BtuB
Created by Collin Conrad
Successful metabolism in gram-negative bacteria, such as Escherichia coli, requires a number of cofactors that cannot be synthesized within the cell. In order to survive, these bacteria must obtain these additional nutrients and metabolites from their surrounding environment. Membrane proteins exist as part of the phospholipid bilayer that composes the outer shell of a given bacteria, and they are important tools in the collection of these essential molecules. Btub is one of these membrane receptor proteins. The primary function of BtuB is that of a translocon; it binds with a number of different molecules in order to carry them across the cell membrane and into the cell. The molecules targeted by BtuB include coblamins (vitamin b12), metals (iron siderophores), various sugars, and other cargo that can be deadly for the bacteria such as bacteriophages and colicins (8).
Colicins are polypeptide toxins that destroy E. coli bacterial cells through a number of different mechanisms. Colicin E3 specifically functions as a ribosomal endoribonuclease, destroying specific phosphodiester bonds in the E. coli's rRNA. This halts protein synthesis within the cell causing it to die. In order to begin the process of cell destruction the Colicin E3 bacteriocin forms a 1:1 complex with BtuB (10). Once the Colicin E3 polypeptide is bound small conformational changes occur in the BtuB structure; however, these transformations are insufficient for the formation of a translocation channel. Instead, the colicin is transferred to a neighboring OmpF membrane protein. This second membrane functions as a cotransporter, opening a path for the completion of the colicin's entry into the cell (10).
The complex formed by BtuB and Colicin E3 has a molecular weight of 81,391.23 and an isoelectric point of 5.50. Analysis of tertiary structure using the Dali server points to a number of proteins with similar functions to that of the colicin-binding membrane protein, BtuB.
One of the proteins containing similarities in tertiary structure is the Colicin 1a receptor protein of Escherichia coli (PDB ID: 2hdf). This protein displays a Z-value of 37.2 and rmsd value of 2.81. Colicin 1a is another bacteriocin that kills E. coli cells upon binding with its respective outer membrane protein. The Colicin 1a receptor protein differs from BtuB in that it does not seem to require a cotransporter in order to complete the destruction of the bacterial cell. Instead, when the ligand binds to the receptor a voltage-dependant ion channel is opened that inhibits energy metabolism (6).
A second protein that demonstrates significant similarity to BtuB is the Serratia marcescens hemophore receptor found in E. coli bacteria (PDB ID: 3ddr). This protein displays a Z-value of 33.5 and rmsd value of 2.81. Much like BtuB, the Serratia marcescens receptor also functions as a scavenger and translocator for molecules important to the survival of the E. coli bacterial cells. However, instead of coblamins or colicins this membrane protein coordinates with iron-rich hemephore proteins in order to transport heme into the bacterial cells. Upon bonding with the receptor protein the heme coordinations are broken and displaced from their hemophore and transferred into the cell (2).
The Escherichia coli protein, BtuB, exists physiologically as a monomer, spanning the outer membrane of the gram-negative bacteria. It is composed of 594 amino acid residues that can be broken into two general regions or domains. The first is the outer, C-terminal portion of the protein, which consists of twenty-two anti-parallel beta-sheets. This outer portion, known as a beta-barrel, surrounds an inner, N-terminal, globular domain. The inner portion of the protein consists of a combination of short alpha-helices and Beta-sheets that form what is described as a cork or hatch (5). The overall secondary structure is largely dominated by beta-sheets. These two domains work together to bind and transport various substrates into the bacterial cell.
The beta-barrel domain of BtuB begins at residue 137 and stretches through the C-terminus at residue 594. The twenty-two, anti-parallel Beta-strands are variable in length, ranging from 9 to 24 residues long. On the periplasmic end of membrane protein the beta-strands are connected by short turns, 2-4 residues in length. On the opposite, extracellular side, each pair of strands is connected by much longer loops, 7-19 residues in length, which participate in substrate binding. Many of the extracellular loops are disordered under normal conditions but undergo complete or partial ordering in the presence of certain bound substrates (9).
Embedded within the lumen of the 22-stranded outer beta-barrel is the N-terminal hatch(5). This inner domain extends from residues 1-132. Residues 6-12 make up the energy-coupling segment termed the Ton box, which is required to couple BtuB with inner membrane protein TonB to provide energy for substrate transport (1). The remainder of the core portion of BtuB is built around a four-stranded beta-sheet that exists at a 30-40 degree angle with respect to the membrane plane (7). This portion contains very little secondary structure with the exception of three short alpha-helices. All of these inner structures are connected by four loops that face the extracellular side of the Beta-barrel lumen. Finally, residues 133-136 serve as a linker between the inner and outer domains of the membrane protein.
BtuB uses a number of its long extracellular loops as well as some of the higher points on the inner hatch to capture a number of different substrates. One of these substrates is the lethal colicin E3. Colicin E3 binds to BtuB using a 135-residue (R135) receptor-binding domain that is described as a coiled coil. This domain stretches from residue 313-447 of colicin E3 and consists of a pair of long (100 Å) alpha-helices (10).
Twenty-seven residues of the R135 domain participate in binding with the extracellular portion of BtuB. This involves most of the residues between Ile-369 and Thr-402. This portion of the colicin polypeptide interacts with18. twenty-nine different residues in the BtuB structure, mostly within the loops of the beta-barrel but also extending to four residues (Thr-55, Asn-57, Leu-63, and Ser-64) within the cork portion of the structure. Only the tip of the coiled-coil structure is involved in the binding process with the remaining residues stretching out away from the cell into the extracellular space until it can be transported (10). Upon binding of the colicin substrate, there are small conformational changes that take place within the hatch of the membrane protein. These changes, however, are not enough to allow for the passage of the colicin into the bacterial cell (10).
Additional ligands bind to the outer membrane protein BtuB causing slight changes in conformation within the structure. This can be seen in the addition of calcium ions to the protein. The ions are captured and held in place by an " aspartate cage" found in the extracellular loops of the beta-barrel (8). This cage consists of five aspartate residues (Asp-179, Asp-193, Asp-195,Asp-230, and Asp-241) that surround the ions holding them in place. Upon the addition of calcium to the protein structure the extracellular loop connecting beta-strands three and four moves from complete disorder to complete order (PDB ID = 1ngg).
Further conformational changes are observed upon the binding of cyanocoblamin (vitamin B12) with BtuB. The vitamin coordinates in a tight fitting pocket, forming eleven hydrogen bonds and eleven van der Waals interactions with the surrounding residues (9). The Ton box, used for energy coupling with the inner membrane protein TonB, undergoes vitamin B12 dependent 26 unfolding in which the box moves 20-30 Å into the periplasmic space1. While this conformation has not been observed in crystal structure, this substrate-dependent unfolding has been observed in bilayers (1).