Permuting the PGF Signature Motif Blocks both Archaeosortase-Dependent C-Terminal Cleavage and Prenyl Lipid Attachment for the Haloferax volcanii S-Layer Glycoprotein

Abstract

For years, the S-layer glycoprotein (SLG), the sole component of many archaeal cell walls, was thought to be anchored to the cell surface by a C-terminal transmembrane segment. Recently, however, we demonstrated that the Haloferax volcanii SLG C terminus is removed by an archaeosortase (ArtA), a novel peptidase. SLG, which was previously shown to be lipid modified, contains a C-terminal tripartite structure, including a highly conserved proline-glycine-phenylalanine (PGF) motif. Here, we demonstrate that ArtA does not process an SLG variant where the PGF motif is replaced with a PFG motif (slg G796F,F797G ). Furthermore, using radiolabeling, we show that SLG lipid modification requires the PGF motif and is ArtA dependent, lending confirmation to the use of a novel C-terminal lipid-mediated protein-anchoring mechanism by prokaryotes. Similar to the case for the ⌬artA strain, the growth, cellular morphology, and cell wall of the slg G796F,F797G strain, in which modifications of additional H. volcanii ArtA substrates should not be altered, are adversely affected, demonstrating the importance of these posttranslational SLG modifications. Our data suggest that ArtA is either directly or indirectly involved in a novel proteolysis-coupled, covalent lipid-mediated anchoring mechanism. Given that archaeosortase homologs are encoded by a broad range of prokaryotes, it is likely that this anchoring mechanism is widely conserved. IMPORTANCEProkaryotic proteins bound to cell surfaces through intercalation, covalent attachment, or protein-protein interactions play critical roles in essential cellular processes. Unfortunately, the molecular mechanisms that anchor proteins to archaeal cell surfaces remain poorly characterized. Here, using the archaeon H. volcanii as a model system, we report the first in vivo studies of a novel protein-anchoring pathway involving lipid modification of a peptidase-processed C terminus. Our findings not only yield important insights into poorly understood aspects of archaeal biology but also have important implications for key bacterial species, including those of the human microbiome. Additionally, insights may facilitate industrial applications, given that photosynthetic cyanobacteria encode uncharacterized homologs of this evolutionarily conserved enzyme, or may spur development of unique drug delivery systems.A variety of protein complexes decorate cell surfaces, where they support cell stability and facilitate interactions between the cell and the extracellular environment (1-3). Determining the mechanisms that allow these proteins to remain associated with the cell envelope is critical for developing a comprehensive understanding of the biosynthesis of cell surface structures. Protein anchoring to the cell membrane was likely first accomplished by means of transmembrane (TM) segments (4). However, as cell membranes became densely packed with protein complexes, protein-anchoring mechanisms that allowed more economic use of membrane surface area emerged (5). One such mechanism anchors...

Conserved residues are critical for Haloferax volcanii archaeosortase catalytic activity: Implications for convergent evolution of the catalytic mechanisms of non‐homologous sortases from archaea and bacteria

“…In previous work, we demonstrated that the H. volcanii ArtA is required for the processing and lipidation of substrates that contain a conserved PGF C‐term tripartite structure (Abdul Halim et al ., ). While conserved amino acids that may be critical for ArtA catalytic activity had been predicted in silico (Haft et al ., ), the catalytic site remained uncharacterized in vivo .…”

“…Recently, a novel surface protein anchoring mechanism in prokaryotes was identified, in which substrates containing a C‐terminal tripartite structure, reminiscent of the sortase substrate tripartite structure, are C‐terminally processed, but unlike sortase A substrates, are then anchored to the cytoplasmic membrane (Abdul Halim et al ., ). The enzymes involved in this process, archaeal archaeosortases and bacterial exosortases, were identified using in silico phylogenetic profiling analysis of genomes that also encode proteins having a C‐terminal tripartite architecture with variations on the LPXTG motif (Navarre and Schneewind, ; Haft et al ., ).…”

“…The enzymes involved in this process, archaeal archaeosortases and bacterial exosortases, were identified using in silico phylogenetic profiling analysis of genomes that also encode proteins having a C‐terminal tripartite architecture with variations on the LPXTG motif (Navarre and Schneewind, ; Haft et al ., ). In vivo studies of the Haloferax volcanii archaeosortase A (ArtA), confirmed that it is indeed required for processing of proteins containing the conserved C‐term motif, including a PGF‐motif followed by an H‐domain and positively charged residues (Abdul Halim et al ., , ). However, unlike sortase substrates, which are anchored to the peptidoglycan cell wall, ArtA substrates are covalently attached to a lipid embedded in the cytoplasmic membrane following C‐terminal processing (Abdul Halim et al ., ).…”

“…In vivo studies of the Haloferax volcanii archaeosortase A (ArtA), confirmed that it is indeed required for processing of proteins containing the conserved C‐term motif, including a PGF‐motif followed by an H‐domain and positively charged residues (Abdul Halim et al ., , ). However, unlike sortase substrates, which are anchored to the peptidoglycan cell wall, ArtA substrates are covalently attached to a lipid embedded in the cytoplasmic membrane following C‐terminal processing (Abdul Halim et al ., ). Moreover, in contrast to sortase A substrates, which appear to be secreted exclusively via the general secretory (Sec) pathway, we recently determined that some ArtA substrates are translocated across the cytoplasmic membrane via the Twin Arginine Transport (Tat) pathway while others are translocated via the Sec pathway (Schneewind and Missiakas, ; Goosens and van Dijl, ; Abdul Halim et al ., ).…”

Multidomain, Surface Layer-associated Glycoside Hydrolases Contribute to Plant Polysaccharide Degradation by Caldicellulosiruptor Species

“…In archaea, S-layer proteins are most often attached to the cell membrane. S-layer proteins can be anchored to the membrane via a C-terminal transmembrane helix domain (3, 9) or covalently attached to cell membrane lipid via an archaeosortase, as shown in Haloferax volcanii (10,11). In most microorganisms, the S-layer is composed entirely of one or two proteins (SLPs), which self-assemble.…”