RBP·VHH5 complex, a previously selected llama VHH domain inhibiting p2 phage adsorption (12), to highlight the different binding mode of these two types of binders.