1.B.24 The Mycobacterial Porin (MBP) Family
Mycobacteria are acid-fast, high G+C, Gram-positive bacteria that have outer mycolic acid-containing membranes, the thickest outer membrane known for bacteria. These membranes serve as an effective permeability barrier but contain porins that serve functions analogous to those observed for Gram-negative bacteria. A 100 kDa protein complex from the outer membrane of Mycobacterium smegmatis has been purified and shown to form cation-selective channels in reconstituted planar lipid bilayers. MspA when expressed in M. tuberculosis increased growth, glucose uptake and sensitivity to many antibiotics (β lactams, isoniazid, streptomycin and ethambutol) as well as to hydrophobic dyes. MspA also increases susceptibility to copper ion toxicity (Speer et al. 2013). It is therefore appears to be nonselective (Mailaender et al., 2004). An identical porin was isolated from Tsukamurella inchonensis (Dörner et al., 2004).
The mspA gene encodes a 211 residue protein with an N-terminal 27 amino acyl residue signal sequence and a 184 residue mature protein (MW = 20 kDa) following the cleavage site. The octomeric porin could be dissociated to inactive monomers by boiling in 80% dimethyl sulfoxide. The membrane boundaries of MspA are defined by aromatic girdles as in porins of Gram-negative bacteria. Loops and a 3.4 nm long part of the rim domain are embedded in the outer membrane. The 3-d structure is known (PDB#1UUN). It is a β-barrel with N- and C-termini of their single hairpins on the outside, and their chains run in an anti-clockwise direction around the central pore. Both of these characteristics are opposite in most gram-negative bacterial β-barrels (Remmert et al., 2010).
The projection structure of MspA has been determined at 17 Å resolution. It forms a cone-like complex with a single central pore of 10 nm in length. This pore is 2.5 x the length of a typical Gram-negative porin channel. This correlates with the greater thickness of the mycobacterial outer membrane, due to the greater length of the mycolic acids as compared with phospholipids (Niederweis, 2003). The channel consists of two hydrophobic β-barrels of 3.7 nm in length and a more hydrophilic globular rim domain (Mahfoud et al., 2006). Thus, its structure differs from those of the trimeric porins found in Gram-negative bacteria (Engelhardt et al., 2002).
The structure of MspA from M. smegmatis has been solved by x-ray analysis (Faller et al., 2004). It has a homooctomeric goblet-like conformation with a single central channel. It contains two consecutive β-barrels with non-polar outer surfaces that form a ribbon around the porin. Passing DNA with a series of double-stranded sections through MspA provides proof of principle of a simple DNA sequencing method using a nanopore (Derrington et al., 2010).
Expression of the mspA gene in E. coli yielded the MspA monomer as well as the channel-forming 100 kDa protein complex. From the molecular weight of the monomer, the 100 kDa protein could be a homopentamer. No homologues could be identified in slow growing Mycobacteria such as M. tuberculosis and M. leprae. However, by hybridization, the genomes of M. fortuitum and M. chelonae, other fast growing Mycobacteria, seemed to include homologues. Based on CD and FT-IR spectra, MspA is composed primarily of β-strands (M. Niederweis, personal communication).
The M. smegmatis genome encodes four paralogues, MspA, MspB, MspC and MspD where MspB (215 aas) and MspC (215 aas) as well as MspD (207 aas) are 93%, ≥98%, and 82% identical to MsbA, respectively. All three exhibit similiar physical and electrophysiological ion conducting properties. However, MspA provides the main hydrophilic pathway through the outer membrane. An mspA deletion mutant shows 4-10-fold reduction in the rates of permeability towards hydrophilic molecules such as glucose and cephaloridine (Stahl et al., 2001). Loss of MspC in addition to MspA resulted in a further 5x drop in porin activity (Bender et al., 2005). Thus, outer membrane porin permeability is a rate limiting step for glucose entry in this Mycobacterial species.
MspA of Mycobacterium smegmatis is used for DNA sequencing. The octameric MspA pore can be isolated from M. smegmatis in milligram quantities, is extremely stable against denaturation and rapidly inserts into lipid membranes. MspA pores composed of different Msp subunits are formed in M. smegmatis, and hetero-oligomers of different Msp monomers exhibit functional heterogeneity. All four msp genes were deleted from the M. smegmatis genome after insertion of an inducible porin gene from M. tuberculosis (Pavlenok and Niederweis 2016). In the msp quadruple mutant, no Msp porins were detected, and mutant MspA proteins could be produced at normal levels. Lipid bilayer experiments demonstrated that some MspA mutant proteins formed functional channels.
The generalized transport reaction catalyzed by MspA is:
Small molecule (out) ⇋ small molecule (in)
M. smegmatis porin, MspA (cation selective due to a high density of negative charges in the constriction zone, but it transports glucose, serine, hydrophilic β-lactams and (slowly) phosphate (Wolschendorf et al., 2007)) The MspC paralogue appears to have the same specificity as MspA. Both can also transport fluoroquinolones and chloramphenicol but not the larger erythromycin, kanamycin, and vancomycin (Danilchanka et al., 2008). Also allows uptake of ferric iron (Jones and Niederweis, 2010). The 3-d structure is known (PDB#1UUN). It is a β-barrel with N- and C-termini of their single hairpins on the outside, and their chains run in an anti-clockwise direction around the central pore. Both of these characteristics are opposite in most gram-negative bacterial β-barrels (Remmert et al., 2010). Forms octameric voltage-gated nanopores where each subunit contributes 2 TMSs to the 16 stranded β-barrel (Faller et al. 2004; Rodrigues et al. 2011; Pavlenok et al. 2012) that can be used for nanopore sequencing (Laszlo et al. 2016).
MspA of Mycobacterium smegmatis
MspA porin (233aas; one N-terminal alpha-helical TMS). This protein is almost identical to a cholate-transporting porin in R. jostii (RjpA; porin A) that is involved in cholate uptake (Somalinga and Mohn 2013).
MspA porin of Rhodococcus opacus (C1B943)
MspA porin homologue (227aas; 1 N-terminal TMS) (shows significant similarity with members of both 1.B.24 and 1.B.58).
MspA porin of Gordonia effusa (H0QY58)
MspA porin homologue (289aas; 1 N-terminal TMS)
MspA porin of Tsukamurella paurometabola (D5UQW2)
RjpA outer membrane porin of 233 aas and 3 TMSs. Transports bile acids such as cholate (Somalinga and Mohn 2013).
RjpA of Rhodococcus jostii
RjpB outer membrane porin of 233 aas and 3 TMSs. Transports bile acids such as cholate (Somalinga and Mohn 2013).
RjpB of Rhodococcus justii
RjpC outer membrane porin of 283 aas and 1 - 3 TMSs. Transports bile acids such as cholate (Somalinga and Mohn 2013).
RjpC of Rhodococcus justii
RjpD outer membrane porin of 233 aas and 3 TMSs. Transports bile acids such as cholate (Somalinga and Mohn 2013).
RjpD of Rhodococcus justii