Revealing the secrets of proteins: A triple-nuclear and multi-dimensional high-resolution NMR spectroscopy study

Abstract

Nuclear magnetic resonance (NMR) spectroscopy is one of the major tools for studying the structure and dynamics of proteins in solution. The still ongoing development of new and improved pulse-sequences for multi-dimensional, heteronuclear NMR experiments has made the field applicable for continuously larger biomolecules. In this study, a variety of NMR experiments was conducted on two rather different proteins: chicken brain α-spectrin repeat 17 (R17) and human Nα-acetyltransferase 50 protein (hNaa50p). The aims were to investigate the thermodynamical and conformational stabilities of the former and the enzyme mechanism of the latter protein, respectively. R17 is a 13 kDa domain of the ubiquitous structural protein spectrin, which is a key component in the cytoskeleton. Its structure consists of a triple-helix bundle, where two loops connect the long helices. Spectrin repeats have shown large diversities in their thermodynamical and conformational stabilities, which probably is associated with function. R17 and its neighbouring repeat, R16, are two thoroughly studied domains. R16 was one of only two spectrin repeats that had been investigated by NMR spectroscopy prior to this study. Because R17 is four times less stable than R16 in terms of ΔG, the latter domain was used for stability comparison. NMR studies, including dynamic measurements and hydrogen-deuterium exchange, in combination with NMR, circular dichroism and fluorescence measurements at stepwise increasing temperatures revealed that the repeat is rigid at room temperature but that both the thermodynamical and conformational stabilities are gradually reduced when the temperature exceeds 40 °C. The destabilization of the domain seems to initiate in the centre of helix C and the amino acids that are close in space to one particular residue, V99, in the triple-helix bundle. A multiple sequence alignment of 35 chicken brain spectrin repeats revealed that this valine is a rare substituent of a moderately conserved tryptophan at that position. Previous studies had shown that the small valine side chain introduces a cavity in the centre of the triple-helix bundle and diminishes the amount of hydrophobic interactions in the core of the repeat. Thus, the substitution of this tryptophan appears to be the most probable reason for the reduced stability of R17 compared to R16. hNaa50p is an almost 20 kDa enzyme that exhibits both Nα- and Nɛ-acetyltransferase activity. In these reactions, the acetyl group of acetyl-Coenzyme A (acetyl-CoA) is transferred to the backbone of the N-terminal amino acid and the side chain of specific lysine residues, respectively. The substrate specificity of hNaa50p’s Nα-acetyltransferase function is preferably directed towards peptides starting with the amino acid sequence MLGP, whereas K34, K37 and K140 are Nɛ-auto acetylated. hNaa50p appears both freely in the cell and associated to two other proteins, hNaa10p and hNaa15p, which together make up the NatE complex. hNaa50p is essential for proper sister chromatid cohesion and chromosome resolution, and has increasingly been linked to cancer development. An inhibitor of its enzyme activity could destabilize chromosome formation and prevent nuclear division, and thus function as anti-cancer drug. In order to get insight into the Nα-acetyltransferase reaction mechanism, the affinities of hNaa50p towards its substrates and products, and the enzyme mechanism itself were studied by enzymatic assays and NMR spectroscopy. According to our results, acetyl-CoA is the first substrate to enter and bind the active site, whereas CoA functions as product inhibitor. The peptide is not able to bind to hNaa50p in the absence of acetyl-CoA and its product, acetylated peptide, showed no affinity for the enzyme. Altogether, this indicates that hNaa50p follows an ordered sequential mechanism, possibly of the Theorell-Chance type. Thus, an inhibitor whose binding leads to the formation of a ternary complex might be a promising anti-cancer drug.