Determination of the three‐dimensional solution structure of the histidine‐containing phosphocarrier protein HPr from Escherichia coli using multidimensional NMR spectroscopy

Abstract

We recorded several types ofheteronuclear three‐dimensional (3D) NMR spectra on 15N‐enriched and 13C/15N‐enriched histidine‐containing phosphocarrier protein, HPr, to extend the backbone assignments [van Nuland, N. A. J., van Dijk, A. A., Dijkstra, K., van Hoesel, F. H. J., Scheek, R. M. & Robillard, G. T. (1992) Eur. J. Biochem. 203, 483–491] to the side‐chain 1H,15N and 13C resonances. From both 3D heteronuclear 1H‐NOE 1H‐13C and 1H‐NOE 1H‐15N multiple‐quantum coherence (3D‐NOESY‐HMQC) and two‐dimensional (2D) homonuclear NOE spectra, more than 1200 NOE were identified and used in a step‐wise structure refinement process using distance geometry and restrained molecular dynamics involving a number of new features. A cluster of nine structures, each satisfying the set of NOE restraints, resulted from this procedure. The average root‐mean‐square positional difference for the Cα atoms is less than 0.12 nm. The secondary structure topology of the molecule is that of an open‐face β sandwich formed by four antiparallel β strands packed against three α helices, resembling the recently published structure of Bacillus subtilis HPr, determined by X‐ray crystallography [Herzberg, O., Reddy, P., Sutrina, S., Saier, M. H., Reizer, J. & Kapafia, G. (1992) Proc. Natl. Acad. Sci. USA 89, 2499–2503). Copyright © 1992, Wiley Blackwell. All rights reserved