3.A.15 The Outer Membrane Protein Secreting Main Terminal Branch (MTB) Family
The MTB family (also called Type II secretion systems) corresponds to the PulEF/OutEF/ExeEF/XpsEF/XcpRS families of Swiss Prot. They are constituents of the pilin/fimbrilin exporters as well as the main terminal branch of the general secretory pathway (GSP; TC #3.A.5) of Gram-negative bacteria McLaughlin et al. 2012. Type IV pilins are probably exported via these systems. Two of these proteins, PulE and PulF, are homologous to the ComGA and ComGB proteins of the competence system in Bacillus subtilis (TC #3.A.14). It has also been reported that components of MTB systems function in natural transformation in Campylobacter jejuni (Wiesner et al., 2003). Specific MTB systems may therefore be capable of DNA uptake as well as (or instead of) protein export.
The MTB is complex, consisting of at least 14 proteins that somehow function in the ATP- and/or pmf-energized transport of exoproteins from the periplasm across the outer membrane to the external milieu. The best characterized MTB system is the pullulanase secretion system of Klebsiella oxytoca, but several other MTB complexes have been characterized. The actual integral outer membrane protein porin of this system is the PulD secretin (the product of the pulD gene), a member of the Secretin family (TC #1.B.22).
All of the other constituents of the MTB are either integral constituents of the inner membrane (PulC, F, G, H, I, J, K, M, N and O), a peripheral constituent of the inner membrane (PulE) or in one case, a peripheral outer membrane lipoprotein which probably functions as a secretin-specific chaperone protein (PulS). One of the inner membrane proteins (PulO) is a peptidase/N-methyl transferase that processes precursors of PulG, H, I and J. PulE is an ATP-binding ATPase/kinase that exhibits an essential zinc-finger motif, while PulL is required for PulE to associate with the membrane. These proteins probably form a trans-periplasmic complex called the 'secreton' that (1) recognizes the substrate proteins in the periplasm, (2) energizes transport across the outer membrane using the pmf and/or ATP, and (3) controls opening of the PulD pore. Others may be involved in secreton assembly. Some reports have led to the suggestion that both ATP and the pmf energize the MTB for exoprotein export. However, other reports have suggested that the pmf alone energizes secretion, and ATP functions in assembly of the complex. Regardless, the substrate protein folds in the periplasm prior to transport across the outer membrane. The secretion signal may be contained in the tertiary conformation of the native protein, or multiple signals may be present.
Membrane-associated ATPases constitute essential elements common to most secretion machineries in Gram-negative bacteria. How ATP hydrolysis by these ATPases is coupled to secretion remains unclear. Shiue et al., (2007) identified R286 as a key residue in the type II secretion system (T2SS) ATPase XpsE of Xanthomonas campestris that plays a pivotal role in coupling ATP hydrolysis to protein translocation. Mutation of R286 to alanine made XpsE hydrolyse ATP at a rate five times that of the wild-type XpsE. Yet the mutant is non-functional in protein secretion via T2SS. Because R286 is conserved among members of the secretion NTPase superfamily, Shiue et al., (2007) speculated that its equivalent in other homologues plays a critical energy coupling role for T2SS, type IV pilus assembly and type IV secretion systems.
PulD homologues are numerous and include outer membrane proteins of Gram-negative bacteria of the Secretin family (TC #1.B.22). These proteins include (1) outer membrane secretins of the Type III secretory pathway (TC #3.A.6) (e.g., YscC of Yersinia enterocolitica; spQ01244), (2) fimbrial export proteins (e.g., PilQ of Pseudomonas aeruginosa; spP34750), (3) DNA uptake competence proteins (e.g., ComE of Haemophilus influenzae; spP31772), and (4) certain bacteriophage-encoded proteins (e.g., Gene IV protein, GP14, of phage f1; spP03666). PulD homologues form homomultimeric ring structures (10-20 subunits/complex, probably as a dodecamer) with large central pores (internal diameters of 50-100 Å).
PulL is 26% identical to EspL of Vibrio cholerae, and PulM is 27% identical to EspM. EspL and M are parts of a 12-component cholerae toxin type II secretion system. EspL (P45782; 407aas; 2TMSs) and EspM (P41851; 165aas; 2TMSs) are both inner membrane proteins that form dimers and interact with each other (Johnson et al., 2007).
ype IV pili are long thin surface-displayed polymers of the pilin subunit that are present in a diverse group of bacteria. These multifunctional filaments are critical to virulence for pathogens such as Vibrio cholerae, which use them to form microcolonies and to secrete the colonization factor TcpF. The type IV pili are assembled from pilin subunits by a complex inner membrane machinery. The core component of the type IV pilus-assembly platform is an integral inner membrane protein belonging to the GspF superfamily of secretion proteins. These proteins convert chemical energy from ATP hydrolysis by an assembly ATPase on the cytoplasmic side of the inner membrane to mechanical energy for extrusion of the growing pilus filament out of the inner membrane (Kolappan and Craig 2013). Most GspF-family inner membrane core proteins are predicted to have N-terminal and central cytoplasmic domains, cyto1 and cyto2, and three transmembrane segments, TM1, TM2 and TM3. Cyto2 and TM3 represent an internal repeat of cyto1 and TM1. The 1.88 Å resolution crystal structure of the cyto1 domain of V. cholerae TcpE has been solved (Kolappan and Craig 2013). It is required for assembly of the toxin-coregulated pilus. This domain folds as a monomeric six-helix bundle with a positively charged membrane-interaction face at one end and a hydrophobic groove at the other end that may serve as a binding site for partner proteins in the pilus-assembly complex.
The generalized reaction catalyzed by the MTB is:
folded protein (periplasm) + pmf + ATP → folded protein (extracytoplasmic space)