Functions of the Periplasmic Loop of the Porin MspA from Mycobacterium smegmatis

Abstract

MspA is the major porin of Mycobacterium smegmatis and mediates diffusion of small and hydrophilic solutes across the outer membrane. The octameric structure of MspA, its sharply defined constriction zone, and a large periplasmic loop L6 represent novel structural features. L6 consists of 13 amino acids and is directly adjacent to the constriction zone. Deletion of 3, 5, 7, 9, and 11 amino acids of the L6 loop resulted in functional pores that restored glucose uptake and growth of a porin mutant of M. smegmatis. Lipid bilayer experiments revealed that all mutant channels were noisier than wild type (wt) MspA, indicating that L6 is required for pore stability in vitro. Voltage gating of the Escherichia coli porin OmpF was attributed to loops that collapse into the channel in response to a strong electrical field. Here, we show that deletion mutants ⌬7, ⌬9, and ⌬11 had critical voltages similar to wt MspA. This demonstrated that the L6 loop is not the primary voltage-dependent gating mechanism of MspA. Surprisingly, large deletions in L6 resulted in 3-6-fold less extractable pores, whereas small deletions did not alter expression levels of MspA. Pores with large deletions in L6 were more permissive for glucose than smaller deletion mutants, whereas their single channel conductance was similar to that of wt MspA. These results indicate that translocation of ions through the MspA pore is governed by different mechanisms than that of neutral solutes. This is the first study identifying a molecular determinant of solute translocation in a mycobacterial porin.Mycobacteria have a unique outer membrane composed of covalently attached mycolic acids and a variety of other loosely associated lipids. Visualized by cryo-electron tomography (1), the outer membrane is an efficient permeability barrier that renders mycobacteria intrinsically resistant to many drugs and toxic compounds. As is the case for Gramnegative bacteria, this permeability barrier must be functionalized by outer membrane proteins. Although over 60 outer membrane proteins are known to exist in Escherichia coli (41), very few have been described in mycobacteria to date.The major protein of Mycobacterium smegmatis is MspA (42), which is the primary member of the nearly identical Msp (M. smegmatis porin) proteins. Msp proteins account for ϳ80% of detergent-extractable proteins at 100°C (2) in wt 4 M. smegmatis mc 2 155. MspA represents the main pathway for small and hydrophilic solutes and nutrients across the outer membrane (3). One hallmark of MspA is its extremely high stability. Functional pores are detectable at temperatures up to 100°C, a pH range from 0 -14, and resist the action of denaturing agents such as 2% SDS (4), thus providing the utility of MspA for nanotechnological applications. However, it is unknown how M. smegmatis utilizes Msp porins to functionalize the outer membrane in varying conditions or what parts of the channel determine substrate translocation. It is our goal to understand the translocation determinants of the MspA channel.The cry...

MspA Nanopores from Subunit Dimers

“…This phenomenon is not well understood on a molecular level [25], [26], but could be utilized as an indicator of the overall structural and dynamic channel properties. To examine whether the linkage of two MspA subunits by a peptide influences the overall structure of the MspA pore we analyzed the voltage gating of M1-MspA and of M1-M1 19 MspA.…”

Expression of the ompATb operon accelerates ammonia secretion and adaptation of Mycobacterium tuberculosis to acidic environments

“…However, it was unknown whether heterologous ompATb expression in M. smegmatis would result in correctly localized protein, which may require other proteins of M. tuberculosis . To determine whether OmpATb correctly inserted in the outer membrane of the triple porin mutant M. smegmatis ML16, an enzyme‐linked immunosorbent assay (ELISA) with whole cells was employed as recently described (Huff et al ., 2009). OmpATb was detected on the cell surface of M. smegmatis ML16 by OmpATb antibodies (Fig.…”

Engineering a Novel Porin OmpGF Via Strand Replacement from Computational Analysis of Sequence Motif

“…However, the overall foldability, stability, and biophysical properties of a β MP are also influenced by other factors. For example, long-range interactions between pore-facing residues may contribute to the stability of β MPs [57]; non-TM regions such as loops may also affect the stability [58, 59, 60]; interactions between loops and pore-facing residues may affect the gating behavior [23]. While further improvement will likely be fruitful, our present work provides the first practical demonstration of the overall feasibility of a novel computational engineering strategy and suggests that additional artificial protein nanopores with desired properties can be engineered.…”