PDBsum entry 1n0s

Binding protein

PDB id: 1n0s

Contents

Protein chains

173 a.a. *

Ligands

FLU ×2

SO4

Waters ×98

* Residue conservation analysis

PDB id:

1n0s

Name:

Binding protein

Title:

Engineered lipocalin flua in complex with fluorescein

Structure:

Bilin-binding protein. Chain: a, b. Synonym: bbp, anticalin flua. Engineered: yes

Source:

Pieris brassicae. Large cabbage white. Organism_taxid: 7116. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.00Å R-factor: 0.196 R-free: 0.243

Authors:

I.P.Korndoerfer,A.Skerra

Key ref:

I.P.Korndörfer et al. (2003). Crystallographic analysis of an "anticalin" with tailored specificity for fluorescein reveals high structural plasticity of the lipocalin loop region. Proteins, 53, 121-129. PubMed id: 12945055 DOI: 10.1002/prot.10497

Date:

15-Oct-02 Release date: 05-Aug-03

Protein chains

P09464  (BBP_PIEBR) -  Bilin-binding protein from Pieris brassicae

Seq: 189 a.a.

Struc: 173 a.a.*

* PDB and UniProt seqs differ at 21 residue positions (black crosses)

DOI no: 10.1002/prot.10497

Proteins 53:121-129 (2003)

PubMed id: 12945055

Crystallographic analysis of an "anticalin" with tailored specificity for fluorescein reveals high structural plasticity of the lipocalin loop region.

I.P.Korndörfer, G.Beste, A.Skerra.

ABSTRACT

The artificial lipocalin FluA with novel specificity toward fluorescein was derived via combinatorial engineering from the bilin-binding protein, BBP by exchange of 16 amino acids in the ligand pocket. Here, we describe the crystal structure of FluA at 2.0 A resolution in the space group P2(1) with two protein-ligand complexes in the asymmetric unit. In both molecules, the characteristic beta-barrel architecture with the attached alpha-helix is well preserved. In contrast, the four loops at one end of the beta-barrel that form the entrance to the binding site exhibit large conformational deviations from the wild-type protein, which can be attributed to the sidechain replacements. Specificity for the new ligand is furnished by hydrophobic packing, charged sidechain environment, and hydrogen bonds with its hydroxyl groups. Unexpectedly, fluorescein is bound in a much deeper cavity than biliverdin IX(gamma) in the natural lipocalin. Triggered by the substituted residues, unmutated sidechains at the bottom of the binding site adopt conformations that are quite different from those observed in the BBP, illustrating that not only the loop region but also the hydrophobic interior of the beta-barrel can be reshaped for molecular recognition. Particularly, Trp 129 participates in a tight stacking interaction with the xanthenolone moiety, which may explain the ultrafast electron transfer that occurs on light excitation of the bound fluorescein. These structural findings support our concept of using lipocalins as a scaffold for the engineering of so-called "anticalins" directed against prescribed targets as an alternative to recombinant antibody fragments.

Selected figure(s)

Figure 2.
Figure 2. The two monomers of FluA in the asymmetric crystal unit. Each polypeptide chain is shown in a ribbon presentation, light- and dark-gray, respectively, with its N- and C-terminus labeled and the fluorescein ligand depicted as a ball-and-stick model. The intermolecular -sheet that is formed via association of the loop #3 segments from the two monomers can be seen at the middle, together with the position of the crystallographically fixed sulfate ion.

Figure 4.
Figure 4. Stereo images with details of the novel ligand-binding site in FluA. (A) 2F[o]-F[c] electron density for the bound fluorescein contoured at 1.0 , together with sidechains of residues closer than 3.8 Å. Amino acids that were mutated in FluA (cf. Fig. 1) are colored dark-green, whereas original residues of BBP are depicted in light-green. (B) The set of 16 randomly mutated sidechains in the FluA crystal structure with the complexed fluorescein. (C) Arrangement of positively charged residues at the entrance to the ligand pocket of FluA. The three characteristically arranged Arg side chains (see text) are colored dark-blue. Note that there is a considerable distance between Arg 95 - whose sidechain is well defined in the electron density - and the bound fluorescein along the view axis (distance between the guanidinium and carboxylate groups: 8.6 Å) such that no direct contact is formed.

The above figures are reprinted by permission from John Wiley & Sons, Inc.: Proteins (2003, 53, 121-129) copyright 2003.

Figures were selected by the author.