Crystal structure and function of a DARPin neutralizing inhibitor of lactococcal phage TP901-1: comparison of DARPin and camelid VHH binding mode

Abstract

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 BppUxBppL 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 A resolution. The DARPinxRBP 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.

FIGURE 1.

Top, single clone crude extract ELISAs. A, DARPin 18. B, DARPin 19. C, DARPin 20. DARPin binding was tested on a well with neutravidin-immobilized (BppU·BppL)_biot and compared with the signal intensity obtained from a control well with only neutravidin and bovine serum albumin (BSA). Bottom, competition ELISAs. DARPins 18 (D), 19 (E), and 20 (F) were preincubated with increasing concentrations of free non-biotinylated BppU·BppL complex for 1 h at 4 °C before the addition to their respective ELISA wells.

FIGURE 2.

Sequence alignment of DARPins 18, 19, and 20. The DARPin consensus is displayed with X, indicating randomized positions (any amino acid residues except glycine, proline, and cysteine) and Z for the partially randomized positions (allowing histidine, tyrosine, or asparagine); all remaining positions are framework residues (13). This figure was created with Multalin (44).

FIGURE 3.

Structure of the RBP·DARPin 20 complex. A, shown is the overall structure with both DARPin (rainbow-colored) and the RBP (the three chains colored in salmon, purple, and yellow) in schematic representation. The inset shows the detail of the interactions involving a central role of Tyr-90 from the DARPin; the small letters refer to DARPin (d) and the a, b, c chains of the RBP. B, shown is the overall structure of the two partners with molecular surface representation according to the same color scheme as in A. C, shown is the interface region with the buried surface area of the DARPin colored red, green, and yellow corresponding to interaction with chain A, B, and C of the RBP, respectively. D, shown is the same view as in C rotated approximately by 90°. E, shown is an illustration of the concave (DARPin)-convex (RBP) type of the interaction interface. Nt, N terminus; Ct, C terminus.

FIGURE 4.

Comparison of the phage TP901-1 RBP·DARPin 20 complex with the phage p2 RBP·VHH5 complex (6). The three VHH5 have been superimposed on the TP901-1 RBP structure for comparison, taking advantage of the three-dimensional similarity of the two RBPs. The blue grid locates the bound glycerol in the receptor binding site (10). Each VHH5 binds in a crevice between two subunits. The structure of the three VHHs was taken from the Protein Data Bank entry 2BSE.