Archaeal binding protein-dependent ABC transporter: molecular and biochemical analysis of the trehalose/maltose transport system of the hyperthermophilic archaeon Thermococcus litoralis

Abstract

We report the cloning and sequencing of a gene cluster encoding a maltose/trehalose transport system of the hyperthermophilic archaeon Thermococcus litoralis that is homologous to the malEFG cluster encoding the Escherichia coli maltose transport system. The deduced amino acid sequence of the malE product, the trehalose/maltose-binding protein (TMBP), shows at its N terminus a signal sequence typical for bacterial secreted proteins containing a glyceride lipid modification at the N-terminal cysteine. The T. litoralis malE gene was expressed in E. coli under control of an inducible promoter with and without its natural signal sequence. In addition, in one construct the endogenous signal sequence was replaced by the E. coli MalE signal sequence. The secreted, soluble recombinant protein was analyzed for its binding activity towards trehalose and maltose. The protein bound both sugars at 85 degrees C with a Kd of 0.16 microM. Antibodies raised against the recombinant soluble TMBP recognized the detergent-soluble TMBP isolated from T. litoralis membranes as well as the products from all other DNA constructs expressed in E. coli. Transmembrane segments 1 and 2 as well as the N-terminal portion of the large periplasmic loop of the E. coli MalF protein are missing in the T. litoralis MalF. MalG is homologous throughout the entire sequence, including the six transmembrane segments. The conserved EAA loop is present in both proteins. The strong homology found between the components of this archaeal transport system and the bacterial systems is evidence for the evolutionary conservation of the binding protein-dependent ABC transport systems in these two phylogenetic branches.

FIG. 1

Constructs for the expression of malE from T. litoralis in E. coli. (A) pRHo1000. The cleavable signal sequence of E. coli MalE replaced the lipid anchor sequence of TMBP. Arrowhead 2 indicates where E. coli MalE is cleaved during secretion; arrowhead 1 shows the N terminus (determined by amino acid sequencing) of the periplasmic hybrid protein expressed in E. coli. The shaded sequence is that of the hybrid protein. (B) pRHo1002. The sequence of the hybrid protein from panel A has been shortened; its N terminus now is after the expected E. coli cleavage site SASALA. The N-terminal lysine (K) has been changed to methionine (M). The protein was soluble and remained in the cytoplasm. The shaded sequence is that of the hybrid protein. (C) pRHo1001. The intact malE gene of T. litoralis, including its lipid anchor-encoding sequence, was cloned after the inducible E. coli promoter. The protein expressed in E. coli (shaded sequence) was lipid modified and tightly membrane bound. The putative site of lipidation and its mature N terminus (cysteine) is indicated by arrowhead 3. Asterisks indicate identity; dots indicate homologous exchanges. Ec, E. coli; Tl, T. litoralis.

FIG. 2

SDS-PAGE of the native binding protein as isolated from the membrane of T. litoralis. Lanes: 1, membrane preparation from the uninduced strain (no yeast extract in the medium); 2, membrane preparation from the induced strain (with yeast extract in the medium); 3, purified protein containing the lipid anchor; 4, water-soluble hybrid protein (the construct carries the cleavable signal sequence of E. coli at its N terminus) isolated from the E. coli periplasm (as a reference); St, molecular mass standards (from top to bottom: 66, 45, 36, 29, 24, 20.1, and 14.2 kDa).

FIG. 3

Fluorescence changes of TMBP in the presence of trehalose and maltose. The recordings were done consecutively. First, the middle trace was obtained after temperature equilibration without addition of substrate. The upper trace was then recorded after the addition of 1 μM (final concentration) maltose, followed by the lower trace after the addition of 100 μM trehalose. Similar tracings in the reverse order were obtained when the order of the additions was reversed, i.e., first 1 μM trehalose and then 100 μM maltose (data not shown). Conditions were as follows: temperature, 55°C; excitation, 280 nm; TMBP concentration, 16 μg/ml; buffer, 50 mM Tris-HCl (pH 7.5) containing 0.6% LDAO.

FIG. 4

Partial nucleotide sequences of the T. litoralis malE, -F, and -G genes and flanking regions. The deduced amino acid sequences are given in one-letter code below the nucleotide sequences, and the nucleotide and amino acid numbers are given on the right. 300/13 indicates the last amino acid of the malF gene product (no. 300) and last amino acid of MalG in this line (no. 13). In the nucleotide sequence, a putative boxA promoter element is boxed, the putative ribosome binding site is in boldface, and a putative terminator sequence is underlined. In the deduced amino acid sequence of TMBP (the malE gene product), the conserved recognition sequence for the cleavage sites of lipoprotein signal peptidases is underlined and the presumably formed N-terminal cysteine of the mature protein is in boldface and marked with an arrowhead. Most of the sequence within the structural genes is omitted, as indicated by points and deletion signs. The complete sequence is deposited in GenBank under accession no. AF012836.

FIG. 5

Alignment of the signature sequences of cluster 1 binding proteins with TMBP of T. litoralis. The different amino acids possible for each position are indicated below the signature sequence. Numbers in parentheses indicate positions. The highly conserved lysine residue (K) is boxed. Residues of T. litoralis that match the signature sequence are in boldface, and residues conserved in other sequences are underlined. The signature sequence is from reference . Tl, T. litoralis; Ec, E. coli (16); St, Salmonella typhimurium (12); Sp, S. pneumoniae (39); Tt, T. thermosulfurigenes (41); MalE, MalX, and AmyE, maltose/maltodextrin-binding proteins; UgpB (35), glycerol-3-phosphate-binding protein.

FIG. 6

Putative MSS in inner membrane proteins. (A) T. litoralis and E. coli MalF proteins. In contrast to E. coli MalF, the T. litoralis protein has only six putative MSS (there are eight in E. coli MalF) and no large periplasmic loop between MSS3 and MSS4. MSS1 and MSS2 from E. coli MalF are missing in T. litoralis MalF. Since the numbering of the MSS corresponds to the E. coli sequence, the most N-terminal MSS of TMBP is termed MSS3. (B) T. litoralis and E. coli MalG proteins. The positions of the MSS appear to be conserved; the numbering is identical to that of the E. coli sequence. Tl, T. litoralis; Ec, E. coli. Asterisks indicate identical residues; dots indicate homologous residues.

FIG. 7

SDS-PAGE analysis of TMBP after expression in E. coli. (A) TMBP with the N-terminal cleavable signal sequence of E. coli (construct A in Fig. 1). Lanes: 1, uninduced E. coli cells harboring pRHo1000; 2, induced cells; 3, cytoplasmic extract of induced cells that were treated by cold osmotic shock; 4, same as lane 3 but the sample was heated to 80°C for 10 min; 5, periplasmic shock proteins of induced cells; 6, same as lane 5 but the sample was heated to 80°C for 10 min; 7, TMBP purified from the sample shown in lane 6; 8, TMBP isolated from T. litoralis membranes; St, molecular mass standards (from top to bottom: 94, 67, 43, and 30 kDa). (B) Cytoplasmic TMBP; its N terminus is the expected cleavage site after the E. coli signal sequence (construct B in Fig. 1). Lanes: 1, uninduced E. coli cells harboring pRHo1002; 2, induced E. coli cells; 3, cytoplasmic extract of induced cells; 4, same as lane 3 but the sample was heated to 80°C for 10 min; 5, purified periplasmic TMBP (construct A in Fig. 1); 6, TMBP isolated from T. litoralis; St, as for panel A. (C) Native precursor TMBP from T. litoralis. The protein was lipid modified and membrane bound (construct C in Fig. 1). Lanes: 1, membranes from E. coli harboring pRHo1001 solubilized in octyl-β-glucoside and induced for the expression of the native precursor TMBP; 2, same as lane 1 but the sample was heated for 10 min to 80°C; 3, purified TMBP from T. litoralis; 4, purified periplasmic TMBP endowed with the E. coli signal sequence; St, as for panel A. (D) Western blot of TMBP from T. litoralis membranes and from the different constructs expressed in E. coli. Lanes: 1, whole E. coli cells harboring pRHo1000 encoding the hybrid protein with the E. coli signal sequence; 2, whole cells harboring pRHo1002 encoding the hybrid protein without the E. coli signal sequence; 3, whole cells harboring pRHo1001 encoding the intact T. litoralis protein; 4, membrane preparations of T. litoralis uninduced for the transport system; 5, membrane preparations of T. litoralis induced for the transport system; St, protein standards; 6, same as lane 1 (whole cells harboring pRHo1000); 7, French pressure cell extract of cells harboring pRHo1000; 8, periplasmic proteins isolated from cells harboring pRHo1000.

FIG. 8

PAGE under nondenaturing conditions and in the presence of radioactively labeled trehalose. Lanes: 1, cytoplasmic extract (not heat treated) of cells containing pRHo1000 (construct A in Fig. 1); 2, purified periplasmic TMBP (construct A in Fig. 1); 3, heterologously expressed wild-type TMBP (construct C in Fig. 1) solubilized in 1% octyl-β-glucoside; 4, purified TMBP solubilized in 1% octyl-β-glucoside. The gel was dried and autoradiographed. Staining was indicative of trehalose binding. Lane 3 contains two forms of the protein, one of which hardly entered the gel.

FIG. 9

Binding affinity of the periplasmic form of TMBP as measured by substrate retention. Exit of substrate (trehalose [A] or maltose [B]) from a dialysis bag containing purified periplasmic TMBP (0.41 μM) (closed symbols) or substrate only (open symbols) is shown. Samples of 20 μl were removed from the dialysis bag at different time intervals, and radioactivity was counted in a scintillation counter. The half-life of internal substrate was calculated after the rate of exit had become first order. The temperature was kept constant at 85°C. The half-life in the presence of TMBP (37 min in the case of trehalose and 41 min in the case of maltose) was larger by the factor 1 + (P/K_d) than that in the absence of the protein (10.1 and 11.5 min, respectively) (P is the concentration of TMBP in molar concentration of binding sites; one binding site per polypeptide chain was assumed).