Crystal Structure of SmcL, a Bacterial Neutral Sphingomyelinase C from Listeria

Abstract

Sphingomyelinases C are enzymes that catalyze the hydrolysis of sphingomyelin in biological membranes to ceramide and phosphorylcholine. Various pathogenic bacteria produce secreted neutral sphingomyelinases C that act as membrane-damaging virulence factors. Mammalian neutral sphingomyelinases C, which display sequence homology to the bacterial enzymes, are involved in sphingolipid metabolism and signaling. This article describes the first structure to be determined for a member of the neutral sphingomyelinase C family, SmcL, from the intracellular bacterial pathogen Listeria ivanovii. The structure has been refined to 1.9-Å resolution with phases derived by single isomorphous replacement with anomalous scattering techniques from a single iridium derivative. SmcL adopts a DNase I-like fold, and is the first member of this protein superfamily to have its structure determined that acts as a phospholipase. The structure reveals several unique features that adapt the protein to its phospholipid substrate. These include large hydrophobic ␤-hairpin and hydrophobic loops surrounding the active site that may bind and penetrate the lipid bilayer to position sphingomyelin in a catalytically competent position. The structure also provides insight into the proposed general base/acid catalytic mechanism, in which His-325 and His-185 play key roles. Sphingomyelinases C (SMases C)4 (EC 3.1.4.12) are phosphodiesterases that catalyze the hydrolysis of the membrane phospholipid sphingomyelin (SM) at the aqueous:lipid interface, generating ceramide and phosphorylcholine. Several types of enzymes with SMase C activity have been identified in eukaryotes and prokaryotes. Eukaryotic SMases C have been classified according to their pH optima and are known as acid SMase (1), alkaline SMase (2), and neutral SMase (nSMase) (3, 4). In prokaryotes, some broad specificity phosphatidylcholine phospholipases C display SM hydrolyzing activity (5, 6) but a number of pathogenic bacteria, such as Staphylococcus aureus (␤-toxin (7)), Bacillus cereus (8), Leptospira interrogans (9), and Listeria ivanovii (10), produce SM-specific phospholipases. These bacterial SMases C share sequence homology with the eukaryotic nSMases, and all currently available data suggests that eukaryotic nSMases and bacterial SMases C (henceforth bacterial nSMases) share a similar catalytic mechanism and overall structure (11,12). A sequence alignment of selected members of the nSMase family is presented in Fig. 1. In contrast to the eukaryotic and bacterial nSMases there is no identifiable sequence conservation within the other types of SMase C. Additionally, these enzymes utilize different catalytic mechanisms, and are predicted to be structurally unrelated to nSMases.Mammalian nSMases are thought to play a key role in sphingolipid metabolism and there is increasing evidence implicating SM metabolites in cell signaling, cell proliferation, and apoptosis (13-16). Two human nSMases have been cloned, nSMase1 (3) and nSMase2 (4). Sequence analysis of these proteins and other e...

Molecular Determinants of Sphingomyelin Specificity of a Eukaryotic Pore-forming Toxin

“…Emerging studies indicate that aromatic amino acids are important for the interaction with membrane SM. Neutral sphingomyelinases, membrane-damaging virulence factors of Gram-positive bacteria, possess a ␤-hairpin with exposed aromatic residues that participate in the binding of membrane SM (69,70). Tryptophans are also involved in SM binding of the earthworm pore-forming toxin, lysenin (72).…”

“…SM-specific proteins are widespread in nature and are found in bacteria (69,70), fungi (71), and animals, i.e. sea anemones (7) and earthworms (58).…”

Structure and Biological Activities of Beta Toxin fromStaphylococcus aureus

“…Data sets for wild-type recombinant beta toxin were collected on Molecular Biology Consortium's beamline 4.2.2 at the Advanced Light Source synchrotron facility at the Lawrence Berkeley National Laboratory with a NOIR1 detector (E. M. Westbrook, J. Morse, R. E. Fischer, W. M. McGuigan, S. K. Onishi, P. Vu, I. Naday, C. Bauer, J. Phillips, T. A. Thorson, and R. D. Durst, presented at X-Ray and Gamma-Ray Detectors and Applications IV, Seattle, WA, 2003). The solution of the structure was found by molecular replacement using the coordinates of a previously solved SMase, Smase, from L. ivanovii (24), as the search model. SHELXL (27) was used for initial structural model refinement, followed by REFMAC (21).…”

Bacterial Sphingomyelinases and Phospholipases as Virulence Factors

“…The molecular masses of bacterial SMase Cs vary from 27 to 39 kDa for most of the enzymes, except for those of Pseudomonas and Leptospira, which have a molecular mass of 58 and 63 kDa, respectively, due to the presence of additional sequences at their C termini (19,20). The three-dimensional structures of enzymes in this group (Bacillus cereus PDB ID 2DDT, Listeria ivanovii PDB ID 1ZWX, Staphylococcus aureus ␤-toxin PDB ID 3I5V, and Streptomyces griseocarneus PDB ID 3WCX) (21)(22)(23) reveal that, although sharing less than 17% sequence identity with mammalian DNase I, they adopt the DNase I fold, as do the mammalian neutral SMases (23). In fact, despite a change in substrate specificity (from an endonuclease to a phosphodiesterase), both enzyme types catalyze the same chemical reaction.…”