The Core of the Tetrameric Mycobacterial Porin MspA Is an Extremely Stable β-Sheet Domain

Abstract

MspA is the major porin of Mycobacterium smegmatis mediating the exchange of hydrophilic solutes across the cell wall and is the prototype of a new family of tetrameric porins with a single central pore of 10 nm in length. Infrared and circular dichroism spectroscopy revealed that MspA consists mainly of antiparallel ␤-strands organized in a coherent domain. Heating to 92 and 112°C was required to dissociate the MspA tetramer and to unfold the ␤-sheet domain in the monomer, respectively. The stability of the MspA tetramer exceeded the remarkable stability of the porins of Gram-negative bacteria for every condition tested and was not reduced in the presence of 2% SDS and at any pH from 2 to 14. These results indicated that the interactions between the MspA subunits are different from those in the porins of Gram-negative bacteria and are discussed in the light of a channel-forming ␤-barrel as a core structure of MspA. Surprisingly, the channel activity of MspA in 2% SDS and 7.6 M urea at 50°C was reduced 13-and 30-fold, respectively, although the MspA tetramer and the ␤-sheet domain were stable under those conditions. Channel closure by conformational changes of extracellular loops under those conditions is discussed to explain these observations. This study presents the first experimental evidence that outer membrane proteins not only from Gram-negative bacteria but also from mycobacteria are ␤-sheet proteins and demonstrates that MspA constitutes the most stable transmembrane channel protein known so far. Thus, MspA may be of special interest for biotechnological applications.

Trichomonas vaginalis Hmp35, a Putative Pore-forming Hydrogenosomal Membrane Protein, Can Form a Complex in Yeast Mitochondria

“…Likewise, Tom40, the major constituent of the urea/salt/carbonate-resistant mitochondrial general import pore (51) is also predicted to have a ␤-barrel structure that forms a voltage-gated ion channel through which proteins are imported into mitochondria (22). Interestingly, Hmp35 is also predicted to be a predominantly ␤-structured protein with a 47% ␤-sheet content, which is comparable with the 45% predicted and the 48 -52% determined experimentally for MspA (53). The Toc75 protein translocase found in chloroplasts likewise exists as a ␤-barrel (54).…”

“…This implies that there may be a chaperone or other helper protein, present in hydrogenosomes but missing in mitochondria, that confers protease resistance upon the Hmp35 protein. Recently, the major porin from Mycobacteria, MspA, has been described as one of the most stable transmembrane proteins (53). This porin exists as a tetramer that is stable in heat up to 90°C in 2% SDS and in 7.6 M urea and at all pH values.…”

Porins limit the intracellular persistence of Mycobacterium smegmatis

“…MspA from M. smegmatis belongs to a novel class of porins present in many fast-growing mycobacteria but apparently absent in slow-growers (Niederweis et al, 1999). MspA is an extremely stable octameric protein composed of 20 kDa monomers (Faller et al, 2004;Heinz et al, 2003). In addition to the mspA gene, M. smegmatis possesses three homologous genes named mspB, mspC and mspD.…”

Identification and characterization of a cell wall porin from <i>Gordonia jacobaea</i>

“…Most bacterial porins, including MspA from M. smegmatis, have antiparallel b-sheet structures conferring stability inside the bacterial wall (Fairman et al, 2011;Heinz et al, 2003). However, there are some microbial pore-forming proteins where a-helix structures are relevant for their stability, such as PorH from Corynebacterium glutamicum, C. efficiens, C. callunae and PorACj from C. jeikeium (Abdali et al, 2013;Hünten et al, 2005a, b).…”

“…The presence of this structure in both gram-negative (Bagos et al, 2004(Bagos et al, , 2005Zhai and Saier, 2002) and gram-positive (Heinz et al, 2003) bacteria can be predicted by several types of algorithms. In our sequence c6/294, JPred algorithm and the GOR program predicted the presence of several a-helical regions located in the aminoterminal region and a low proportion of b-sheet, mainly located in the intermediate region.…”