Sucrose Specific Porin
Created by Vikram Pole
Sucrose-specific porin from Salmonella typhimurium is a transmembrane protein that allows for the uptake of sucrose under growth-limiting conditions (1). All cells must transport molecules into and out of themselves for survival, and the rate of transport is determined by the concentration difference between the external environment and the internal environment. The role of nonspecific porins is to maintain a high permeability so that molecules can get where they are needed quickly (2). Nonspecific porins work well at medium-to-high concentrations of molecules; however, under growth-limiting conditions, nutrients are at very low concentrations. The cell cannot simply increase the number of nonspecific porins in the cell membrane, because doing so would damage the protective abilities of the membrane (3). Instead, the cell can synthesize specific porins, which target specific molecules, under growth-limiting conditions (1). For example, maltoporin maltose complex is specific for maltose and maltooligosaccharides and is synthesized when these sugars are at concentrations below 100 uM (4). Another specific porin, sucrose-specific porin, allows bacteria to grow on sucrose as the sole source of carbon under growth-limiting conditions (5). The molecular mass of sucrose-specific porin is 136122.55 Da, and its isoelectric point (pI) is 5.13.
There are a series of steps that sucrose must take to enter the cell. First, sucrose must diffuse from the external environment to a cavity in sucrose-specific porin. Then the sucrose molecule collides with the walls of the cavity until it reaches the binding site on the end of the greasy slide of sucrose-specific porin. Next, it diffuses along the greasy slide. Finally, sucrose passes through the greasy slide and enters the cell (1). The rate at which this process occurs can be optimized depending on the concentration of sucrose on each side of the membrane (6).
The structure of sucrose-specific porin directly influences its function as a sucrose uptake system. It is a trimer with identical subunits. Each subunit has 413 amino acids, and there are a series of 18 anti-parallel beta-strands surrounding a hydrophilic pore (1). In contrast, nonspecific porins have only 16 beta-strands (7). The protein's shape is a beta-barrel consisting of many strands and loops. Loop 3 constricts the cross-sectional area of the channel so that large molecules cannot pass through. When the subunits are bound, they form a channel in the center. Only sucrose can properly fit in the channel; larger molecules are too big to fit, and smaller molecules are too small to interact properly with the protein. Thus, sucrose can travel through the channel and enter the cell (1).
In maltoporin, there are six aromatic residues in the greasy slide, and these residues constitute binding sties for maltose and maltooligosaccharides (7). In sucrose-specific porin, four of these aromatic amino acids are conserved, one is replaced, and the last one is missing. Therefore, the critical aromatic residues that bind sucrose are Trp-151, Tyr-118, Tyr-78, Trp-482, and Phe-435 (8). Sucrose-specific porin can actually bind two molecules of sucrose at once. However, sugars bind only in a certain direction - with the nonreducing end towards the periplasm (9). The calcium ion is a ligand of sucrose-specific porin. Calcium ion binding to the protein aids in the binding of sucrose to the protein. So, calcium ions facilitate sucrose binding to the protein (10).
Sucrose-specific porin shows some similarity to BenF-like porin (pdb id = 3JTY) from Pseudomonas fluorescens. The results of Dali (Z score = 19.5) and protein Blast (E value = 1e-87) are significant. Therefore, both sucrose-specific porin and BenF-like porin have similar primary and tertiary structures. Just as the calcium ion helps with sucrose binding to sucrose-specific porin, the ligand lauryl dimethylamine-N-oxide facilitates the binding of BenF-like porin to its substrate.
However, sucrose-specific porin shows much better similarity to maltoporin (pdb id = 1MPM) from Escherichia coli. The results of Dali (Z score = 36.8) and protein Blast (E value = 4e-173) are significant. Since the Z score is greater than 2, both proteins have similar tertiary structures; since the E value is less than 0.05, both structures also have similar primary structures (11). Both are beta-barrels and contain 18 beta-strands surrounding a hydrophilic pore (1). 411 amino acids are aligned between them together. But, sucrose-specific porin has 72 N-terminal residues that are not present in maltoporin (12). The two proteins are not highly conserved in their looping segments. For example, loops 4, 6, and 9 in maltoporin are not conserved in sucrose-specific porin, because they are generally shorter (1). However, four out of the six sugar binding sites (Trp-151, Tyr-118, Tyr-78, and Trp-482) in maltoporin are highly conserved in sucrose-specific porin. Moreover, the residues surrounding the constriction site (78-80, 110, 114, 118, 120-121, 140, 142, 161, 187-188, 194-201, 204, 207, and 322-323) are very similar (1). There are three major differences between these two. Maltoporin has Arg-109, Tyr-118, and Asp-121, while sucrose-specific porin has Asn-192, Asp-201, and Phe-204, respectively. These changes in sucrose-specific porin allow it to form a relatively large cross-sectional channel mouth so that channel conductance is higher (13). As a result, sucrose-specific porin is a more efficient sugar uptake system than maltoporin. In fact, experiments have shown that under conditions of low concentrations of sucrose, bacteria cannot grow unless they have sucrose-specific porin. This finding clearly demonstrates that sucrose-specific porin has an incredibly high permeability for sucrose (12).
The primary structure of sucrose-specific porin has approximately 72 N-terminal residues that are not present in maltoporin. The majority of the differences in amino acid sequence are in the looped sections, but there are also a few major changes in the binding sites of the two proteins (1).
The secondary structure of maltoporin has 4 alpha-helices (2% helical) and 28 beta-strands (60% beta sheets) per chain, while sucrose-specific porin has relatively more alpha helices (8 alpha-helices, 6% helical) and fewer beta-strands (24 beta-strands, 55% beta sheets). Both share the calcium ion as a ligand; however, this ligand binds each protein at different residues. Also, maltoporin has two additional ligands that sucrose-specific porin does not have; they are beta-D-glucose and alpha-D-glucose.
The tertiary structure describes the folding pattern of proteins. Both sucrose-specific porin and maltoporin consist of beta-barrels. Moreover, both proteins have 18 beta-strands in their beta-barrels, which is in contrast to the 16 beta-strands that nonspecific porins have (1). These proteins also have numerous loops of varying lengths. In particular, loop 3 of sucrose-specific porin attaches to the interior barrel wall via polar and nonpolar interactions. This action squeezes the channel in the middle, much like a belt. Thus, the cross-sectional area of the channel at the center becomes constricted to 8 x 11 Angstroms. The shape of the channel looks like an hour glass, with a wide external portion (top) and a relatively narrow center (1). The cross-sectional area of a slice taken from top-to-bottom resembles a human kidney. The dimensions of this cross section are approximately 50 x 28 Angstroms (7).
Since sucrose-specific porin is a transmembrane protein, it contains three regions, corresponding to where the regions are in relation to the plasma membrane. The middle portion of the protein is within the hydrophobic region of the plasma membrane. Therefore, a hydrophobic zone made up of aliphatic nonpolar amino acids, such as alanine, valine, leucine, and isoleucine, surround the protein. The two ends of sucrose-specific porin are in contact with a hydrophilic environment. As a result, hydrophilic zones surround the protein at its ends. Aromatic amino acids mark the borders between the three regions (1). Overall, the amphipathic nature of sucrose-specific porin is essential for its function as a trasmembrane protein.
Sucrose-specific porin undergoes alternate conformations, depending on whether it is bound to something or not. The torsion angles of sucrose are phi = 108 degrees and psi = -45 degrees in the unbound state. But when sucrose binds to the first binding site on sucrose-specific porin, these angles change to phi = 75 degrees and psi = -31 degrees. When the second sucrose molecule is added (recall that sucrose-specific porin can bind two molecules of sucrose at once), the torsion angles become phi = 90 degrees and psi = -61 degrees (1). In addition, there are conformational changes in sucrose-specific porin when the calcium ion ligand binds to it. When a calcium ion binds to the protein, it alters the conformation of the residues involved in sugar binding. The change in conformation allows those amino acids to form hydrogen bonds more efficiently with the hydroxyl groups of the sugars (14).
In conclusion, specific porins, such as sucrose-specific porin and maltoporin, allow bacteria to survive under growth-limiting conditions (i.e. when the concentration of nutrients is very low). This ability gives these bacteria a growth advantage over bacteria that have only nonspecific porins, which function only when nutrient concentrations are higher. Thus, specific porins provide an extremely valuable source of carbon for growth (15).