PDBsum entry 2p81

Transcription

PDB id: 2p81

Contents

Protein chain

44 a.a. *

* Residue conservation analysis

PDB id:

2p81

Name:

Transcription

Title:

Engrailed homeodomain helix-turn-helix motif

Structure:

Segmentation polarity homeobox protein engrailed. Chain: a. Fragment: helix-turn-helix motif. Engineered: yes. Mutation: yes

Source:

Drosophila melanogaster. Fruit fly. Organism_taxid: 7227. Gene: en. Expressed in: escherichia coli. Expression_system_taxid: 562.

NMR struc:

25 models

Authors:

T.L.Religa

Key ref:

T.L.Religa et al. (2007). The helix-turn-helix motif as an ultrafast independently folding domain: The pathway of folding of Engrailed homeodomain. Proc Natl Acad Sci U S A, 104, 9272-9277. PubMed id: 17517666 DOI: 10.1073/pnas.0703434104

Date:

21-Mar-07 Release date: 12-Jun-07

Protein chain

P02836  (HMEN_DROME) -  Segmentation polarity homeobox protein engrailed from Drosophila melanogaster

Seq: 552 a.a.

Struc: 44 a.a.*

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

Enzyme reactions

• Enzyme class: E.C.?

DOI no: 10.1073/pnas.0703434104

Proc Natl Acad Sci U S A 104:9272-9277 (2007)

PubMed id: 17517666

The helix-turn-helix motif as an ultrafast independently folding domain: The pathway of folding of Engrailed homeodomain.

T.L.Religa, C.M.Johnson, D.M.Vu, S.H.Brewer, R.B.Dyer, A.R.Fersht.

ABSTRACT

Helices 2 and 3 of Engrailed homeodomain (EnHD) form a helix-turn-helix (HTH) motif. This common motif is believed not to fold independently, which is the characteristic feature of a motif rather than a domain. But we found that the EnHD HTH motif is monomeric and folded in solution, having essentially the same structure as in full-length protein. It had a sigmoidal thermal denaturation transition. Both native backbone and local tertiary interactions were formed concurrently at 4 x 10(5) s(-1) at 25 degrees C, monitored by IR and fluorescence T-jump kinetics, respectively, the same rate constant as for the fast phase in the folding of EnHD. The HTH motif, thus, is an ultrafast-folding, natural protein domain. Its independent stability and appropriate folding kinetics account for the stepwise folding of EnHD, satisfy fully the criteria for an on-pathway intermediate, and explain the changes in mechanism of folding across the homeodomain family. Experiments on mutated and engineered fragments of the parent protein with different probes allowed the assignment of the observed kinetic phases to specific events to show that EnHD is not an example of one-state downhill folding.

Selected figure(s)

Figure 1.
Fig. 1. Comparison of chemical shifts between EnHD L16A and its 16–59 fragment. (a) The ^1H-^15N HSQC spectra of EnHD residues 16–59 (red) overlayed with that of L16A (blue). Peaks for L16A residues up to 15 are shown as black contours. (b) The natural abundance ^1H-^13C HSQC spectra of EnHD residues 16–59 (red) overlayed with that of L16A (blue). The methyl groups in the fragment were not stereospecifically assigned.

Figure 4.
Fig. 4. Relaxation kinetics of EnHD residues 16–59 monitored by fluorescence (a, b, and f) and IR (c and d) and equilibrium properties (e). (a) Kinetic time course for EnHD residues 16–59 at 25°C, 2 M [NaCl]. The raw data (red) were fitted after subtracting from the NATA trace (blue), giving the rate constant of 206,000 s^–1 (4.8 µs). (b) Kinetic time course for EnHD L16A at 25°C, 2 M [NaCl]. As for the fragment, the raw data were subtracted from the NATA trace and fitted to both single (gray) and double (brown) exponentials. The single-exponential fit gave a rate of 44,000 s^–1, and the double-exponential fit produced rates of 34,000 s^–1 and 205,000 s^–1. (c) Folding of EnHD L16A (red) and 16–59 fragment (blue), monitored by IR at 25°C, 100 mM [NaCl]. (d) As in c, but measured at 500 mM [NaCl]. Two kinetic phases were observed in the folding of L16A and 16–59 fragment at low salt concentrations when monitored by IR: the fast phase and the (previously unidentified) ultrafast phase. In addition to these phases, a slow phase was observed in the folding kinetics of L16A at 500 mM. No slow phase was observed in the folding kinetics of the fragment at high salt, as shown in Table 1. (e) Thermal denaturation of the fragment at increasing salt concentrations, followed by CD at 222 nm. (f) Rates extracted from independent traces at multiple temperatures for the fragment (red) and the L16A mutant (blue/gray) at 2 M [NaCl]. The spread in the data shows the inaccuracy of the measurement and/or data fitting. The L16A data were fitted to a single and a double exponential. At higher temperatures, it was not possible to fit the L16A data to a double exponential.

Figures were selected by an automated process.