Selective transport by SecA2: an expanding family of customized motor proteins

Abstract

The SecA2 proteins are a special class of transport-associated ATPases that are related to the SecA component of the general Sec system, and are found in an increasingly large number of Gram-positive bacterial species. The SecA2 substrates are typically linked to the cell wall, but may be lipid-linked, peptidoglycan-linked, or non-covalently associated S-layer proteins. These substrates can have a significant impact on virulence of pathogenic organisms, but may also aid colonization by commensals. The SecA2 orthologues range from being highly similar to their SecA paralogues, to being distinctly different in apparent structure and function. Two broad classes of SecA2 are evident. One transports multiple substrates, and may interact with the general Sec system, or with an as yet unidentified transmembrane channel. The second type transports a single substrate, and is a component of the accessory Sec system, which includes the SecY paralogue SecY2 along with the accessory Sec proteins Asp1-3. Recent studies indicate that the latter three proteins may have a unique role in coordinating post-translational modification of the substrate with transport by SecA2. Comparative functional and phylogenetic analyses suggest that each SecA2 may be uniquely adapted for a specific type of substrate. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.

Keywords: Accessory Sec system; Asp1; Asp2; Bacterial glycoprotein; Glycoprotein transport; S-layer.

Figure 1. Phylogenetic relatedness of the SecA and SecA2 proteins

Phylogenetic analysis of the SecA and SecA2 proteins from selected Gram-positive bacterial species was performed using DARWIN [96]. Species are presented more than once if there are strains that encode significantly different SecA2 orthologues. The unrooted tree shows three main branches of SecA2 divergence: 1) SecA2s of the accessory Sec system (red), 2) SecA2s of the Actinobacteria species (green), and 3) other SecA2s (cyan) and SecA proteins of the general Sec system (black), with E. coli SecA included as a point of reference. SecA2 homologues from Arabidopsis thaliana and the unicellular red alga Cyanidioschyzon merolae are included for comparison.

Figure 2. Comparative domain organization among SecA2 proteins

SecA2 domains were identified by alignment with E. coli SecA. Domain boundaries are based on those determined by Papanikolau et al [4]. NBD, nucleotide binding domain; PPXD, preprotein cross-linking domain; HWD, helical wing domain; HSD, helical scaffold domain; IRA, intramolecular regulator of ATPase activity; CTD, C-terminal domain. IRA1 is a functional domain that overlaps with the two-helix finger structural domain [6, 97]. Numbers shown below the SecA2 proteins indicate the percent similarity/identity with the corresponding domain of the SecA paralogue.

Figure 3. Genetic loci encoding the multi-substrate SecA2 proteins

The chromosomal regions flanking secA2 (blue) in Mycobacterium tuberculosis, Listeria monocytogenes, Clostridium difficile and Bacillus anthracis are shown. A component required for SecA2 transport in B. anthracis is also in blue. Genes encoding defined SecA2 substrates are shown in red, and presumed substrates are indicated in pink. Substrates encoded elsewhere in the chromosome are not shown.

Figure 4. Gene organization within the accessory Sec loci

Comparison of the aSec locus in selected Gram-positive bacterial species. Genes encoding the aSec system components are colored blue, and the export susbstrate is shown in red. Glycosyl transferases and other enzymes involved solely in carbohydrate modifications are shown in green. Genes of unknown function are shown in grey, while genes encoding potential regulatory proteins are depicted in black. Two different variations found in Streptococcus agalactiae strains are shown. The Staphylococcus locus is representative of most Staphylococcal species. A slightly different arrangement, in which secA2 is immediately upstream of secY2 and the asp123 genes, is found in Pediococcus, Enterococcus and some Lactobacillus species.

Figure 5. SRR glycoprotein domain organization and conserved signal peptide features

Upper diagram: The domains identified in GspB are characteristic of the SRR glycoprotein family. SP, signal peptide; AST, accessory Sec transport domain; SRR1 and SRR2, serine-rich repeat regions 1 and 2, respectively; BR, ligand binding region; CWA, cell wall anchoring domain (including an LPxTG motif). Lower diagram: Amino-terminal sequence of the SRR glycoproteins from S. gordonii (GspB), S. parasangunis (Fap1), S. aureus (SraP), and P. acidilactici (SRRpp).

Figure 6. Impact of signal peptides and post-translational modifications on trafficking through the general Sec or aSec system

A signal sequence with a relatively strong Sec avoidance motif is indicated in cyan, and signal sequences that can support transport by the general Sec system are indicated in red. A: In most streptococci, the aSec locus encodes four or more putative glycosyl transferases, and the SRR glycoprotein signal peptide is not optimal for general Sec transport. B: GspB variants with one or more substitutions of critical glycine residues in the signal peptide H region, or wild-type Fap1, may be inefficiently transported by the general Sec system (dashed line indicates transport of partially or incorrectly glycosylated substrates). C: In staphylococci, the aSec locus includes just the core GtfA/B glycosyl transferase, and the SRR glycoprotein signal peptide can facilitate transport via either the Sec or aSec pathway.

Figure 7. SecY versus SecY2 topology and structural features

A: The predicted topology of SecY2 is very similar to that of SecY. B: The inactive SecY channel is impermeable to small molecules, due to the constriction by a ring of hydrophobic residues near the center of the channel along with a plug formed by residues of TM2. In the active channel, the central pore widens and the plug is displaced. Some prl mutations in E. coli SecY result in a partially or more readily opened channel.

Figure 8. Model for SRR glycoprotein biogenesis

A: A partially glycosylated preprotein arrives at the translocon, and one or more of the Asps facilitate an interaction between SecA2 and the preprotein AST domain that trigger opening of the SecY2 channel. B: An Asp123 complex further modifies the glycan composition as translocation of the SRR glycoprotein proceeds through SecA2/Y2. The Asp123 modifications could include trimming, replacement, or addition of carbohydrate moieties. SecA2 and SecY2 are thought not to have a direct enzymatic role in glycan modification, but instead facilitate the interaction between the Asp complex and the substrates.