Topology of the Porin MspA in the Outer Membrane of Mycobacterium smegmatis

Abstract

MspA is the major porin of Mycobacterium smegmatis mediating the exchange of hydrophilic solutes across the outer membrane (OM). It is the prototype of a new family of octameric porins with a single central channel of 9.6 nm in length and consists of two hydrophobic ␤-barrels of 3.7 nm in length and a more hydrophilic, globular rim domain. The length of the hydrophobic domain of MspA does not match the thicknesses of mycobacterial OMs of 5-12 nm as derived from electron micrographs. Further, the membrane topology of MspA is unknown as it is for any other mycobacterial OM protein. We used MspA as a molecular ruler to define the boundaries of the OM of M. smegmatis by surface labeling of single cysteine mutants. Seventeen mutants covered the surface of the rim domain and were biotinylated with a membrane-impermeable reagent. The label efficiencies in vitro were remarkably similar to the predicted accessibilities of the cysteines. By contrast, six of these mutants were protected from biotinylation in M. smegmatis cells. Tryptophan 21 defines a horizontal plane that dissects the surfaceexposed versus the membrane-protected residues of MspA. The 8 phenylalanines at position 99 form a ring at the periplasmic end of the hydrophobic ␤-barrel domain. These results indicated that (i) the membrane boundaries of MspA are defined by aromatic girdles as in porins of Gram-negative bacteria and (ii) loops and a 3.4-nmlong part of the hydrophilic rim domain are embedded into the OM of M. smegmatis. This is the first report suggesting that elements other than hydrophobic ␣-helices or ␤-sheets are integrated into a lipid membrane.Mycobacteria are surrounded by an inner membrane and a giant macromolecule consisting of peptidoglycan, arabinogalactan, and very long chain fatty acids, the mycolic acids (1). Minnikin (2) originally proposed that the mycolic acids, which are covalently bound to the arabinogalactan-peptidoglycan co-polymer, form the inner leaflet of an unique outer membrane (OM).3 Experimental evidence for this model was provided by x-ray diffraction studies, which showed that the mycolic acids are oriented in parallel and perpendicular to the plane of the cell envelope (3). The presence of an organized structure of extremely low fluidity in an isolated mycobacterial cell wall was confirmed by differential scanning calorimetry (4). The vast abundance of non-covalently bound lipids in mycobacteria is assumed to constitute the outer leaflet of a supported asymmetric lipid bilayer. The analysis of spin-labeled fatty acids inserted into isolated mycobacterial cell walls by electron paramagnetic resonance supported the existence of a moderately fluid outer leaflet, whereas the inner leaflet had an extremely low fluidity (4, 5). This interpretation is consistent with observations that mutants with defects in the production of some of the major extractable lipids (glycopeptidolipids, phthiocerol dimycocerosate) showed an increased OM permeability (6, 7). Thus, the mycobacterial OM resembles a supported asymmetric lipid bilayer ...

Disclosure of the mycobacterial outer membrane: Cryo-electron tomography and vitreous sections reveal the lipid bilayer structure

“…However, the outer membrane has a measured total thickness of Ϸ8 nm or less, in perfect agreement with the porin MspA that serves as a molecular ruler. The hydrophobic surface of MspA is only 3.7 nm in height (17), and the porin (total length: 9.8 nm) is inaccessible to surface labeling over 7 nm from the periplasmic end to the middle of the hydrophilic rim (18). These distances correspond to the observed membrane dimensions, including headgroup regions.…”

“…An electron-transparent zone in stained thin sections (13,14), which was shown to comprise lipids (i.e., predominantly mycolic acids) (15), was covered by a stained outer layer thought to represent carbohydrates, peptides, and other lipids (4). The whole structure is of considerable thickness, which poses the problem of understanding how the Mycobacterium smegmatis porin MspA with its comparatively much shorter hydrophobic surface is inserted (16)(17)(18).…”

Direct Visualization of the Outer Membrane of Mycobacteria and Corynebacteria in Their Native State

“…Porins are not drawn to scale. Of note, the porin of M. smegmatis MspA is expected to protrude out of the OM (27). The porin of corynebacteria has been proposed to be made by a stack of short proteins (6 kDa) (43).…”

Surface Proteome of “Mycobacterium avium subsp. hominissuis” during the Early Stages of Macrophage Infection

“…For infectious bacteria, surface proteins are essential components of many aspects of pathogenesis (47,48). Host specificity, adhesion, invasion, molecular transport, and antimicrobial resistance are processes known to be directly mediated by proteins present at the surface interface (16,17,50,81). These proteins are also the primary target of the host immune system.…”