Membrane
Fusion Protein CusB (3OW7) from Escherichia
coli
Created by: Brogan Jones
Cation Efflux System Protein CusB (PDB
ID: 3OW7) is a membrane fusion protein found in Escherichia coli. Escherichia
coli is an anaerobic enteric bacterium that lives in the digestive tract of
warm-blooded animals (1). Digestive tracts can be toxic environments for the
bacterium because there are usually high concentrations of bactericides, such
as copper (1). Escherichia coli uses copper homeostatic mechanisms to regulate
toxic concentrations of copper. One mechanism that Escherichia coli uses is a heavy-metal efflux pump system that
recognizes and exports copper ions. Escherichia
Coli’s efflux system spans the inner and outer membranes of the cell to
export the substrates directly out (2).
Its efflux system has one heavy-metal transporter, CusA, which
recognizes and confers silver and copper ions, and an outer membrane channel,
CusC (2). The purpose of CusB is to bridge the inner membrane efflux pump CusA
and outer membrane channel CusC to mediate resistance to silver and copper ions
(2). The structure of CusB allows it to bind to the toxic ions and interact
with other efflux proteins so that the levels of copper can be regulated within
the Escherichia coli cell.
CusB has a molecular weight of
45127.74 Da and an isoelectric point 6.47 (3). The secondary structure of CusB consists of 379 amino acids
and has four linearly arranged domains (4). The first three domains consist
mostly of beta strands and the fourth domain consists entirely of alpha helices
and is folded into a three-helix bundle structure (4). Multiple-wavelength
anomalous dispersion method of the crystal structure of CusB showed that CusB
crystal is an asymmetric unit of two elongated protomers. Each protomer
consists of the four different domains mentioned above (2). In both protomers, domain 1 is a beta barrel
domain with six beta strands (2). In protomer A, domain 2 consists of six beta
strands and one short alpha helix. In the protomer B, domain 2 has six beta
strands and two alpha helices (2). The
two protomers interact antiparallel to each other, with domains 1 and 2
contacting domains 2 and 1 of the other, respectively (5). Thus, it is
suspected that these domains form the interaction site for CusB
oligomerization. Domain 3 consists of eight beta strands in each of the
protomers. Domain 4 in both protomers has alpha helices folded into an
anti-parallel, three-helix bundle (2).
As stated above, the two distinct
conformations of CusB are closely related and differ by ~21° overall (5). The
conformations interact through hydrogen bonds and pack side by side to form a
funnel-like structure (6). It was found that the linker region composed of the
two loops (residues 116-120 and 240-242) between domains 2 and 3 forms a flexible
hinge in the protein (5). The two molecules of CusB found in a single crystal
suggest that the membrane fusion protein has conformational flexibility with
two transient states. (5). Protomer A adopts a more open conformation while
protomer B has a compact form of the structure. Thus, these two molecules may
correspond to the open and closed states of CusB within the efflux system and it
is very likely that these linker regions provide the flexibility for CusB to
carry out its biological function in the copper efflux system (2).
The secondary structure of CusB
is also critical for its function in the heavy- metal efflux pump. The domains
of CusB allow it to interact with CusA and CusC proteins, which are the heavy
metal transporter and the outer membrane channel proteins respectively. Mass
spectral data suggests that domain 1 of CusB should interact with the
periplasmic domain of the CusA transporter. The lysine residue, Lys-95, of the
polypeptide beta chain originating from the N-terminal of CusB
directly interacts with the lysine residue of peptide alpha in the periplasmic
domain (residues 148-157) of CusA (2). In addition, Thr-89, Asn-91, and Arg-292 of CusB form hydrogen bonds with Lys-594, Arg-147, and Gln-198 of CusA. Furthermore, domain 1 and the lower half
of domain 2 of CusB create a cap that fits closely on the periplasmic domain of
CusA. Domains 3, 4, and the upper half of domain 2 forms an elongated, central
channel (6). Because the lower half of the channel is primarily beta barrels
and the upper half consists entirely of alpha helical tunnels, the channel
gradually constricts and dilates as it approaches the outer membrane (6). The
inner surface of the channel is mostly negatively charged, which suggests that
it binds to the positively charged copper ions (6). These interactions and
structural features form the crystal structure of the CusBA complex (PDB ID:
3NE5), which is shaped like a funnel (6).
Two identical binding sites for
copper are found in each protomer of CusB, making four copper binding
sites in the asymmetric crystal unit. The first site is found in domain 1 and
is coordinated with the residues, Met-324, Phe-358, and Arg-368 (2). This site
is located near the bottom of the elongated CusB molecule, and so is believed
to interact with the periplasmic domain of the CusA efflux pump (6). The second copper binding site is located near the center of the helix bundle in domain 4,
and is bound by the critical residues, Met-190, Trp-158, and Glu-162. This site
may make direct contact with the outer membrane channel CusC (6). The binding
sites in the other protomer of CusB are the same. Due to the position of these copper-binding
sites, it is believed that CusA may capture the copper ions that are released
from CusB. Furthermore, since the second binding site is located in the domain
that may interact with the outer membrane channel CusC, CusB may deliver the
ions to CusC that are eventually exported out of the cell (6).
The Dali Server and PSI-BLAST
program revealed that the membrane fusion protein, ZneB (PDB ID: 3LNN), found
in Cpriavidus metallidurans is
comparable to CusB. The Dali Server finds proteins with similar tertiary
structures to a query. It uses a sum-of-pairs method to measure the similarity
of intramolecular distances. It takes Z-scores as measurements, and a score
above 2 means the protein has similar folds (7). ZneB and CusB have a Z score
of 14.6. The PSI-BLAST program finds proteins with similar primary structure to
the protein query. The program evaluates
the gaps and the total sequence homology of the sequence, and assigns an E
value to the subjects. A gap is an amino acid or group of amino acids that
exist in the subject’s sequence but not in the query’s sequence. Gaps increase
the E value and total sequence homology decreases the E value. Thus, an E value
of less than 0.05 is considered significant for proteins (8). ZneB has an E
value of 2x10-5 in comparison with CusB.
Like CusB, ZneB is a membrane fusion protein that helps the bacterium regulate toxic compounds. ZneB is a
component of the ZneCAB heavy-metal efflux system in Cpriavidus metallidurans and ZneB is specific for zinc ions (9).
Its crystal structure also reveals there is flexible interface between proximal
domains that play a role in the substrate efflux through metal binding and
release (9). This is very similar to the flexibility of the two conformations
of CusB that allow it to help transport copper ions in the CusABC transport
system. Furthermore, both membrane proteins have four different domains of
which three mostly consist of beta strands and the last is helical. However,
ZneB has a different amphiphile transporter in the membrane protein domain and
a different length of the helix in the beta barrel domain that faces the
membrane protein (9).