PDBsum entry 1mep

Biotin-binding protein

PDB id: 1mep

Contents

Protein chains

119 a.a. *

Ligands

BTN ×4

Waters ×293

* Residue conservation analysis

PDB id:

1mep

Name:

Biotin-binding protein

Title:

Crystal structure of streptavidin double mutant s45a/d128a with biotin: cooperative hydrogen-bond interactions in the streptavidin- biotin system.

Structure:

Streptavidin. Chain: a, b, c, d. Fragment: core streptavidin (residues 13-139). Engineered: yes. Mutation: yes

Source:

Streptomyces avidinii. Organism_taxid: 1895. Gene: core streptavidin. Expressed in: escherichia coli bl21. Expression_system_taxid: 511693.

Biol. unit:

Tetramer (from PQS)

Resolution:

1.65Å R-factor: 0.204 R-free: 0.294

Authors:

D.E.Hyre,I.Le Trong,E.A.Merritt,R.E.Stenkamp,N.M.Green,P.S.Stayton

Key ref:

D.E.Hyre et al. (2006). Cooperative hydrogen bond interactions in the streptavidin-biotin system. Protein Sci, 15, 459-467. PubMed id: 16452627 DOI: 10.1110/ps.051970306

Date:

08-Aug-02 Release date: 02-Sep-03

Protein chains

P22629  (SAV_STRAV) -  Streptavidin from Streptomyces avidinii

Seq: 183 a.a.

Struc: 119 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.?

DOI no: 10.1110/ps.051970306

Protein Sci 15:459-467 (2006)

PubMed id: 16452627

Cooperative hydrogen bond interactions in the streptavidin-biotin system.

D.E.Hyre, I.Le Trong, E.A.Merritt, J.F.Eccleston, N.M.Green, R.E.Stenkamp, P.S.Stayton.

ABSTRACT

The thermodynamic and structural cooperativity between the Ser45- and D128-biotin hydrogen bonds was measured by calorimetric and X-ray crystallographic studies of the S45A/D128A double mutant of streptavidin. The double mutant exhibits a binding affinity approximately 2x10(7) times lower than that of wild-type streptavidin at 25 degrees C. The corresponding reduction in binding free energy (DeltaDeltaG) of 10.1 kcal/mol was nearly completely due to binding enthalpy losses at this temperature. The loss of binding affinity is 11-fold greater than that predicted by a linear combination of the single-mutant energetic perturbations (8.7 kcal/mol), indicating that these two mutations interact cooperatively. Crystallographic characterization of the double mutant and comparison with the two single mutant structures suggest that structural rearrangements at the S45 position, when the D128 carboxylate is removed, mask the true energetic contribution of the D128-biotin interaction. Taken together, the thermodynamic and structural analyses support the conclusion that the wild-type hydrogen bond between D128-OD and biotin-N2 is thermodynamically stronger than that between S45-OG and biotin-N1.

Selected figure(s)

Figure 1.
Structural superposition of bound wild-type (white), S45A (blue), D128A (red), and S45A/D128A double-mutant (green) streptavidin structures. (A) Stereoview of the overall structures. (B). Details of the binding pocket described in the text. The bound structures were superimposed by least-squares fit of the 65 C[alpha] atoms in the [beta]-barrel core of each monomer. The nearly identical structure of these cores can be seen in the overlay of the [beta]-sheet backbone in the [beta]-barrel core. The protein adjusts to the two mutations by a combination of the shifts found in the individual mutants, with the main-chain near S45A shifting toward biotin, the main-chain near D128A shifting away from biotin, a water molecule replacing the missing D128 carboxylate, and the biotin shifting away from the bottom of the pocket (the new water in the D128A structure is hidden by that in the double mutant due to nearly complete overlap). Hydrogen bonds involving Q24, S45, D128, and biotin in wild-type streptavidin are shown by dotted lines.

Figure 4.
Thermodynamic cycles for stepwise mutation of wild-type streptavidin to the S45A/D128A double mutant, showing [Delta]G[298], [Delta]H[298], and T[Delta]S[298]. The cycle starts with wild type in the upper left corner and proceeds to the single mutants, with the thermodynamic perturbation shown next to the arrows. The cycle continues to the double mutant, with the difference in thermodynamic parameters between each single mutant and the double mutant shown next to those arrows. The units on the quantities in the figure are kcal/mol.

The above figures are reprinted from an Open Access publication published by the Protein Society: Protein Sci (2006, 15, 459-467) copyright 2006.

Figures were selected by an automated process.