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1.1. Antibiotic Resistance
Antibiotic resistance has increased significantly in the recent years, making treatment of bacterial infections harder. The development of antibiotic resistance in bacteria can be due to several factors. Antibiotic resistance can develop as an evolutionary process, based on the selection of bacteria with an enhanced ability to survive doses of antibiotics produced by other bacteria within the environment. This process has been accelerated by the over prescription and inaccurate use of antibiotics in medicine. This has contributed to a significant increase in the adaptive mutations in E. coli in the order of 10-5 per genome per generation (1).
Genes encoding antibiotic resistance may also be transferred from one bacterium to another and between different bacterial species. This occurs via horizontal gene transfer, which is the uptake of DNA or RNA between bacteria, via the transfer of plasmids that carry genes encoding antibiotic resistance. Uptake of DNA or RNA occurs by transduction and transformation. Point mutations within the bacterial genome may also occur naturally during chromosomal replication, or induced when a virus gene is incorporated within the host genome causing, genetic rearrangements. Plasmids can carry different genes with diverse resistance mechanisms to unrelated antibiotics. Resistance to multiple antibiotics within the bacteria may result when a transferred resistance mechanism is capable of providing resistance to more than one antibiotic .
Antibiotic resistant genes from E. coli can therefore be transferred to Staphylococcus aureus (2). This can lead to multidrug resistant bacteria such as MRSA (Methicillin Resistant Staphylococcus aureus). MRSA can lead to many people being hospitalized each year, after infection, some may die as a result.
There are four main molecular mechanisms that bacteria exhibit to inhibit antimicrobials; direct inactivation or modification to inactivate the antimicrobial (e.g. ¢ - lacatamases, enzymes that phosphorylate, adenylate, or acetylate aminoglycoside antibiotics), alteration of the antibiotic target site of action, alteration of the metabolic pathway affected, possessing an alternative pathway, bypassing the affected pathway. The accumulation of antimicrobials may also be reduced by increasing antibiotic active efflux . Proteins that cause active efflux of antibiotics will be the central point of my interest in my research.
Antibiotics are vital in the fight against bacterial infection. Antibiotics are characterised into narrow-spectrum and broad-spectrum antibiotics based on their target specificity. Narrow -spectrum antibiotics target particular types of bacteria such as gram-negative or gram-positive bacteria, whereas broad-spectrum antibiotics affect a wide range of bacteria. Antibiotics work using various mechanisms of action. The majority of antibiotics target bacterial functions or growth processes . Antibiotics target; the bacterial cell wall (penicillins (Fig 1.7.), cephalosporins), cell membrane (polymixins), interferes with essential bacterial enzymes (quinolones, sulfonamides). Those that target protein synthesis, such as the aminoglycosides, macrolides, and tetracyclines, are usually bacteriostatic .
Figure 1.7. Chemical structure of penicillin (A) and chloramphenicol (B).
1.3.1. Bacteria Cell Structure
Bacteria can be classed in to two main groups; gram positive and gram negative. Classification is due to how they react to gram stain (crystal violet stain). Gram-positive bacteria have a thick cell wall made of peptidoglycan (fig) (50-90% of cell wall), which stains purple, while gram-negative bacteria have a thinner layer (10% of cell wall), which stains pink. Gram-negative bacteria are also morphologically distinct from gram-positive bacteria, due to the presence of the outer membrane that contains lipids and is separated from the cell wall by the periplasmic space (Fig 1.1.).
Figure 1.1. Generalized structure of gram-positive and gram-negative bacteria, illustrating the difference in the bacterial composition.
1.3.2. Bacterial Outer membrane
The outer membrane is the outermost surface and is a double layered lipid membrane (Fig 1.2.) that encloses the peptidoglycan and inner membrane. The outer membrane is unique due to the outer layer being primarily composed of lipopolysaccharide (LPS) (Fig 1.2.). The LPS layer confers gram-negative bacteria with an increased resistance to environmental hazards such as toxic agents, host-defence proteins or digestive enzymes, due to its low permeability of hydrophobic and charged compounds. This effect is caused by strong interactions between LPS molecules in the prescence of ions such as Mg2+ or Ca2+ and the limited intramolecular mobility of their hydrocarbon chains (ref). Porins are one of the main constituent proteins of the outer membrane in gram-negative bacteria and some gram-positive. Porins are beta barrel proteins that cross the outer membrane and act as a pore through which substances can passively diffuse. Typical substances include small metabolites (e.g. sugars, ions and amino acids). Other proteins of in the outer membrane include outer membrane factors (e.g. tolC) that are involved in a variety of functions.
1.3.3. Structure of outer membrane porins
Porins are composed of beta sheets; the beta sheets are usually linked by beta turns on the cytoplasmic face and long loops of amino acids on the outer face. The beta sheets lie antiparallel and form a cylindrical tube called a beta barrel. The beta sheets amino acids alternate between polar and non-polar residues. The non-polar residues face outwards to interact with the non-polar lipid membrane, where the polar residues face into of the pore allowing interaction with the aqueous channel. The porin channel is partially blocked by a loop, called the eyelet, which projects into the cavity. General, it is found between strands 5 and 6 of each barrel and it defines the size of solute that can traverse the channel. The channel is lined almost exclusively with negatively charged amino acids arranged on opposite sides of the channel, creating a transversal electric field across the pore. The eyelet has a negative charge that is partially compensated for by two bound calcium atoms. This asymmetric arrangement of molecules is thought to have an influence in the selection of molecules that can traverse the channel.
Many gram negative bacteria are known to express ompF. ompF is known to contribute to the survival of bacteria under different osmolarity conditions. Under low osmolarity conditions the gene encoding (mar gene) ompF is up regulated. OmpF also is known to play a significant role in the drug resistance of bacteria. ompF plays a role in drug resistance as it provides a channels for drugs (e.g. quinolones, tetracyclines, and β-lactams) to be transported into the bacteria, therefore a reduction in the expression of ompF reduces the permeability to certain drugs. It has been suggested that the decreased expression of ompF is a key step to the development of bacterial mutants that are multidrug resistance.
The porin ompF is a trimeric integral membrane protein responsible for the passive transport of small hydrophilic molecules, such as nutrients and waste products, across the outer membrane of Escherichia coli. ompF porin forms tight homotrimers, with each monomer folded as a 16-stranded antiparallel 3 barrel. There are eight short turns that resemble at the periplasmic and eight long loops at the other, extracellular. Six of the extracellular loops (loops 1 and 4-8) pack together and partially close the entrance to the barrel. Loop 2 is involved in monomer-monomer interactions and loop 3 folds inside the barrel where it constricts the size of the pore. The pore lumen is polar due to charged residues; this produces a strong electrostatic field across the channel.
Figure Visualization of ompF topology in bacterial gram-negative inner membrane. The three protomers of ompF which form the homotrimer are shown in green, red and blue.
tolC belongs to the OMF (outer membrane factor) and is a constituent of the outer membrane. tolC is central in the efflux of diverse range of compounds, from antibacterial drugs , small inhibitory molecules to large proteins, in each case interacting with a specific inner membrane translocase in response to substrate engagement. This provides substrate specificity and regulated access through the pore. Many species of bacteria encode homologues of E.coli TolC. tolC is assembled as a trimer of a 428-residue protomer. The long axis measures 140Å and for nearly 100Å the body of the trimer forms a uniform cylinder of about 35Å internal diameter. The distal end provides wide solvent access, as it is open while the proximal end is tapered and so nearly closed, the large interior cavity present is mostly solvent-filled. The tolC trimer can be separated into a b-domain (¡ - helical domain) (distal end) and a mixed a/b-domain. The peptide chain of each monomer goes from distal to proximal along the long axis four times, and passes from b-strands at the distal end of the structure to ¡ - helices. In the trimer the peptide strands of each monomer in the b-domain associate in an antiparallel orientation to form a 12-stranded ¢-barrel that has a right handed twist. The ¡ -helices form the main proportion of tolC, these also form a 12-stranded antiparallel barrel, in contrast has a left hand twist.
The structure of the tolC reveals a mechanism to facilitate the direct passage of substrates across two membranes and the intervening periplasmic space. For the efflux of small molecules the tapered proximal end must open to release the molecule. Yet in the case of the efflux of proteins the proximal end must be at least partially unfolded.
A proposed mechanism is that the inner pair of coiled coils rotates around its neighbouring partner to dilate the entrance. The inner and outer sets of coiled coils have similar sequences. Allowing the inner pair could re-pack to become compatible with the outer pair by an, untwisting movement. The uncoiling mechanism proposed is triggered via protein - protein interactions. Interactions occur on the recruitment of tolC by the inner membrane translocase. The equatorial domain is one possible recognition site for such interaction. Its strands and helices pack against the inner set of coiled coils, and any change in this relationship, induced by interactions with a partner protein, might activate an allosteric transition in the coiled coils to couse the unfolding.
Figure Visualization of tolC topology in bacterial gram-negative inner membrane. The three protomers of tolC that form the homotrimer are shown in green, red and blue.
Show acrb tolc and acra structure
1.3.4. Bacterial Inner membrane
The inner membrane consists of a phospholipid bilayer (Fig 1.2.) and is the major physiological barrier between the outside of the cell and the cytoplasm, due to its inherent impermeability to many nutrients and waste products of metabolism. The inner membrane is therefore the site of various biochemical reactions (Fig 1.2.). Reactions are carried out by various proteins including; transport of substances into or out of the cytoplasm, transmission of signals into the cytoplasm and the generation of energy via the use of a proton motive force that is maintained by proteins in the inner membrane. Proteins within the inner membrane are also involved in cell division. Transport proteins comprise 3% to 15% of genomic potential in all organisms. Transport activities are usually energy-linked, this allows transport against the prevailing electrochemical gradient of the solute. Drugs/antibiotics, which often are able to penetrate the cell membrane are actively secreted from the cell via integral membrane proteins (Fig 1.2.), thereby conferring resistance.
Figure 1.2. Representation of the gram-negative bacterial cell membranes. The location of various proteins within the cell membranes is shown and the interaction between the outer and inner membrane (cytoplasmic membrane) proteins and examples of their functions (Faraldo-Gomez and Sansom, 2003).
1.4.1. Classes of MDR efflux pumps
Multiple families of membrane proteins expressed within the inner membrane of gram-negative bacteria confer drug/antibiotic resistance. Proteins that confer drug resistance of two or more drugs are classed as multidrug resistance (MDR) transporters (Fig 1.3.). On the basis of bioenergetic and structural criteria, multidrug resistance transporters can be divided into two major classes; adenosine 5'- triphosphate (ATP)-binding cassette (ABC) proteins that directly couple drug efflux to ATP hydrolysis and secondary multidrug transporters that utilize the transmembrane electrochemical gradient of protons or sodium ions to drive the extrusion of drugs from the cell. (Fig 1.3.) (e.g. lmrA, sav1866, msbA). Secondary transporters include four families; resistance/ nodulation/ division superfamily (RND) (e.g. acrB, acrF, acrD, yhiV), multiple antimicrobial toxin extrusion (MATE) family (e.g. norM), small multidrug resistance (SMR) family (e.g. emrE, tehA), and the major facilitator superfamily (MFS) (e.g. emrD, mdfA, emrB) (Fig1.3.). These families of transporters are prevalent within microbial genomes. MDR transporters are diverse in there structure; this allows them to be diverse in their substrate specificity (1).
Figure 1.3. Families of multidrug transporters. The families of multidrug-resistance transporters: the ATP-binding cassette (ABC) superfamily, the major facilitator superfamily (MFS), the multidrug and toxic-compound extrusion (MATE) family, the small multidrug resistance (SMR) family and the resistance nodulation division (RND) family. A diagrammatic representation of the structure and membrane location of efflux pumps from each of these families is shown. Examples of individual proteins from each class are shown. Antibiotic substrates and examples of other substrates are also shown.
MDR transporters are classified by a range of criteria; the number of components that the pump has (single or multiple), the number of transmembrane-spanning regions that the transporter protein has, the energy source that the pump uses and the types of substrate that the pump exports.
A single organism can express MDR transporters from more than one family and/or more than one type of efflux pump belonging to the same family. For example, E. coli can express more than one type of Acr efflux pump.
Transporters of the RND family, which are expressed by gram-negative bacteria and are associated with clinically significant MDR, are organized as tripartite systems (Fig 1.3.). These efflux pumps comprise the following: a transporter protein (e.g. acrB (acriflavine resistance protein B)), which is located in the inner membrane of the bacterium; an accessory protein (e.g. acrA), which is located in the periplasmic space; and an outer-membrane protein (e.g. tolC), which is located in the outer membrane of the bacterium. Efflux through RND-family pumps is driven by the proton motive force, an electrochemical gradient in which the movement of hydrogen ions drives transport of the substrate.
ABC Multidrug Transporters
ABC multidrug transporters contain two ATP binding cassettes and are driven by the free energy of ATP hydrolysis. All ATP-dependent drug efflux proteins known to date are members of the ABC superfamily. In general, ABC transporters require four distinct domains; two highly hydrophobic membrane domains, which usually consist of six putative transmembrane ¡-helices each, and two hydrophilic nucleotide-binding domains, containing the Walker A and B motifs and the ABC signature. Most bacterial ABC drug transporters mediate the export of specific antibiotics an example of an ABC multidrug transporters is lmrA that is from Lactococcus lactis. As for proton antiporters, a conformational change of the ABC protein is necessary for drug extrusion and probably is triggered by drug binding and ATP hydrolysis.
ABC transporter Sav1866 is expressed within host organism Staphylococcus aureus. sav1866 is able to transport a broad spectrum of structurally very diverse cytotoxic drugs and endogenous lipids. Sav1866 is expressed a homodimeric protein, consisting of 12 transmembrane helices. Sav1866 is 120 long, 65wide and 55 deep, Each Sav1866 subunit consists of two domains an amino-terminal transmembrane domain and a carboxyterminal nucleotide binding domain. The two subunits twist around one another along the entirety of the protein, interacting with one another. Considerable structural homology between the nucleotide binding domain of sav1866 and other ABC transporters nucleotide binding domains. Sav1866 possess a large internal cavity, it is not yet clear how substrates are transported. One proposed mechanism of transport involves two states, an inward facing conformation where the channel is open t the cell interior allowing the substrate to enter. The second conformation being outward open, where the cavity is open to the exterior. When ATP is bound to the nucleotide binding domains causes a tight interaction between the two domains brining a conformational change to the outward open state, allowing substrates to be exported. The hydolysis of ATP and its release from the nucleotide binding domain will release the tight interaction between the two domains aiding a conformational change back to the resting state (inward open conformation).
MATE multidrug transporters
The bacterial MATE-type transporters function as exporters of cationic drugs, such as norfloxacin and ethidium, through H+ or Na+ exchange. MATE transporter proteins are common constituents of many living organisms. Phylogenetic analysis of known sequences has led to division of the MATE family into three large subfamilies comprising 14 smaller subgroups. Family 1 comprises bacterial MATE transporters and includes Vibrio parahaemolyticus NorM. Length of proteins in the MATE family ranges from ~400 to ~700 amino acids, most members consist of 400-550 residues with 12 transmembrane helices. No apparent consensus sequence has been identified to be conserved in all MATE proteins; however, all MATE proteins share ~40% sequence similarity.
SMR proteins exhibit low substrate specificities; they are capable of conferring clinically significant resistance to large hydrophobic, cationic molecules, dyes, sanitizing agents, detergents, lipophilic compounds and antibiotics. SMRs act as antiporters, coupling the efflux of one drug molecule from the cytosol to the import of protons using the proton motive force. The structural and mechanistic details of this process, however, may exhibit substrate-specific difference.
Drug efflux has not been demonstrated for all identified SMR proteins and this characteristic resulted in the divergence of this family into three classes: small multidrug pumps (SMP) and suppressor of groEL mutation proteins (SUG) and finally paired SMR (PSMR) proteins which require co-expression of two separate SMR genes
Individual SMR molecules must assemble into homo-oligomeric structures to transport substrates, a requirement likely related to the relatively small size of the individual SMR molecule. A minimum of 12 transmembrane segments seem to be required for activity, meaning SMR transporters are probably organized in trimers.
The putative mechanism of drug transport, as established by site-directed mutagenesis of a SMR transporter, could involve the following steps: 1) exchange between the drug and a proton fixed on a charged residue, 2) translocation of the drug by a series of conformational changes driving it through a hydrophobic pathway, 3) replacement of the drug by a proton in the external medium and return to the initial conformational state. Two proposed mechanisms are shown in Fig XX. The overall result of the transport is therefore an exchange between the drug and a proton.
Efflux pumps of the RND superfamily play an important role in multidrug resistance (both intrinsic and elevated) in Gram-negative bacteria. This is because these pumps become associated with two other classes of proteins, the outer membrane channel such as tolC of E. coli and the periplasmic protein such as AcrA of E. coli, classified into the MFP (membrane fusion protein) family. Importantly, each of these three component proteins is essential for drug efflux, and the absence of even one component makes the entire complex totally nonfunctional. The construction of this tripartite complex suggested that the drugs are here exported directly into the external medium, rather than into the periplasm. This is a huge advantage for bacterial cells, because once exported into the external space, drug molecules must traverse the outer membrane barrier to reenter the cells. Thus these pumps work synergistically with the outer membrane barrier. Wild-type strains of most Gram-negative bacteria are resistant to most lipophilic antibiotics. The inactivation of the major and onstitutively expressed RND pump. The characteristic intrinsic resistance of gram-negative bacteria owes as much to the RND pumps as to the outer membrane barrier. It was obvious that there is no structural similarity between most of these compounds.
The MFS consists of membrane transport proteins that are involved in the symport, antiport, or uniport of various substrates (e.g. sugars, Krebs cycle intermediates, phosphate esters, oligosaccharides, and antibiotics). Hydropathy analysis and alignment of conserved motifs of the resistance-conferring drug efflux proteins revealed that these proteins can be divided into two separate clusters, with either 12 or 14 transmembrane segments (TMS).
Structure and Function of RND transporter acrB
RND drug exporter of E. coli, acrB crystal structure has been solved at 2.8 Å resolution (Murakami et al., 2002, 2006). The crystal structure has given new insights into the function and mechanism of acrB.
An acrB monomer contains 12 transmembrane ¡ helices. Three acrB subunits are organized into a homotrimer (Fig 1.4.). acrB subunits are comprised of a 70 Å headpiece protruding from the external membrane surface and a 50 Å thick transmembrane domain (Fig 1.4.). The top of the periplasmic domain opening is similar I shape to a funnel. The periplasmic part of acrB consists of the tolC docking domain, located farthest from the membrane plane, and the pore domain. tolC has been shown to dock coaxially to the docking domain. Three α-helices form the pore that connects the funnel with a central cavity at the bottom of the headpiece. The central cavity leads to a further 35 Å wide transmembrane hole defined by the ring like arrangement of the transmembrane helices of the trimer, this is proposed to be filled with phospholipids. Three chambers at the monomer interfaces located just above the membrane plane lead toward the central cavity. Several different hydrophobic and amphipathic ligands can bind in different positions within the acrB homotrimer cavity simultaneously. Binding involves hydrophobic forces, aromatic stacking and van der Waals interactions (Yu et al., 2003).
Figure 1.4. Visualization of multidrug efflux transporter acrB topology in bacterial gram-negative inner membrane. The three protomers of acrB which form the homotrimer are shown in green, yellow and blue at a resolution of 2.9 Å. The red line represents the periplasmic face of the inner membrane and the blue line the cytoplasmic facee.
The acrAB-tolC system (the efflux pump comprising acrA, acrB and tolC), is thought that the transporter protein acrB, captures its substrates either from within the phospholipid bilayer of the inner membrane or from the cytoplasm and then transports the substrate to the extracellular medium through tolC, which forms a channel in the outer membrane. The cooperation between acrB and tolC is mediated by the periplasmic accessory protein acrA. A proposed mechanism of transport suggests the diffusion of substrates via the transmembrane domains and chambers into the central cavity, then opening of the central pore to allow the transport of the substrates through acrB toward tolC and export to the external medium. During transport each monomer of the acrB-drug complex has been proposed to have a different conformation corresponding to one of the three functional states of the transport cycle. Substrates are exported by a three-step functionally rotating mechanism; substrates undergo ordered binding changes (Seeger et al., (2006). One of the monomers is constrained by the interaction with its neighbours, its conformation is termed loose (L). Another monomer exhibits an opening from the inside of the pore domain toward the funnel of the AcrB trimer, and its conformation is designated as open (O). This conformation allows one of the subdomains to tilt and interact with a neighbouring monomer's subdomains. This interaction imposes a constraint on the conformation of the neighboring monomer, which is designated tight (T). The drug-transport mechanism proposed suggests cycling of each monomer through the conformations L, T, O, and back to L (Fig 1.5.). A sequence of interaction between subdomains of the monomers and the sequence of substrate binding events, allowing for efflux of drugs through the tunnels, with a transport mechanism that is analogous to that of a peristaltic pump (Fig 1.5.) (Takatsuka and Nikaido, 2007; Takatsuka et al., 2010). The unspecific nature of transport implied by this mechanism could account for the broad substrate specificity as well as for the transport of small molecules.
Fig 1.5. Representation of the AcrB monomers in rotational transport mechanism of substrates. The conformational states loose (L), tight (T), and open (O).
(A). Side-view representation of two of the three monomers of the acrB trimer. acrA and tolC.
(B). The lateral grooves in the L and T monomer indicate the substrate binding sites. In the first state of the cycle, a monomer binds a substrate (acridine) in its transmembrane domain (L conformation), subsequently transports the substrate from the transmembrane domain to the hydrophobic binding pocket (conversion to T conformation) and finally releases the substrate in the funnel toward tolC (O conformation). Then conversion from the O-monomer to the L-monomer conformation.
Another proposed mechanism for transport of substrates by acrB is through the central pore, the elevator mechanism (14).
Structure and function of a MFS transporter emrD
MFS transporter emrD (Multidrug resistant protein D) is a multidrug transporter from E. coli. emrD acts as an efflux pump for uncouplers of oxidative phosphorylation. Oxidative phosphorylation can rapidly arrest growth in bacteria by reducing the proton gradient. emrD has been also shown to transport detergents (e.g. benzalkonium and sodium dodecylsulfate) and export a broad spectrum of hydrophobic compounds. The overall structural topology of emrD is similar to that of lacY and glpT. Twelve transmembrane helices of emrD form a compact structure that span ~50 Å in the plan of the phospholipid bilayer and ~45 Å along the membrane normal (Fig 1.6). Four transmembrane helices face away from the interior, while the remaining helices form the internal cavity. The internal cavity of emrD is comprised by mostly of hydrophobic residues. This is consistent with emrD's function of transporting lipophillic compounds (Yin et al., 2006).
Figure 1.6. Visualization of multidrug efflux transporter emrD in bacterial gram-negative inner membrane. The red line represents the periplasmic side of the inner membrane and the blue line the cytoplasmic side.
Based on the structural homology of emrD to other MFS transporters, certain assumtions can be made about its mechanism of action. Several MDR MFS systems have an adaptor protein that facilitates the transport of substrate through the periplasmic space, possibly using an apparatus similar to the tolC-adaptor RND efflux systems. However, no such adaptor protein has been identified that is associated with emrD. emrD may act alone, as do lmrP and bmr in gram-positive bacteria. The intracellular loop region of emrD is reminiscent of the intracellular domain of msbA, which is a bacterial homolog of MDR ABC transporters. In msbA, these helices are thought to recognize head groups of the substrates as well as to transmit structural changes caused by ATP hydrolysis and substrate binding.
Importance of 3D Structure
For the designing of newer and safer drugs with good potency it is necessary to understand drug receptor interactions in detail. Drug receptor interactions are governed by the stereochemistry of the drugs and the receptors. Enhanced knowledge of drug design specifics will increase the chances of obtaining a safer drug with good therapeutic effects. Information required for this can be obtained via the resolution of the 3D structure of proteins.
The resolution of the 3D structure of proteins is a key factor in gaining more detailed understanding of the function of a protein, for example by showing ligand binding sites involved in inhibition or activation of a protein. The acquirement of the 3D structure can also show information on the mechanism of the protein.
With concerns to antibiotic resistance within bacteria a more detailed knowledge of the 3D structure can aid the development of new drugs to target these structures, as new ligand binding sites can be identified that may inhibit the action of the protein. Modified antibiotics may also be developed which are effective against bacteria with antibiotic resistant phenotypes. Patients infected with an antibiotic resistant strain of bacteria being can then be treated more efficiently.
Challenges of gaining membrane protein structure
The low abundance and the hydrophobic nature of membrane proteins conferring antibiotic resistance can make them difficult to isolate in amounts required for the elucidation of their three-dimensional structures. The determination of inner membrane protein structures is therefore the major bottleneck in the quest to understand the detailed molecular mechanisms of membrane transport.
One of the main difficulties associated with the structural determination of membrane proteins is the solubilisation of the target protein using various detergents.dsfsfsdfdsf
Protein targets of interest
Due to antibiotic resistance being a growing problem the proteins that I will be investigating are from a range of families conferring antibiotic resistance.
rarD is part of a new multidrug transporter family (eamA transporters), this family has not yet been characterised. There is a limited amount of information on the function of rarD, apart from that it is responsible for chloramphenicol resistance.
tehA SMR family transporter
tehA is part of the SMR family. Research has suggested that resistance to most lipophilic cationic dyes and related compounds in E. coli is due to tehA and does not appear to require tehB. Additionally, tehA confers hypersensitivity to those compounds with two quaternary cations (dequalinium and methyl viologen) and resistance to those compounds containing only a single quaternary cation (tetraphenylarsonium, ethidium, crystal violet, and proflavin).
Localized homology to a core region composed of transmembrane sequences (TMS) II through to V of tehA has been shown between tehA and other multidrug transporters of the SMR family. TMS II to V may be primarily responsible for the resistance conferred and the transport observed. Deletion of the C-terminal region of tehA (TMS VIII to X) does not decrease transport significantly. The observation that the tehA chromosomal deletion mutant shows a reduced rate of ethidium efflux implies that tehA may be a contributor to the observed drug efflux in E. coli. H. influenzae tehA homolog, does not contain the key homologous sequence to that of other SMR transporters, is not resistant, and shows no transport activity.
tehB is a soluble protein which associates weakly with the membrane. tehB possesses some amino acid sequence similarity to many SAM-dependent non-nucleic acid methyltransferases. These proteins have three shared motifs, and the tehB proteins show homologies within all regions with comparable sequence interval distances. tehB likely has a methyltransferase activity, a detoxification mechanism for tellurite resistance by tehAB is proposed. The chemical modification of tellurite with methyl groups would provide a means for detoxification. However the final product of the reaction is not a volatile form of methylated tellurium. tehAB determinant was found to continuously remove tellurite from the growth medium. This is consistent with the occurrence of a modification of tellurite within the cell. The presence of tehB appears to inhibit tehA's transport of ethidium.
The biochemical mechanism of tellurite resistance mediated by tehAB is not yet well understood. tehA is capable of utilizing the membrane energetics. Since tehB inhibits ethidium resistance and transport, this suggests that tehB interacts with tehA in some manner to change the affinity of tehA for these compounds.
bcr MFS transporter
bcR belongs to the same family as emrD the major facilitator superfamily. bcR is involved in sulfonamide and bicyclomycin resistance. Bcr performs this function as it is an efflux pump for; bicyclomycin, L-cysteine and sulfonamides. bcR may also be a membrane translocase (9).
baeS and components it regulates
baeS is a member of the two-component regulatory system baeS/baeR. baeS may activate baeR by phosphorylation and act as a regulator of genes that confer antibiotic resistance, including mdtABC (10).
mdt B/C are part of the same superfamily as acrB (RND superfamily), where mdtA is part of the membrane fusion protein family (MFP). mdtA/B/C form the mdtABC tripartite complex, conferring resistance against novobiocin and deoxycholate (10).
opmN and nmpC are general outer membrane porins. Porins are beta barrel transmembrane proteins. Porins act as a pore through the membrane through which small metabolites (sugars, amino acids and ions) can diffuse often by passive transportation.
The research to be carried out is to crystallize and obtain the 3D structures of the proteins in table 1 using x-ray crystallography. The aims of the project are; 1) Express outer membrane targets. 2) Purify outer membrane targets. 3) Crystalise outer membrane targets. 4) Increase expression of inner membrane targets to produce sufficient quantities, that purification can occur to obtain adequate pure protein. 5) Perform FSEC and stability assays on all inner membrane targets. 6) Crystalise remaining inner membrane targets. 7) Reproduce highly diffracting rarD crystals. 8) Characterise rarD using isothermal titration calorimetry (ITC). 9) Improve tehA diffraction.
Table 1. Progress of target proteins so far.
Progress So Far
~ 4.3 Å
~ 2.6 Å
~ 6.0 Å
The outer membrane proteins will also be investigated in conjunction to inner membrane proteins. Outer membrane proteins will be included as they are more stable compared to inner membrane proteins, so in theory should be easier to crystallize. Outer membrane proteins can be expressed in inclusion bodies and then refolded as well as being produced in a functional form within the membrane, making them easier to crystallize.