Solution structure of the active-centre mutant I14A of the histidine-containing phosphocarrier protein from Staphylococcus carnosus.

Publisher: Blackwell Science Ltd. on behalf of the Federation of European Biochemical Societies Country of Publication: England NLM ID: 0107600 Publication Model: Print Cited Medium: Print ISSN: 0014-2956 (Print) Linking ISSN: 00142956 NLM ISO Abbreviation: Eur J Biochem Subsets: MEDLINE

Publication: -2004: Oxford, UK : Blackwell Science Ltd. on behalf of the Federation of European Biochemical Societies
Original Publication: Berlin, New York, Springer.

Mutation*; Bacterial Proteins/*chemistry ; Phosphoenolpyruvate Sugar Phosphotransferase System/*chemistry ; Staphylococcus/*chemistry; Bacterial Proteins/genetics ; Binding Sites ; Hydrogen Bonding ; Models, Molecular ; Nuclear Magnetic Resonance, Biomolecular ; Phosphoenolpyruvate Sugar Phosphotransferase System/genetics ; Protein Binding ; Protein Conformation ; Xenon/metabolism

High-pressure NMR experiments performed on the histidine-containing phosphocarrier protein (HPr) from Staphylococcus carnosus have shown that residue Ile14, which is located in the active-centre loop, exhibits a peculiarly small pressure response. In contrast, the rest of the loop shows strong pressure effects as is expected for typical protein interaction sites. To elucidate the structural role of this residue, the mutant protein HPr(I14A), in which Ile14 is replaced by Ala, was produced and studied by solution NMR spectroscopy. On the basis of 1406 structural restraints including 20 directly detected hydrogen bonds, 49 1H(N)-15N, and 25 1H(N)-1Halpha residual dipolar couplings, a well resolved three-dimensional structure could be determined. The overall fold of the protein is not influenced by the mutation but characteristic conformational changes are introduced into the active-centre loop. They lead to a displacement of the ring system of His15 and a distortion of the N-terminus of the first helix, which supports the histidine ring. In addition, the C-terminal helix is bent because the side chain of Leu86 located at the end of this helix partly fills the hydrophobic cavity created by the mutation. Xenon, which is known to occupy hydrophobic cavities, causes a partial reversal of the mutation-induced structural effects. The observed structural changes explain the reduced phosphocarrier activity of the mutant and agree well with the earlier suggestion that Ile14 represents an anchoring point stabilizing the active-centre loop in its correct conformation.

0 (Bacterial Proteins)
3H3U766W84 (Xenon)
EC 2.7.1.- (Phosphoenolpyruvate Sugar Phosphotransferase System)
EC 2.7.1.- (phosphocarrier protein HPr)

Date Created: 20041221 Date Completed: 20050706 Latest Revision: 20161017

20221213

10.1111/j.1432-1033.2004.04447.x

15606769