Structure of the type IVa major pilin from the electrically conductive bacterial nanowires of Geobacter sulfurreducens - PubMed (original) (raw)

Structure of the type IVa major pilin from the electrically conductive bacterial nanowires of Geobacter sulfurreducens

Patrick N Reardon et al. J Biol Chem. 2013.

Abstract

Several species of δ proteobacteria are capable of reducing insoluble metal oxides as well as other extracellular electron acceptors. These bacteria play a critical role in the cycling of minerals in subsurface environments, sediments, and groundwater. In some species of bacteria such as Geobacter sulfurreducens, the transport of electrons is proposed to be facilitated by filamentous fibers that are referred to as bacterial nanowires. These nanowires are polymeric assemblies of proteins belonging to the type IVa family of pilin proteins and are mainly comprised of one subunit protein, PilA. Here, we report the high resolution solution NMR structure of the PilA protein from G. sulfurreducens determined in detergent micelles. The protein is >85% α-helical and exhibits similar architecture to the N-terminal regions of other non-conductive type IVa pilins. The detergent micelle interacts with the first 21 amino acids of the protein, indicating that this region likely associates with the bacterial inner membrane prior to fiber formation. A model of the G. sulfurreducens pilus fiber is proposed based on docking of this structure into the fiber model of the type IVa pilin from Neisseria gonorrhoeae. This model provides insight into the organization of aromatic amino acids that are important for electrical conduction.

Keywords: Bacterial Metabolism; Bioenergetics; Electron Transport; Membrane Proteins; NMR; Pili; Structural Biology.

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Figures

FIGURE 1.

FIGURE 1.

Alignment of various major pilin subunit amino acid sequences. Species whose pilin subunit amino acid sequence length is >66 were truncated to 67 amino acids. Eight species capable of EET and two species that are not capable of EET were included in the alignment.

FIGURE 2.

FIGURE 2.

Overview of the solution NMR structure of G. sulfurreducens PilA. A, overlay of the ensemble of 18 structures that did not contain NOE violations >0.5 Å or dihedral angle violations >5°. B, ribbon diagram of the selected conformer (see “Materials and Methods”) with the highly conserved core domain corresponding to amino acids 1–22 colored in orange and the rest of the protein colored blue. C, ribbon diagram of the selected conformer with the aromatic residues shown in blue space filling. The amino terminus and carboxyl terminus are indicated by N and C, respectively. D, 15N TROSY spectrum of GSu PilA with backbone amide assignments. Data were collected at 750 MHz on an Agilent VNMR spectrometer. E, overlay of the homology model of GSu PilA (38) and the experimentally determined GSu PilA structure. The arrows indicate where the degree of bend differs between the structures near residues 22 and 42. To emphasize the differences, the structures were aligned using residues 23–41.

FIGURE 3.

FIGURE 3.

Summary of H-N heteronuclear NOE data. Spectra with and without proton saturation (3s) were acquired. The plot shows the ratio of cross-peak intensities with and without proton saturation. The data were collected at 800 MHz. Asterisks indicate resonances that could not be clearly identified and quantitatively characterized.

FIGURE 4.

FIGURE 4.

Summary of paramagnetic relaxation by Gd-DTPA. The site-specific reduction in peak intensity upon addition of 4 m

m

Gd-DTPA to an 15N-labeled sample of GSu PilA is shown. Values near 1 indicate no loss of intensity and protection from Gd-DTPA. No correction was made for the small sample dilution (∼3% maximum) that occurred during the titration. The data were collected at 750 MHz. Asterisks indicate resonances that could not be clearly identified and quantitatively characterized.

FIGURE 5.

FIGURE 5.

Model of a nanowire fiber based on the structure of G. sulfurreducens PilA. A, superimposition of PilA from G. sulfurreducens on to the homologous type IV pilin from N. gonorrhoeae (Protein Data Bank code 2HIL) (12). The amino terminus and carboxyl terminus are indicated by N and C, respectively. B, model of the bacterial nanowire, based on the pilus assembly of N. gonorrhoeae (Protein Data Bank code 2HIL) (12). Aromatic side chains are shown in space filling. A single cluster of aromatic side chains is shown in blue space filling, whereas all others are shown in gray. Ribbons of each subunit were colored individually. C, schematic diagram showing the progression of the aromatic clusters up the pilus structure. The aromatic band is colored blue, and the aromatic devoid band is colored yellow.

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