2K0D

NMR structure of a mutant colicin e7 immunity protein im7 with an extended helix III

Summary for 2K0D

• Entry DOI: 10.2210/pdb2k0d/pdb
• NMR Information: BMRB: 15645
• Descriptor: Colicin-E7 immunity protein (1 entity in total)
• Functional Keywords: protein, toxin inhibitor
• Biological source: Escherichia coli
• Total number of polymer chains: 1
• Total formula weight: 11094.28

Authors

Figueiredo, A.M.,Whittaker, S.B.,Knowling, S.E.,Spronk, C.A.,Radford, S.E.,Moore, G.R. (deposition date: 2008-02-01, release date: 2009-01-20, Last modification date: 2024-05-29)

Primary citation

Knowling, S.E.,Figueiredo, A.M.,Whittaker, S.B.,Moore, G.R.,Radford, S.E.
Amino acid insertion reveals a necessary three-helical intermediate in the folding pathway of the colicin E7 immunity protein Im7.
J.Mol.Biol., 392:1074-1086, 2009

PubMed Abstract: The small (87-residue) alpha-helical protein Im7 (an inhibitor protein for colicin E7 that provides immunity to cells producing colicin E7) folds via a three-state mechanism involving an on-pathway intermediate. This kinetic intermediate contains three of four native helices that are oriented in a non-native manner so as to minimise exposed hydrophobic surface area at this point in folding. The short (6-residue) helix III has been shown to be unstructured in the intermediate ensemble and does not dock onto the developing hydrophobic core until after the rate-limiting transition state has been traversed. After helix III has docked, it adopts an alpha-helical secondary structure, and the side chains of residues within this region provide contacts that are crucial to native-state stability. In order to probe further the role of helix III in the folding mechanism of Im7, we created a variant that contains an eight-amino-acid polyalanine-like helix stabilised by a Glu-Arg salt bridge and an Asn-Pro-Gly capping motif, juxtaposed C-terminal to the natural 6-residue helix III. The effect of this insertion on the structure of the native protein and its folding mechanism were studied using NMR and varphi-value analysis, respectively. The results reveal a robust native structure that is not perturbed by the presence of the extended helix III. Mutational analysis performed to probe the folding mechanism of the redesigned protein revealed a conserved mechanism involving the canonical three-helical intermediate. The results suggest that folding via a three-helical species stabilised by both native and non-native interactions is an essential feature of Im7 folding, independent of the helical propensity of helix III.

PubMed: 19651139
DOI: 10.1016/j.jmb.2009.07.085

Experimental method

SOLUTION NMR