PDBsum entry 1n4j

Biotin-binding protein

PDB id: 1n4j

Contents

Protein chain

113 a.a. *

Waters ×63

* Residue conservation analysis

PDB id:

1n4j

Name:

Biotin-binding protein

Title:

Streptavidin mutant n23a at 2.18a

Structure:

Streptavidin. Chain: a. 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 PDB file)

Resolution:

2.18Å R-factor: 0.190 R-free: 0.253

Authors:

I.Le Trong,S.Freitag,L.A.Klumb,V.Chu,P.S.Stayton,R.E.Stenkamp

Key ref:

I.Le Trong et al. (2003). Structural studies of hydrogen bonds in the high-affinity streptavidin-biotin complex: mutations of amino acids interacting with the ureido oxygen of biotin. Acta Crystallogr D Biol Crystallogr, 59, 1567-1573. PubMed id: 12925786 DOI: 10.1107/S0907444903014562

Date:

31-Oct-02 Release date: 02-Sep-03

Protein chain

P22629  (SAV_STRAV) -  Streptavidin from Streptomyces avidinii

Seq: 183 a.a.

Struc: 113 a.a.*

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

Enzyme reactions

• Enzyme class: E.C.?

DOI no: 10.1107/S0907444903014562

Acta Crystallogr D Biol Crystallogr 59:1567-1573 (2003)

PubMed id: 12925786

Structural studies of hydrogen bonds in the high-affinity streptavidin-biotin complex: mutations of amino acids interacting with the ureido oxygen of biotin.

I.Le Trong, S.Freitag, L.A.Klumb, V.Chu, P.S.Stayton, R.E.Stenkamp.

ABSTRACT

An elaborate hydrogen-bonding network contributes to the tight binding of biotin to streptavidin. The specific energetic contributions of hydrogen bonds to the biotin ureido oxygen have previously been investigated by mapping the equilibrium and activation thermodynamic signatures of N23A, N23E, S27A, Y43A and Y43F site-directed mutants [Klumb et al. (1998), Biochemistry, 37, 7657-7663]. The crystal structures of these variants in the unbound and biotin-bound states provide structural insight into the energetic alterations and are described here. High (1.5-2.2 A) to atomic resolution (1.14 A) structures were obtained and structural models were refined to R values ranging from 0.12 to 0.20. The overall folding of streptavidin as described previously has not changed in any of the mutant structures. Major deviations such as side-chain shifts of residues in the binding site are observed only for the N23A and Y43A mutations. In none of the mutants is a systematic shift of biotin observed when one of the hydrogen-bonding partners to the ureido oxygen of biotin is removed. Recent thermodynamic studies report increases of DeltaDeltaG(o) of 5.0-14.6 kJ mol(-1) for these mutants with respect to the wild-type protein. The decreasing stabilities of the complexes of the mutants are discussed in terms of their structures.

Selected figure(s)

Figure 2.
Figure 2 Stereoview of the structures of the N23A mutant superposed on the biotin complex of wild-type streptavidin. The biotin-bound structure of N23A is shown in dark blue, the unbound structure is in light blue and the wild-type structure is in yellow. Figure drawn with MOLSCRIPT (Kraulis, 1991[Kraulis, P. J. (1991). J. Appl. Cryst. 24, 946-950.]) and Raster3D (Merritt & Bacon, 1997[Merritt, E. A. & Bacon, D. J. (1997). Methods Enzymol. 277, 505-524.]).

Figure 6.
Figure 6 Stereoview of the superposition of Y43F on wild-type streptavidin. Unbound Y43F in light blue, bound Y43F in dark blue and wild-type streptavidin in yellow. The binding site in the unbound Y43F structure is occupied by a water molecule and an MPD molecule (Freitag et al., 1999[Freitag, S., Le Trong, I., Klumb, L. A., Stayton, P. S. & Stenkamp, R. E. (1999). Acta Cryst. D55, 1118-1126.]). Figure drawn with MOLSCRIPT (Kraulis, 1991[Kraulis, P. J. (1991). J. Appl. Cryst. 24, 946-950.]) and Raster3D (Merritt & Bacon, 1997[Merritt, E. A. & Bacon, D. J. (1997). Methods Enzymol. 277, 505-524.]).

The above figures are reprinted by permission from the IUCr: Acta Crystallogr D Biol Crystallogr (2003, 59, 1567-1573) copyright 2003.

Figures were selected by the author.