Type I Protein Secretion-Deceptively Simple yet with a Wide Range of Mechanistic Variability across the Family

Abstract

A very large type I polypeptide begins to reel out from a ribosome; minutes later, the still unidentifiable polypeptide, largely lacking secondary structure, is now in some cases a thousand or more residues longer. Synthesis of the final hundred C-terminal residues commences. This includes the identity code, the secretion signal within the last 50 amino acids, designed to dock with a waiting ATP binding cassette (ABC) transporter. What happens next is the subject of this review, with the main, but not the only focus on hemolysin HlyA, an RTX protein toxin secreted by the type I system. Transport substrates range from small peptides to giant proteins produced by many pathogens. These molecules, without detectable cellular chaperones, overcome enormous barriers, crossing two membranes before final folding on the cell surface, involving a unique autocatalytic process.Unfolded HlyA is extruded posttranslationally, C-terminal first. The transenvelope "tunnel" is formed by HlyB (ABC transporter), HlyD (membrane fusion protein) straddling the inner membrane and periplasm and TolC (outer membrane). We present a new evaluation of the C-terminal secretion code, and the structure function of HlyD and HlyB at the heart of this nanomachine. Surprisingly, key details of the secretion mechanism are remarkably variable in the many type I secretion system subtypes. These include alternative folding processes, an apparently distinctive secretion code for each type I subfamily, and alternative forms of the ABC transporter; most remarkably, the ABC protein probably transports peptides or polypeptides by quite different mechanisms. Finally, we suggest a putative structure for the Hly-translocon, HlyB, the multijointed HlyD, and the TolC exit.

Figure 1

Organization of the hly, the hasA, and the slaA/lipA/prtA operons. The hly promoter and the binding site of the transcriptional regulators RfhA (213) or Fur are indicated. In the case of the has operon, the surface receptor HasR (gray), and, for the hly operon, the acyltransferase HlyC (green), are also encoded within the operon. The slaA gene encodes a surface protein, lipA a lipase, and prtA a metalloprotease. The allocrite or transport substrate genes are indicated in dark blue, the ABC transporters in brown, the MFPs in red, and the OMP, if present in the operon, in light blue. Please note that the outer membrane protein (TolC in the case of HlyA) is frequently not encoded in the corresponding T1SS operon. CHP in the rtx operon encodes an additional ABC transporter, but how these two transporters function independently or together is unknown (see the text on page 20).

Figure 2

Summary of structural information for the hemolysin A T1SS. (Left) Homology model of dimeric HlyC based on the crystal structure of ApxC (53). The TAAT-specific insertion, which is unique and not present in the GNAT family, is highlighted in red. The Arg residue together with the catalytic triad (composed of a Ser, His, and Asn) that interacts with ACP is shown in ball-and-stick representation. (Right) The NMR structure of the CLD (54) and the crystal structure of the ATP-bound dimer of the NBDs (175) are shown in green and yellow, respectively. The TMD of HlyB and for HlyD are shown schematically as blue and red cylinders, respectively. The trimeric crystal structure of TolC (green/cyan/yellow) (138) is shown in cartoon representation. No structural information for the substrate, HlyA, is available. Please note that the presentation here for the structure, oligomeric state, and the extent of HlyD overlap with TolC is arbitrary. Note, while Koronakis et al. (145) suggested a trimeric arrangement for HlyD, more and more crystal structures and modeling evidence suggest that a closely packed hexameric state, as in other MFP analogues, is essential to maintain a tightly sealed structure. Evidence also suggests a tip-to-tip interaction or a small overlap between the ends of the MFP and TolC as in the AcrAB complex (169). The indicated contact between HlyB and TolC is arbitrary and remains controversial for the analogous tripartite AcrAB-TolC efflux pump (169). See the text for more details.

Figure 3

Known structures of substrates of T1SS. Ca²⁺ ions are highlighted as blue spheres and proteins are displayed in cartoon representation. From top to bottom, a fragment of the giant adhesion SiiE that has non-RTX Ca²⁺ binding sites (121), alkaline protease from P. aeruginosa (5), T1SS RTX lipase LipA (113), and HasA (110), which lacks a Ca²⁺ binding domain. The β-rolls are highlighted. The N and C termini are indicated. Please note that only a single domain of SiiE is shown. The domain architecture is provided above the structure. SiiE contains 53 so-called Big domains shown as black boxes. The N-terminal coiled coil and the C-terminal insertion are shown as white, rectangular boxes. The three Big domains of SiiE that have been crystallized are highlighted by a yellow box. The molecular architecture of a calcium binding site is summarized in the central black box. One Ca²⁺ ion (blue sphere) is coordinated by two GG repeats through interactions of the Ca²⁺ ion with the carboxylate side chain and two carbonyl oxygens of the peptide bond per GG repeat.

Figure 4

Cartoon representation of the HlyA constructs mentioned in the text. The length of the constructs is scaled to their number of amino acids, which are provided for each construct. The major pore-forming domain of HlyA is shown in red, and the secretion signal is in blue. The individual nona repeats in the RTX domain are shown as vertical green bars. N and C termini are indicated. The fragment of HlyA, A1 contains 208 amino acids, A2 contains 160, and A3 contains 102.

Figure 5

Models for the nature of the secretion signal code. (A) Predicted secondary structure of the C-terminal secretion sequence of HlyA. (B) The Ling model derived from directed combinatorial mutagenesis (104) emphasizes the functional importance of the proximal helix I plus the extreme terminal residues. (C) The linear code model (100, 101) emphasizing individual key residues (highlighted in red) essential for function in the hemolysin subfamily, as derived from single-substitution mutagenesis. (D) Secondary structure predictions for the C-terminal region containing the secretion signal sequence of representatives from other subfamilies of T1SS transport substrates. Note in (D) that β-sheet or α-helical regions are predicted for the C-terminal signal region with little conservation of the primary sequence.

Figure 6

Crystal structures of TolC in (A) the closed (PDB entry 1EKP, [138]) and (B) the open state (PDB entry 2XMN [107]). Each monomer of the TolC trimer is colored differently. The length of the periplasmic helices is highlighted. The maximal opening of the closed and open states of TolC is also indicated below the figure.

Figure 7

Structural and functional features of EmrA and its homologue HlyD. (Top) The rectangles represent the distinct conserved regions of EmrA from E. coli (involved in multidrug transport) and HlyD from E. coli, indicating the location of the cytosolic domains, the transmembrane helices, the coiled-coil, the lipoyl and β-barrel domains. Scaling is based on the number of amino acids that contribute to the individual parts. The transmembrane helices are shown in green. The two helical regions of the coiled-coil domain are highlighted by orange boxes and labeled 1 and 2. The two parts of the lipoyl domain are indicated by blue boxes labeled, respectively, N and C for the N- and C-terminal parts of this domain, while the β-barrel domain is represented by brown boxes. The asterisk marks the position of the C terminus of EmrA from A. aeolicus, because this protein has no TMD. The zoom-in shows the sequences of the N-terminal cytoplasmic domains of HlyD, and other MFPs involved in type I secretion, PrtE (protease secretion), LipC (lipase), and LktD (another hemolysin). The putative amphipathic helix (letters in red) and the charged cluster (letters in blue) in HlyD, implicated in interaction with HlyA and consequent recruitment of TolC (145), are highlighted. The positions of the domains of EmrA are derived from the crystal structure obtained for EmrA from A. aeolicus. The corresponding positions in HlyD are estimated from the predicted secondary structures (including the coiled coil). (Bottom) Cartoon representation of the crystal structure of monomeric EmrA from A. aeolicus, which lacks a TMD and an N-terminal cytoplasmic extension (154), with a zoom into the compact form of the associated lipoyl subdomains. The recently determined crystal structure of parts of HlyD (152) is superimposed on the EmrA structure to emphasize the similarity of both proteins (cyan). Black residues in the α-helical hairpin represent the predicted position of the heptad repeats important for coiled-coil formation. In the lipoyl domain, blue residues are identical amino acids in more than 50% of the sequences analyzed in reference , while red residues are similar amino acids in more than 50% of the sequences analyzed. Note the closely adjacent N and C termini made possible by the flexible nature of the α-helical hairpin in EmrA. This would also allow movement to facilitate interaction with the cognate outer membrane protein, TolC.

Figure 8

Mapping the position of mutations on a topology map of HlyD. The topology is based on membrane fractionation experiments and beta-lactamase insertions giving antibiotic resistance (46, 147). Each 50th residue is marked by a black box. Residues 150 to 246 and 251 to 327 define the approximate position of the coiled coils, residues 97 to 128 and 328 to 360 for the two half-lipoyl domains, while the major β-barrel occupies the C-terminal domain. The lipoyl domains, in particular, are likely to be involved in tightly packing the HlyD protomers, while the β-barrel by analogy with other MFPs could be involved in interaction with HlyB. Orange residues encompass the position (close to the middle of the helical hairpin) of the reportedly conserved RLT motif proposed to interact with TolC (214). At the N terminus, key regions required for binding HlyA and consequent triggering of TolC recruitment were defined (145) by the N-terminal deletions discussed in the text: deletion 1 removes the first 20 residues (the majority of the putative amphiphilic helix); deletion 2, the first 40 residues; deletion 3, the first 45 residues; and the internal deletion 4 removes the charged cluster, which is especially important for recruitment of TolC. Blue residues indicate secretion-defective mutations from different groups. * marks mutations blocking secretion, conditional on high calcium ion concentrations in the medium (147). These include mutations having possible effects on HlyD packing and map mainly in the lipoyl domain and the β-barrel.

Figure 9

Mutations localized to the HlyB topology model. This is a composite based on beta-lactamase, beta-galactosidase, and alkaline phosphatase insertions, as described in the text. Each 50th residue in the sequence is marked by the black box. The crystal structures of the CLD and NBD were derived from purified fragments, residue 1 to 130 and residue 467 to the terminus, respectively. The conserved motifs Walker A, C-loop, Walker B, and the histidine of the H-loop, all in the NBD, are highlighted in blue, red, green, and brown, respectively. Letters indicate mutations identified in different genetic screens. Green letters highlight those mutations that affected the level of secretion, including three temperature sensitive mutations marked *, one of which (marked **) also grew poorly at 42°C. Letters in blue boxes are mutations in HlyB suppressing one or other of two deletion mutations in the HlyA secretion signal. Residues additionally marked by a # affected the oligomerization of HlyD, while triangles are secretion-defective insertions. Note the “hotspot” for mutations in the relatively well-conserved region, predicted to be periplasmic domain P3. In the case of ambiguities, for the periplasm the residue substituted is to the right. See text for other details.

Figure 10

(A) ATP/Mg²⁺ bound H662A structure for the head-to-tail dimer of the HlyB-NBD (174). The RecA core of the NBD is shown in green and pale green, respectively, while the α-helical subdomain in yellow/pale yellow and the β-subdomain in gray. The conserved motifs Walker A, Walker B, and C-loop are highlighted in blue, magenta, and red, respectively. Conserved residues interacting with the nucleotide (arrowed) sandwiched between the two monomers are highlighted in ball-and-stick representation and labeled. The bound cofactor Mg²⁺ is shown as a green sphere. (B) Zoom into the ATP binding site. Please note that S606 (from the conserved C-loop) resides in the opposite monomer.