Abstract
The H-NS protein plays a key role in condensing DNA and modulating gene expression in bacterial nucleoids. The mechanism by which this is achieved is dependent, at least in part, on the oligomerization of the protein. H-NS consists of two distinct domains; the N-terminal domain responsible for protein oligomerization, and the C-terminal DNA binding domain, which are separated by a flexible linker region. We present a multidimensional NMR study of the amino-terminal 64 residues of H-NS (denoted H-NS 1-64) from Salmonella typhimurium, which constitute the oligomerization domain. This domain exists as a homotrimer, which is predicted to be self-associated through a coiled-coil configuration. NMR spectra show an equivalent magnetic environment for each monomer indicating that the polypeptide chains are arranged in parallel with complete 3-fold symmetry. Despite the limited resonance dispersion, an almost complete backbone assignment for 1H N, 1H α, 15N, 13CO and 13C α NMR resonances was obtained using a suite of triple resonance experiments applied to uniformly 15N-, 13C/ 15N- and 2H/ 13C/ 15N-labelled H-NS 1-64 samples. The secondary structure of H-NS 1-64 has been identified on the basis of the analysis of 1H α, 13C α, 13C β and 13CO chemical shifts, NH/solvent exchange rates, intra-chain H N-H N and medium-range nuclear Overhauser enhancements (NOEs). Within the context of the homotrimer, each H-NS 1-64 protomer consists of three α-helices spanning residues 2-8, 12-20 and 22-53, respectively. A topological model is presented for the symmetric H-NS 1-64 trimer based upon the combined analysis of the helical elements and the pattern of backbone amide group 15N nuclear relaxation rates within the context of axially asymmetric diffusion tensor. In this model, the longest of the three helices (helix 3, residues 22-53) forms a coiled-coil interface with the other chains in the homotrimer. The two shorter N-terminal helices fold back onto the outer surface of the coiled-coil core and potentially act to stabilise this configuration.
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