Structures of wild-type and mutant human spermidine/spermine N1-acetyltransferase, a potential therapeutic drug target

Abstract

Spermidine/spermine N1-acetyltransferase (SSAT) is a key enzyme in the control of polyamine levels in human cells, as acetylation of spermidine and spermine triggers export or degradation. Increased intracellular polyamine levels accompany several types of cancers as well as other human diseases, and compounds that affect the expression, activity, or stability of SSAT are being explored as potential therapeutic drugs. We have expressed human SSAT from the cloned cDNA in Escherichia coli and have determined high-resolution structures of wild-type and mutant SSAT, as the free dimer and in binary and ternary complexes with CoA, acetyl-CoA (AcCoA), spermine, and the inhibitor N1,N11bis-(ethyl)-norspermine (BE-3-3-3). These structures show details of binding sites for cofactor, substrates, and inhibitor and provide a framework to understand enzymatic activity, mutations, and the action of potential drugs. Two dimer conformations were observed: a symmetric form with two open surface channels capable of binding substrate or cofactor, and an asymmetric form in which only one of the surface channels appears capable of binding and acetylating polyamines. SSAT was found to self-acetylate lysine-26 in the presence of AcCoA and absence of substrate, a reaction apparently catalzyed by AcCoA bound in the second channel of the asymmetric dimer. These unexpected and intriguing complexities seem likely to have some as yet undefined role in regulating SSAT activity or stability as a part of polyamine homeostasis. Sequence signatures group SSAT with proteins that appear to have thialysine Nepsilon-acetyltransferase activity.

Fig. 1.

Structure of apoSSAT. (A) Ribbon diagram showing two orthogonal views of the asymmetric dimer colored according to the topology diagram in B. (B) Topology of the secondary structure elements in chain A. From the N terminus, secondary structure elements are colored green (β1, η1, α1, η2, and α2), yellow (β2, η3, β3, and β4), red (η4, α3, and β5), cyan (α4, β6, and η5), and deep blue (β7 and α5). The C-terminal elements β7 and α5 (enclosed in a broken black line) form part of the core structure of the opposing monomer. The structure represented is that of PDB ID 2B5G5, in which density was not observed for residues 28–31 and 170–171 of chain A nor for residues 2–3, 47–50, 61–63, or 171 of chain B.

Fig. 2.

Alignment of SSAT with known TLAs and the PaiA polyamine N¹-acetyltransferase of Bacillus subtilis, showing positions of secondary structure elements in the two protein structures. Dots above the SSAT sequence mark every tenth amino acid; helices of PaiA are numbered to correspond to those of SSAT, except that αC of PaiA has no corresponding helix in SSAT. The alignment shown summarizes the much more extensive alignment in Fig. 5, which is published as supporting information on the PNAS web site. Symbols above the SSAT sequence are as follows: A, residues involved in AcCoA binding; B, residues forming the alternate pocket for the acetyl group of AcCoA; K, residues involved in directing the acetyl group of AcCoA bound in channel 2 toward K26; P, residues implicated in binding polyamine or BE-3-3-3; W, residues that are simply part of the wall of the surface channel (as discussed in the text or shown in Figs. 3 and 4). Symbols that indicate similarities and differences in amino acid character between the SSAT-like sequences and PaiA-related sequences are as follows: *, the same amino acid in all proteins of the extensive alignment; C, conservation; S, similarity; D, difference. Potentially significant differences between conserved residues in vertebrate SSATs and in known or putative TLAs (apparent in the extensive alignment) occur at SSAT positions K26, Y29, M76, D82, P83, and A130.

Fig. 3.

Space-filling view of the two channels on opposite surfaces of the asymmetric dimer. The channels are at the dimer interface, with chain A mostly to the upper left in the view of channel 1 and to the lower right in the view of channel 2. The two views are related by a 180° rotation about the noncrystallographic 2-fold axis. Negatively charged residues that line the channels are colored red, positively charged residues are colored blue, and hydrophobic residues are colored yellow. The suffix a or b on the amino acid designations indicates whether the residue is from chain A or B. Arrows point to the parts of channel 1 that house AcCoA and polyamine. Channel 2 is occluded by residues 27–29 of chain B, which prevents polyamine but not CoA from binding.

Fig. 4.

Simulated-annealing omit maps showing the electron density and interactions of bound CoA and BE-3-3-3. The electron density corresponding to CoA (A and B) and BE-3-3-3 (C) is drawn as light blue chicken wire. Oxygen atoms are red spheres, and nitrogen atoms are blue spheres. (A) Interactions of CoA that occur in both channel 1 and 2 of the asymmetric dimer. Backbone nitrogens from P-loop residues G102, F103, and G104 interact with the pyrophosphate moiety of CoA. R101, Y140, R142, and R143 side chains from the same monomer are drawn in ball-and-stick representation. (B) Hydrogen bond network with chain A residues that houses the pantetheine moiety of CoA in channel 1 of the asymmetric dimer. Side-chain residues of 94 and 95 beyond C_β have been omitted for clarity. Hydrogen bonds are drawn as dashed lines. (C) Interactions of BE-3-3-3 in channel 1 of the asymmetric dimer. Ball-and-stick representations of residues from chain A and B of the asymmetric dimer are drawn in purple and green, respectively.