Charge asymmetry in the proteins of the outer membrane

Abstract

Motivation: Outer membrane beta-barrels (OMBBs) are the proteins found in the outer membrane of bacteria, mitochondria and chloroplasts. There are thousands of beta-barrels reported in genomic databases with ∼2-3% of the genes in gram-negative bacteria encoding these proteins. These proteins have a wide variety of biological functions including active and passive transport, cell adhesion, catalysis and structural anchoring. Of the non-redundant OMBB structures in the Protein Data Bank, half have been solved during the past 5 years. This influx of information provides new opportunities for understanding the chemistry of these proteins. The distribution of charges in proteins in the outer membrane has implications for how the mechanism of outer membrane protein insertion is understood. Understanding the distribution of charges might also assist in organism selection for the heterologous expression of mitochondrial OMBBs.

Results: We find a strong asymmetry in the charge distribution of these proteins. For the outward-facing residues of the beta-barrel within regions of similar amino acid density for both membrane leaflets, the external side of the outer membrane contains almost three times the number of charged residues as the internal side of the outer membrane. Moreover, the lipid bilayer of the outer membrane is asymmetric, and the overall preference for amino acid types to be in the external leaflet of the membrane correlates roughly with the hydrophobicity of the membrane lipids. This preference is demonstrably related to the difference in lipid composition of the external and internal leaflets of the membrane.

Fig. 1.

Charged residues prefer the external side of beta-barrels. (a) Four OMBBs with roughly average numbers of external-half charged residues per strand are shown in gray. The strands of the barrel are shown as with sheet secondary structure and the rest of the protein as loops. OmpX (PDBID 1QJ8) is an 8-stranded OMBB; Porin (PDBID 2POR) is a 16-stranded OMBB; LamB (PDBID 1AF6) is an 18-stranded OMBB; BtuB (PDBID 2GUF) is a 22-stranded OMBB. Outward-facing charged residues in the barrel are colored, glutamate and aspartate are red, lysine and arginine are blue. Proteins drawn using pymol (http://www.pymol.org/). (b) Histogram showing the distribution of the number of external charges among the OMBB proteins studied herein. The numbers of external-half outward-facing charged residues in the barrel per strand of the barrel are shown. Bins are inclusive of the bin minimum. (c) External region preference by amino acid. A preference score based on the frequency of occurrence in the external versus the internal region of the outer membrane is calculated for each amino acid. Red amino acids are charged, yellow are polar and green are aliphatic. The more hydrophilic an amino acid is the more it prefers the external side of the outer membrane

Fig. 2.

Amino acid location preference within the membrane. The distribution of the proportion of each residue at nine positions in the outer membrane demonstrates how different types of amino acids localize in the outer membrane. (a) Charged amino acids Arg, Asp, Glu and Lys are shown. The average of the four charged types was fit to a polynomial curve of order 2 with an R² of 0.96. (b) Polar amino acids Asn, Gln, His and Ser are shown. The average of the four polar types was fit to a polynomial curve of order 2 with an R² of 0.97. (c) Aliphatic amino acids Ala, Gly, Ile, Leu and Val are shown. The average of the five aliphatic types was fit to a polynomial curve of order 2 with an R² of 0.92. (d) Aromatic amino acids Phe, Trp and Tyr are shown. The average of the three aromatic types was fit to a polynomial curve of order 4 with an R² of 0.91. The curve of each residue sums to 1

Fig. 3.

Average hydrophobicity correlates with the structure of the membrane. (a) The average hydrophobicity for each 2 Å of outward-facing barrel amino acids as a function of position in the membrane ±1 SEM. (b) A MDs simulation of the outer membrane (Shroll and Straatsma, 2002) displaying how the membrane is configured as a function of z. (c) Schematic of the composition of the outer membrane. Phospholipid shown in light gray—head groups are circles and acyl chains are zigzag lines. Most of the LPS is shown in dark gray except for the phosphates, which are shown in yellow. Sugars are shown as hexagons and acyl chains as zigzag lines

Fig. 4.

Correlation between a shorter lipid A and a left-shifted drop of average hydrophobicity at the core region. (a) Average hydrophobicity ± 1 SEM for each 2 Å of the outer membrane divided by organism. Gray circles for E.coli, black diamonds for P.aeruginosa. Curves shown are a local polynomial fit with a bandwidth of 2 Å, gray for E.coli, black for P.aeruginosa. (b) Molecular structure of lipid A in E.coli. (c) Molecular structure of lipid A in P.aeruginosa