PDBsum entry 2ck2

Signaling protein

PDB id: 2ck2

Contents

Protein chains

94 a.a. *

96 a.a. *

Ligands

ACE ×2

Waters ×109

* Residue conservation analysis

PDB id:

2ck2

Name:

Signaling protein

Title:

Structure of core-swapped mutant of fibronectin

Structure:

Human fibronectin. Chain: a, b. Fragment: residues 1447-1542. Synonym: fn, cold-insoluble globulin, cig. Engineered: yes. Mutation: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008. Expression_system_variant: c41.

Resolution:

2.00Å R-factor: 0.201 R-free: 0.268

Authors:

S.P.Ng,K.S.Billings,T.Ohashi,M.D.Allen,R.B.Best,L.G.Randles, H.P.Erickson,J.Clarke

Key ref:

S.P.Ng et al. (2007). Designing an extracellular matrix protein with enhanced mechanical stability. Proc Natl Acad Sci U S A, 104, 9633-9637. PubMed id: 17535921 DOI: 10.1073/pnas.0609901104

Date:

10-Apr-06 Release date: 10-Apr-07

Protein chain

P02751  (FINC_HUMAN) -  Fibronectin from Homo sapiens

Seq: 2477 a.a.

Struc: 94 a.a.*

Protein chain

P02751  (FINC_HUMAN) -  Fibronectin from Homo sapiens

Seq: 2477 a.a.

Struc: 96 a.a.*

* PDB and UniProt seqs differ at 30 residue positions (black crosses)

DOI no: 10.1073/pnas.0609901104

Proc Natl Acad Sci U S A 104:9633-9637 (2007)

PubMed id: 17535921

Designing an extracellular matrix protein with enhanced mechanical stability.

S.P.Ng, K.S.Billings, T.Ohashi, M.D.Allen, R.B.Best, L.G.Randles, H.P.Erickson, J.Clarke.

ABSTRACT

The extracellular matrix proteins tenascin and fibronectin experience significant mechanical forces in vivo. Both contain a number of tandem repeating homologous fibronectin type III (fnIII) domains, and atomic force microscopy experiments have demonstrated that the mechanical strength of these domains can vary significantly. Previous work has shown that mutations in the core of an fnIII domain from human tenascin (TNfn3) reduce the unfolding force of that domain significantly: The composition of the core is apparently crucial to the mechanical stability of these proteins. Based on these results, we have used rational redesign to increase the mechanical stability of the 10th fnIII domain of human fibronectin, FNfn10, which is directly involved in integrin binding. The hydrophobic core of FNfn10 was replaced with that of the homologous, mechanically stronger TNfn3 domain. Despite the extensive substitution, FNoTNc retains both the three-dimensional structure and the cell adhesion activity of FNfn10. Atomic force microscopy experiments reveal that the unfolding forces of the engineered protein FNoTNc increase by approximately 20% to match those of TNfn3. Thus, we have specifically designed a protein with increased mechanical stability. Our results demonstrate that core engineering can be used to change the mechanical strength of proteins while retaining functional surface interactions.

Selected figure(s)

Figure 2.
Fig. 2. FNoTNc retains the structure of its parents. (a) Stereo view showing an overlay of the backbone traces of FNoTNc (green), FNfn10 (blue), and TNfn3 (red). The only regions where FNfn10 and FNoTNc differ significantly are in the C–C' and F–G loops, both regions known to be flexible in FNfn10 (7). (b) Stereo view showing an overlay of FNoTNc (green) and TNfn3 (red). The core residues have the same conformation.

Figure 3.
Fig. 3. Unfolding forces. (a) Unfolding forces of TNfn3 (blue), FNoTNc (red), and FNfn10 (black). FNoTNc unfolds at the same force as TNfn3. Only FNfn10 shows double peaks, indicting the presence of an unfolding intermediate. These traces were collected at a retraction speed of 1,000 nm/s. (b) Histograms of unfolding forces of TNfn3 (blue), FNoTNc (red), and FNfn10 (black) at a retraction speed of 1,000 nm/s. The modal unfolding forces are 127 ± 3 pN (n = 305), 125 ± 3 pN (n = 332), and 104 ± 5 pN (n = 172), respectively [see also supporting information (SI) Fig. 5].

Figures were selected by an automated process.