PDBsum entry 1swt

Binding protein

PDB id: 1swt

Contents

Protein chains

118 a.a. *

Ligands

BTN ×2

Waters ×104

* Residue conservation analysis

PDB id:

1swt

Name:

Binding protein

Title:

Core-streptavidin mutant d128a in complex with biotin at ph 4.5

Structure:

Protein (streptavidin). Chain: a, b. Engineered: yes. Mutation: yes. Other_details: ph 4.5

Source:

Streptomyces avidinii. Organism_taxid: 1895. Expressed in: escherichia coli. Expression_system_taxid: 562. Other_details: t7 expression system (pet-210, novagen, inc., Madison,wi. Synthetic gene)

Biol. unit:

Homo-Tetramer (from PDB file)

Resolution:

2.00Å R-factor: 0.214 R-free: 0.308

Authors:

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

Key ref:

S.Freitag et al. (1999). A structural snapshot of an intermediate on the streptavidin-biotin dissociation pathway. Proc Natl Acad Sci U S A, 96, 8384-8389. PubMed id: 10411884 DOI: 10.1073/pnas.96.15.8384

Date:

22-Oct-98 Release date: 30-Jul-99

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)

DOI no: 10.1073/pnas.96.15.8384

Proc Natl Acad Sci U S A 96:8384-8389 (1999)

PubMed id: 10411884

A structural snapshot of an intermediate on the streptavidin-biotin dissociation pathway.

S.Freitag, V.Chu, J.E.Penzotti, L.A.Klumb, R.To, D.Hyre, I.Le Trong, T.P.Lybrand, R.E.Stenkamp, P.S.Stayton.

ABSTRACT

It is currently unclear whether small molecules dissociate from a protein binding site along a defined pathway or through a collection of dissociation pathways. We report herein a joint crystallographic, computational, and biophysical study that suggests the Asp-128 --> Ala (D128A) streptavidin mutant closely mimics an intermediate on a well-defined dissociation pathway. Asp-128 is hydrogen bonded to a ureido nitrogen of biotin and also networks with the important aromatic binding contacts Trp-92 and Trp-108. The Asn-23 hydrogen bond to the ureido oxygen of biotin is lengthened to 3.8 A in the D128A structure, and a water molecule has moved into the pocket to replace the missing carboxylate interaction. These alterations are accompanied by the coupled movement of biotin, the flexible binding loop containing Ser-45, and the loop containing the Ser-27 hydrogen bonding contact. This structure closely parallels a key intermediate observed in a potential of mean force-simulated dissociation pathway of native streptavidin, where the Asn-23 hydrogen bond breaks first, accompanied by the replacement of the Asp-128 hydrogen bond by an entering water molecule. Furthermore, both biotin and the flexible loop move in a concerted conformational change that closely approximates the D128A structural changes. The activation and thermodynamic parameters for the D128A mutant were measured and are consistent with an intermediate that has traversed the early portion of the dissociation reaction coordinate through endothermic bond breaking and concomitant gain in configurational entropy. These composite results suggest that the D128A mutant provides a structural "snapshot" of an early intermediate on a relatively well-defined dissociation pathway for biotin.

Selected figure(s)

Figure 1.
Fig. 1. Superposition of the wild-type (green) and D128A (red) biotin complexes in the region of the biotin binding site. (A) Only the C chain is depicted for clarity. Compared with wild-type streptavidin, three loops and biotin display a concerted shift away from the mutation site. For the hydrogen binding residues 23 and 128, the side chains are also depicted. A water molecule replaces an Asp-128 oxygen (OD2) in the mutant and interacts with biotin. (B) The hydrogen bonding network shows little deviations at residues Ser-88, Thr-90, and Trp-92, but there is no direct interaction of Ala-128 with biotin and the Asn-23-biotin hydrogen bond length is increased. (C) The tryptophan residues involved in hydrophobic interactions show only minor deviations.

Figure 2.
Fig. 2. A Connolly surface representing the PMF-calculated dissociation pathway of biotin from streptavidin, color-coded by energy (red highest), is overlaid on backbone ribbons representing the crystal structures of wild-type (purple) and D128A (blue) streptavidin. The D128A mutation causes the biotin to shift outward from the binding pocket allowing a water molecule to enter, mimicking features observed in the transition state of the PMF calculations.

Figures were selected by an automated process.