Tomografía de coherencia óptica (OCT)

The ATP-binding cassette (ABC) transporters form a superfamily in both prokaryotes and eukaryotes, which couples the energy from ATP hydrolysis to the translocation of substrates across a membrane (116). Their substrates include polypeptides, sugars, lipids, other hydrophobic compounds, and amino acids (116). All known eukaryotic ABC transporters are exporters while the majority of prokaryotic ABC transporters act as importers. In this Chapter, the LptB2FGC protein complex transports LPS from the

lateral periplasmic leaflet of the IM to the periplasmic LptA protein, representing a different type of ABC exporter indicative of a novel subfamily.

The archetypal ABC transporter is made up of four core domains: two hydrophobic transmembrane domains (TMDs), and two nucleotide-binding domains (NBDs). These can be arranged in a variety of topologies (117). The TMDs consist of multiple transmembrane α-helices which span across the lipid bilayer and form the binding site/path for substrates. It is speculated that the TMDs may possess one or more pairs of high affinity and low affinity binding sites, with substrate transferring from one to the other after entering the transporter (118). There are 48 ABC transporters in humans and 80 in the E. coli (119). The ABC transporter family is currently further divided into 22 subfamilies of prokaryotic importers, 24 subfamily of prokaryotic exporters, and 10 subfamilies of eukaryotic proteins. Despite the relatively high diversity in the TMDs, 25%-30% sequence identity is shared through most of the NBDs in ABC transporter

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superfamily, suggesting a similar mechanism for utilising of ATP power to drive the machinery.

In the bacterial uptake systems, the TMDs of bacterial ABC importers typically interact with a periplasmic binding domain/protein, which delivers the substrate afterwards and ensures specificity. These binding protein-dependent transporters take up a wide variety of substrates, range from small sugars, amino acids, anions, iron chelatros vitamin B12

and etc. In Gram-negative bacteria, small substrates enter the periplasm via diffusion through the outer membrane OMP porins (120). Large compounds, such as vitamin B12

and iron-siderophore complexes, are transported across the outer membrane through high-affinity transporters by consuming energy from electrochemical gradient across the cytoplasmic membrane (121). A typical example of this kind of domain/protein is in the MalFGK2 transporter which transport maltose from the periplasm to cytoplasm

(122). The periplasmic protein MBP usually had low affinity with MalFGK2

transporter, however when the binding affinity of MBP with MalFGK2 transporter

increased significantly when ADP vanadate (a kind of ATP mimic) was trapped inside the NBDs of MalFGK2 transporter (122). This kind of mechanism allow the periplasmic

protein to search for substrate and promotes the efficiency of substrate transporting. ABC transporters also functions in efflux mode in some cases, including surface components of the bacterial cell (such as capsular polysaccharides, lipopolysaccharides, and techoic acid), proteins involved in bacterial pathogenesis (such as hemolysin, heme-binding protein, and alkaline protease), peptide antibiotics, heme, drugs and siderophores (123). In this cases periplasmic protein/domain usually fuse with the TMDs. As an example, the inner membrane ABC tranporter HlyB secret HlyA with an inner membrane protein HlyD (124) and an outer membrane facilitator TolC (125). TolC forms a channel spanning both outer membrane and the periplasm of E. coli (126,127). Both TMDs and NBDs of HlyB and HylD are responsible for substrate recognition. Following the recognition, ABC transporter HlyB presumably transport the unfolded HlyA across the inner membrane (128-131) and into the tunnel formed by TolC.

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transporting substrate by binding and hydrolysing of ATP. ATP hydrolysis is thought to drive a conformational change in the NBDs which propagate to the TMDs resulting in an energy consumable substrate translocation. Unlike TMDs, the NBDs are highly conserved, suggesting their universal role in powering the transport process. The structure of a NBD monomer can be divided into two subdomains: a larger RecA-like subdomain consisting of two β-sheets and six α-helices and a smaller helical subdomain formed by three to four α-helices (119). They are comprised of an ATP binding cassette domain, which contains a Walker A and Walker B motif, the Signature sequence (C motif) and several possible functional sites which together defines the ABC transporter. The Walker A and B motifs are supposed to bind phosphates of ATP and the Mg2+ _ion

when ATP binds in the NBDs (Figure 12, Figure 24 and Table 1). The Walker A motif, also known as the P loop, follows β-strand 3 and forms a loop that binds to the phosphates of ATP or ADP. The aspartic acid residue in Walker B coordinates the Mg2+ ion in the nucleotide binding through H2O (132-134). A glutamic acid residue binds to

the attacking water molecule and the Mg2+ ion (135,136). This glutamic acid residue is the catalytic base for hydrolysis functionality. In LptB E163 is the catalytic site. The Q loop, also known as the lid (137), contains a glutamine residue that binds to the Mg2+ ion and attacking water (134,135). The structure of the Q loop is part of the Rec-like domain and appears to be highly flexible (119). The H loop following the β-strand 8 is referred to as a switch and also contains conserved histidine residue that interact with γ-phosphate of ATP (135,138) (Figure 2). The signature motif is also named as LSGGQ motif, linker peptide or C motif. It has been used to identify ABC transporter and is the only major highly conserved motif that does not have direct interaction with nucleotides in the monomer structure (119).

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Figure 12 E. coli MalK structure in ATP bound form (119).

MalK is the NBD domain of the MalFGK2 ABC transporter that is responsible

for maltose transport (a) Stereo view of the monomer E. coli MalK in ATP bound form. (b) The homodimer of MalK, viewed down the local twofold axis. The RecA-like subdomain is green, and the helical subdomain is cyan. Different colors further distinguish the conserved segments: Walker A motif (red), LSGGQ motif (magenta), Walker B motif (blue), and the Q loop (yellow). The ATP is represented in ball-and-stick model [O atom (red), N atom (blue)]. (c)

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Schematic diagram of the interaction between one of the two ATPs bound to the homodimer. Black lines represent van der Waals contacts, and blue lines correspond to hydrogen bonds and salt bridges. (138).

Motif

Consensus

sequence Function Referencing structures

Walker A or P loop GXXGXGKST ATP binding HisP, MJ0789, MJ1267,

Rad50, TAP1, GlcV, MalK

Q loop or lid Q a. TM subunit

interacion a. BtuCD b. Q H-bond to Mg2+ b. MJ0796 (E171Q), GlcV- ADP c. Binding to the attacking water c. MJ0796 (E171Q) LSGGQ or linker peptide or signature motif

LSGGQXQR ATP binding Rad50, MJ0796 (E171Q),

MalK

Walker B hhhhD D makes a water-

bridged contact with Mg2+ GlcV (Mg2+_ADP, Mg2+_{AMP-PNP), MJ1267} (Mg2+_{ADP) MJ0796} (Mg2+_ADP) E following Walker B a.Binds to attacking water a. MJ0796 (E171Q) b.Binds to Mg2+ through a water b. GlcV (Mg2+_ADP, Mg2+_AMP-PNP) H motif or switch region H His H-bond to γ- phosphate MJ0796 (E171Q), MalK

Table 1 Function of conserved motifs in the nucleotide-binding domain NBDs

This table described the conserved motifs in the NBDs of the ABC transporter, references for the structure mentioned above are: HisP (139) , MJ0796 (132), MJ1267 (133), Rad50 (138), TAP1 (140), GlcV (141), MalK (138), BtuCD (142), MJ0796 (E171Q) (135).

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