PDBsum entry 1rxh

Unknown function

PDB id: 1rxh

Contents

Protein chains

118 a.a. *

Ligands

BNI ×2

Waters ×16

* Residue conservation analysis

PDB id:

1rxh

Name:

Unknown function

Title:

Crystal structure of streptavidin mutant l124r (m1) complexed with biotinyl p-nitroanilide (bni)

Structure:

Streptavidin. Chain: a, b. Engineered: yes. Mutation: yes

Source:

Streptomyces avidinii. Organism_taxid: 1895. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Tetramer (from PDB file)

Resolution:

2.90Å R-factor: 0.212 R-free: 0.309

Authors:

Y.Eisenberg-Domovich,Y.Pazy,O.Nir,B.Raboy,E.A.Bayer,M.Wilchek, O.Livnah

Key ref:

Y.Eisenberg-Domovich et al. (2004). Structural elements responsible for conversion of streptavidin to a pseudoenzyme. Proc Natl Acad Sci U S A, 101, 5916-5921. PubMed id: 15079055 DOI: 10.1073/pnas.0308541101

Date:

18-Dec-03 Release date: 11-May-04

Protein chains

P22629  (SAV_STRAV) -  Streptavidin from Streptomyces avidinii

Seq: 183 a.a.

Struc: 118 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.?

DOI no: 10.1073/pnas.0308541101

Proc Natl Acad Sci U S A 101:5916-5921 (2004)

PubMed id: 15079055

Structural elements responsible for conversion of streptavidin to a pseudoenzyme.

Y.Eisenberg-Domovich, Y.Pazy, O.Nir, B.Raboy, E.A.Bayer, M.Wilchek, O.Livnah.

ABSTRACT

Avidin enhances the hydrolysis of biotinyl p-nitrophenyl ester (BNP) under mild alkaline conditions, whereas streptavidin prevents hydrolysis of BNP up to pH 12. Recently, we imposed hydrolytic activity on streptavidin by rational mutagenesis, based on the molecular elements responsible for the hydrolysis by avidin. Three mutants were designed, whereby the desired features, the distinctive L124R point mutation (M1), the L3,4 loop replacement (M2), and the combined mutation (M3), were transferred from avidin to streptavidin. The crystal structures of the mutants, in complex with biotinyl p-nitroanilide (BNA), the stable amide analogue of BNP, were determined. The results demonstrate that the point mutation alone has little effect on hydrolysis, and BNA exhibits a conformation similar to that of streptavidin. Substitution of a lengthier L3,4 loop (from avidin to streptavidin), resulted in an open conformation, thus exposing the ligand to solvent. Moreover, the amide bond of BNA was flipped relative to that of the streptavidin and M1 complexes, thus deflecting the nitro group toward Lys-121. Consequently, the leaving group potential of the nitrophenyl group of BNP is increased, and M2 hydrolyzes BNP at pH values >8.5. To better emulate the hydrolytic potential of avidin, M3 was required. The combination of loop replacement and point mutation served to further increase the leaving group potential by interaction of the nitro group with Arg-124 and Lys-121. The information derived from this study may provide insight into the design of enzymes and transfer of desired properties among homologous proteins.

Selected figure(s)

Figure 1.
Fig. 1. Electron-density maps of M1, M2, and M3 in complex with BNA. Shown are stereoviews of F[o] - F[c] electron-density maps calculated at respective resolutions indicated in Table 1, after the initial cycle of refinement with no ligand in the model. The initial model included leucine at position 124, and for the M1 and M3 complexes the extended electron density clearly suggests the existence of an arginine residue at this position. The maps were constructed at 2.0 with superimposed coordinates from the final models.

Figure 3.
Fig. 3. Tube representation of the tertiary structures of M3 (blue) and streptavidin (magenta) complexes with BNA (shown in black). The L3,4 loop in both molecules is shown in gold. In streptavidin, the loop is in the closed conformation, thus burying the BNA ligand almost completely. In M3, where the loop has three more residues, L3,4 adopts an open conformation. Note the flipping of the amide bond of the ligand and the altered conformation of the p-nitroanilide group.

Figures were selected by an automated process.