Crystal structure of RimI from Salmonella typhimurium LT2, the GNAT responsible for N(alpha)-acetylation of ribosomal protein S18.

The bacterial ribosome is a complex biological machine composed of three ribosomal RNA (rRNA)1 molecules and 55 proteins. However, it is poorly appreciated that the complexity of the ribosome is increased by post-translational modifications to both the rRNA and ribosomal proteins. While these modifications, which include methylation and acetylation, have been known for decades, a complete understanding of how, when, and why these modifications are made and their contribution to the structure and function of the ribosome remain unknown. A better understanding of how ribosomal modifications are made, at what stage in ribosome biosynthesis they are made, and their influence on the structure or function of the ribosome would be useful for a complete understanding of ribosome assembly and function. Acetylation of the alpha amino group of protein N-terminal amino acids is very common in eukaryotes. In some cells, estimates have shown more than half of all proteins are Nα-acetylated (Driessen et al. 1985; Bradshaw et al. 1998). However, the number of Nα-acetylated proteins in prokaryotes is limited to seven known examples (Table 1). It is not known why ribosomal proteins are overrepresented in this list, nor is the function of any of the acetylations known. Furthermore, except for RimI, RimJ, and RimL, which Nα-acetylate the ribosomal proteins S18, S5, and L12, respectively, the acetyltransferases responsible for the other Nα-acetylations are not known. Table 1. Prokaryotic proteins known to be Nα-acetylated RimI, RimJ, and RimL were all initially identified using a nitrosoguanidine mutagenesis strategy yielding bacteria with a temperature-sensitive growth phenotype and alterations in the ribosomal proteins as assessed by two-dimensional SDS-PAGE gels (Isono and Isono 1978). The Isono group later determined that the temperature-sensitive growth phenotype was independent of the acetylation of S5, S18, and L12, and when cultured in rich media, Escherichia coli can survive without each of the Nα-acetyltransferases; however, these post-translational modifications may have other functions that are not essential for viability. Furthermore, using their strains with mutations in the acetyltransferases, the Isono group determined that each acetyltransferase is specific for only one ribosomal protein (Cumberlidge and Isono 1979; Isono and Isono 1980, 1981). The structural and biochemical data on ribosomal proteins sheds little light on the possible function of their Nα-acetylation. Several mutations in the 16S ribosomal RNA were found to impair acetylation of S5, and since these ribosomal RNA mutations are proximal to S5 on the ribosome, it suggests that S5 may be acetylated while assembled in the 70S ribosome or 30S subunit (Poot et al. 1997). Furthermore, underacetylation of S5 was also observed when ribosomal protein S4, a protein S5 associates with on the ribosome, was altered by drastic gene mutations (Cumberlidge and Isono 1979). Ribosomal protein L12 and its Nα-acetylated version, L7, are commonly referred to as L7/L12. Interestingly, the relative proportion of L7 to L12 is known to vary with growth phase, with L12 predominant in early log phase, whereas L7 predominates at the stationary phase (Ramagopal and Subramanian 1974). In general, L7/L12 appears to be involved in all ribosome reactions where GTP is hydrolyzed (Brot et al. 1974), and is the binding site for EF-G and the antibiotic thiostrepton (Joseph 2003). The ribosomal protein S18, the substrate for RimI, is found in the central domain of the 30S subunit of the ribosome, and interacts so tightly with the ribosomal protein S6 (8.7 nM) that it is effectively a functional heterodimer (Recht and Williamson 2001). The N-terminal 15 amino acids of S18 are not seen in the crystal structure of the 30S subunit of the bacterial ribosome, and are likely to be solvent exposed (Brodersen et al. 2002). The prokaryotic RIM Nα-acetyltransferases and eukaryotic Nα-acetyltransferases, by sequence comparisons, all appear to belong to the GCN5-related N-acetyltransferase (GNAT) family, which acetylate a diverse set of substrates with primary amines, ranging from small molecules to large proteins (Vetting et al. 2005a). One hallmark of GNAT proteins is the very low sequence homology between family members with different target substrates. While RimJ and RimL appear to exhibit a distant sequence homology, RimI appears to be unique especially in light of its smaller size. For example, in the case of Salmonella typhimurium LT2, the sequence identity between RimI (148 residues) and RimJ (194 residues) and RimL (178 residues) is 19% and 20%, respectively, while the sequence identity between RimJ and RimL is 23%. This is despite the fact that RimI and RimJ are termed ribosomal alanine Nα-acetyltransferases, while RimL is termed a serine Nα-acetyltransferase. Based on the low sequence identity it is most likely that the RIM proteins do not have a common ancestor, and evolved independent of each other. In addition, the designation of the RIM proteins as serine or alanine Nα-acetyltransferases may be an artifact of the organism from which they were first purified, and RIM proteins from other organisms may have different Nα-terminal amino acid targets. The structure of RimL from Salmonella typhimurium LT2 was recently determined in complex with CoA and AcCoA; however, only a model of its interaction with its substrate L12 was proposed (Vetting et al. 2005b). Determination of enzyme functional groups, which are crucial to substrate specificity and catalysis require three-dimensional structures of all relevant substrates. It is often the case that structures of these complexes are not possible due to protein dynamics, limitations in “crystallization space,” and the requirement that the substrates are not turned over. Bisubstrate inhibitors can be utilized to overcome these difficulties as they are often good mimics of the active substrates but do not exhibit turnover and have additive or synergistic binding characteristics when compared to the individual substrates. Bisubstrate inhibitors have been successfully used to obtain structural information with other GNAT family members (Poux et al. 2002; Wolf et al. 2002; Hegde et al. 2007; Magalhaes et al. 2008). In the case of the research presented here, the bisubstrate analog was enzymatically synthesized using RimIST, chloroacetyl-CoA, and a peptide substrate. We demonstrate that the structure of an enzyme–bisubstrate complex produced in this manner is useful for providing intermolecular details between conserved residues of the enzyme and its target substrate. This is the first description of the interactions of an Nα-acetyltransferase with its peptide substrate.