A Highly Selective Oligopeptide Binding Protein from the Archaeon Sulfolobus Solfataricus

SSO1273 of Sulfolobus solfataricus was identified as a cell surface-bound protein by a proteomics approach. Sequence inspection of the genome revealed that the open reading frame of sso1273 is associated in an operon-like structure with genes encoding all the remaining components of a canonical protein-dependent ATP-binding cassette (ABC) transporter. sso1273 gene expression and SSO1273 protein accumulation on the cell surface were demonstrated to be strongly induced by the addition of a peptide mixture (tryptone) to the culture medium. The native protein was obtained in multimeric form, mostly hexameric, under the purification conditions used, and it was characterized as an oligopeptide binding protein, named S. solfataricus OppA (OppA_Ss). OppaA_Ss possesses typical sequence patterns required for glycosylphosphatidylinositol lipid anchoring, resulting in an N-linked glycoprotein with carbohydrate moieties likely composed of high mannose and/or hybrid complex carbohydrates. OppA_Ss specifically binds oligopeptides and shows a marked selectivity for the amino acid composition of substrates when assayed in complex peptide mixtures. Moreover, a truncated version of OppA_Ss, produced in recombinant form and including the putative binding domain, showed a low but significant oligopeptide binding activity.Sulfolobus solfataricus is an obligate aerobe that grows in hot and acidic environments either chemolithotrophically by oxidizing metal cations (Fe²⁺ or S⁻) or heterotrophically on simple sugars. It originates from a solfataric field with temperatures between 75°C and 90°C and pH values of 1.0 to 3.0 (9, 15). Within its environment, Sulfolobus can interact with a complex ecosystem consisting of a variety of primary producers and decomposers of organic matter. Moreover, biotopes such as the solfataric field of Sulfolobus contain decomposing materials of higher plants, including cellulose, starch, and proteinaceous compounds, that can act as potential carbon sources. Although S. solfataricus has been reported to grow on a wide variety of reduced organic compounds as the sole carbon and energy source (15), the nutrient utilization by this microorganism requires complex mechanisms of uptake and metabolism that are not yet well defined.Numerous efforts have been directed toward the identification of the carbohydrate utilization strategy in this hyperthermophilic archaeon (18, 23). The metabolic pathways for the degradation of a variety of sugars have been studied in detail and provide evidence that S. solfataricus predominantly uses binding-protein-dependent ABC transporters for the uptake of carbohydrate compounds (1, 2, 13).Archaeal ABC uptake systems are divided into two main classes: the carbohydrate (CUT) and the di-/oligopeptide uptake transporter classes (2). These transporter families use ATP hydrolysis to drive a unidirectional accumulation of solutes into the cytoplasm. The translocator components are composed of two integral membrane proteins, two peripheral membrane proteins that bind and hydrolyze ATP, and an extracellular substrate-binding protein (SBP). The SBP subunit captures and delivers the substrate to the translocon, and it is therefore considered to be one of the determinants of the transport specificity (2, 7, 10).All sequenced genomes of archaea and thermophilic bacteria contain a large number of genes encoding putative ABC transport systems involved in the uptake of organic solutes. The preference of hyperthermophiles for ABC-type transporters could be important for the survival strategy in their natural habitat. In the nutrient-poor environments, such as hydrothermal vents or sulfuric hot springs, in which these organisms thrive, ABC transporters have the advantage that they can scavenge solutes at very low concentrations due to the high binding affinities of their SBP components. Furthermore, these transporters can catalyze translocation at a high rate, resulting in high internal concentrations of solutes. In contrast, secondary transport systems exhibit lower binding affinities, which make these systems less suitable for growth in extreme environments.So far, attempts to predict the functional specificity of the ABC transporters using computational tools have been largely unsuccessful (2, 13, 20). For example, some characterized archaeal sugar transporters, based on the sequence identity and domain organization, were predicted to be di-/oligopeptide transporters (13, 20). These include the cellobiose/β-glucoside transporter system of Pyrococcus furiosus (20) and the maltose/maltodextrin and cellobiose/cello-oligomer transporters of S. solfataricus (13). However, genes encoding sugar-metabolizing enzymes are located in the vicinity of all these transport systems, suggesting that the location of the ABC operon can support the specific transport function.Like oligopeptide binding proteins, MalE and CbtA bind a broad range of polymeric substrates (13, 20). In contrast, sugar-binding proteins usually exhibit a narrow substrate specificity that is often limited to monosaccharides. Therefore, it may well be that the substrate binding pocket of CbtA and MalE resembles that of the OppA family of binding proteins that can accommodate a range of short and long oligopeptides.S. solfataricus contains 37 putative ABC transporters at the genome level (TransportDB, Genomic Comparisons of Membrane Transport systems [http://www.membranetransport.org/index.html]), but only a few of these systems have been functionally characterized. It is interesting that all of these are implicated in the uptake of mono-/oligosaccharides (1, 13, 20, 25).The present work describes the isolation and characterization of the first functional ABC substrate binding protein from S. solfataricus belonging to the di-/oligopeptide transporter family, named S. solfataricus OppA (OppA_Ss). We demonstrate that OppA_Ss is an outer-cell-surface-anchored protein and that its expression is highly induced in the presence of a source of peptides in the culture broth. Furthermore, in vitro substrate specificity studies using complex oligopeptide mixtures indicate that OppA_Ss is highly selective in peptide recognition.