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

J Biol Chem. 2009 Oct 30;284(44):30718-26. doi: 10.1074/jbc.M109.037812. Epub 2009 Sep 9.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Ankyrin Repeat / genetics*
  • Antiviral Agents / chemistry*
  • Bacteriophages / chemistry*
  • Binding Sites
  • Crystallography, X-Ray
  • Lactococcus lactis / virology*
  • Multiprotein Complexes / chemistry
  • Peptide Library
  • Protein Conformation
  • Protein Engineering
  • Receptors, Virus / metabolism
  • Viral Proteins / antagonists & inhibitors
  • Viral Proteins / chemistry*
  • Viral Proteins / metabolism
  • Virus Diseases / prevention & control*

Substances

  • Antiviral Agents
  • Multiprotein Complexes
  • Peptide Library
  • Receptors, Virus
  • Viral Proteins

Associated data

  • PDB/3HG0