Rational Design of an Anticalin-Type Sugar-Binding Protein Using a Genetically Encoded Boronate Side Chain

Abstract

The molecular recognition of carbohydrates plays a fundamental role in many biological processes. However, the development of carbohydrate-binding reagents for biomedical research and use poses a challenge due to the generally poor affinity of proteins toward sugars in aqueous solution. Here, we describe the effective molecular recognition of pyranose monosaccharides (in particular, galactose and mannose) by a rationally designed protein receptor based on the human lipocalin scaffold (Anticalin). Complexation relies on reversible covalent cis-diol boronate diester formation with a genetically encoded l-boronophenylalanine (Bpa) residue which was incorporated as a non-natural amino acid at a sterically permissive position in the ligand pocket of the Anticalin, as confirmed by X-ray crystallography. Compared with the metal-ion and/or avidity-dependent oligovalent lectins that prevail in nature, our approach offers a novel and promising route to generate tight sugar-binding reagents both as research reagents and for biomedical applications.

A Tetrahedral Boronic Acid Diester Formed by an Unnatural Amino Acid in the Ligand Pocket of an Engineered Lipocalin

“…Its associated hydroxide ion from the solvent is engaged in a polar contact with the Asn134 side chain (2.7 Å distance, with non‐ideal H‐bond angle), which appears to stabilize the tetrahedral configuration at the crystallization pH of 8.5 (Figure C). It is notable that this side chain is the result of a substitution of the original residue Lys134 in wild‐type Lcn2 as part of the design of the Borocalin . The polar interaction causes a slight shift of the Bpa side chain towards Asn134 in relation to the previously described structure of the ligand‐free protein .…”

“…In addition to the Bpa residue, three conventional side‐chain exchanges were introduced in order to enhance the steric accessibility and chemical reactivity of the boronic acid group [Lcn2(36Bpa‐NFW), alias Borocalin], thus providing a basis for the engineering of biomedically relevant sugar specificities by employing Anticalin ® technology . For Lcn2(36Bpa‐NFW), we have demonstrated binding of pyranose monosaccharides, in particular galactose and mannose, as well as of the aromatic diol 4‐nitrocatechol, with dissociation constants in the millimolar and micromolar ranges, respectively …”

“…Apart from the obvious influence of the pH on the acid/base‐catalyzed addition/condensation reactions, the tetrahedral cycloadduct is usually preferred for steric reasons because its smaller O−B−O bond angle leads to less ring strain. It is notable that 4‐nitrocatechol readily forms much tighter complexes with boronic acid than with pyranose sugars (see above) . This could be due to 1) the particular geometry of vicinal OH groups as substituents on a planar unsaturated carbon ring, 2) its aromatic character, and/or 3) the electron‐withdrawing and resonance‐stabilizing nitro group .…”

“…The crystal structure of the Borocalin with bound 4‐nitrocatechol was solved from an X‐ray synchrotron data set at 1.98 Å resolution in space group C 2 , with two protein molecules in the asymmetric unit, by molecular replacement with the apo‐Borocalin as search model. After refinement to a final R factor of 20.7 % (Table ), electron density was visible for the two entire polypeptide chains (residues Ser5–Lys188 in chain A, discussed below due to its lower B factors) including Bpa at position 36 and, in particular, the covalently bound ligand (Figure ).…”

“…The just slightly higher RMSD of 0.445 Å for all 174 equivalent Cα atoms (Ser5–Gly178, without the Strep‐tag II) indicates that, beyond the β‐barrel core, the conformations of all loop segments and the α‐helix were also conserved, including the set of those four structurally variable loops that shape the ligand pocket at the open end . Only the residues of the C‐terminal Strep‐tag II peptide (SAWSHPQFEK) were shifted by up to 4.1 Å (at the Cα position), due to an intimate interaction with the neighboring molecule in the crystal lattice in the case of the apo‐protein as explained in our previous study …”