PDBsum entry 2cbq

Antimicrobial

PDB id: 2cbq

Contents

Protein chains

(+ 0 more) 111 a.a. *

Ligands

SO4 ×5

TH2 ×8

Waters ×87

* Residue conservation analysis

PDB id:

2cbq

Name:

Antimicrobial

Title:

Crystal structure of the neocarzinostatin 1tes15 mutant bound to testosterone hemisuccinate.

Structure:

Neocarzinostatin. Chain: a, b, c, d, e, f. Fragment: residues 35-147. Synonym: ncs, mitomalcin, mmc. Engineered: yes. Mutation: yes

Source:

Streptomyces carzinostaticus. Organism_taxid: 1897. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Dimer (from PDB file)

Resolution:

2.60Å R-factor: 0.207 R-free: 0.257

Authors:

A.Drevelle,M.Graille,B.Heyd,I.Sorel,N.Ulryck,F.Pecorari,M.Desmadril, H.Van Tilbeurgh,P.Minard

Key ref:

A.Drevelle et al. (2006). Structures of in vitro evolved binding sites on neocarzinostatin scaffold reveal unanticipated evolutionary pathways. J Mol Biol, 358, 455-471. PubMed id: 16529771 DOI: 10.1016/j.jmb.2006.02.002

Date:

06-Jan-06 Release date: 22-Mar-06

Protein chains

P0A3R9  (NCZS_STRCZ) -  Neocarzinostatin from Streptomyces carzinostaticus

Seq: 147 a.a.

Struc: 111 a.a.*

* PDB and UniProt seqs differ at 7 residue positions (black crosses)

DOI no: 10.1016/j.jmb.2006.02.002

J Mol Biol 358:455-471 (2006)

PubMed id: 16529771

Structures of in vitro evolved binding sites on neocarzinostatin scaffold reveal unanticipated evolutionary pathways.

A.Drevelle, M.Graille, B.Heyd, I.Sorel, N.Ulryck, F.Pecorari, M.Desmadril, H.van Tilbeurgh, P.Minard.

ABSTRACT

We have recently applied in vitro evolution methods to create in Neocarzinostatin a new binding site for a target molecule unrelated to its natural ligand. The main objective of this work was to solve the structure of some of the selected binders in complex with the target molecule: testosterone. Three proteins (1a.15, 3.24 and 4.1) were chosen as representative members of sequence families that came out of the selection process within different randomization schemes. In order to evaluate ligand-induced conformational adaptation, we also determined the structure of one of the proteins (3.24) in the free and complexed forms. Surprisingly, all these mutants bind not one but two molecules of testosterone in two very different ways. The 3.24 structure revealed that the protein spontaneously evolved in the system to bind two ligand molecules in one single binding crevice. These two binding sites are formed by substituted as well as by non-variable side-chains. The comparison with the free structure shows that only limited structural changes are observed upon ligand binding. The X-ray structures of the complex formed by 1a.15 and 4.1 Neocarzinostatin mutants revealed that the two variants form very similar dimers. These dimers were observed neither for the uncomplexed variants nor for wild-type Neocarzinostatin but were shown here to be induced by ligand binding. Comparison of the three complexed forms clearly suggests that these unanticipated structural responses resulted from the molecular arrangement used for the selection experiments.

Selected figure(s)

Figure 8.
Figure 8. Schematic representation of the evolutionary pathway. Positions randomized in the three libraries are indicated in green, those randomized only in library 4 are in purple. NNK and RVY refer to the degenerated nucleotide sequence used to randomize the indicated amino acid during library construction (NNK code for 20 amino acids, RVY code for:A,S,T,N,D,G). The more constrained positions 35 and 96 are indicated in red and cyan. THS molecules are in blue.

Figure 9.
Figure 9. Model of the ternary complex streptavin/testobiotin–3.24 variant. The experimental structure of 3.24 variant complexed with THS and the biotin-bound streptavidin tetramer (PDB code: 1STP) were used to build this model. The model shows two streptavidin monomers displaying biotinylated testosterone (orange) on the same face of streptavidin tetramer (yellow/orange). The 3.24 variant is in purple with the bound biotinylated testosterone molecules in dark grey. This model suggest that the concomitant binding of two streptavidin-bound testosterone molecules is compatible with the linker length and known structures of the 3.24 variant and streptavidin-binding sites.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2006, 358, 455-471) copyright 2006.

Figures were selected by an automated process.