2.A.1 The Major Facilitator Superfamily (MFS)
The MFS is a very old, large and diverse superfamily that includes millions of sequenced members. They catalyze uniport, solute:cation (H+, but seldom Na+) symport and/or solute:H+ or solute:solute antiport. Most are of 400-600 amino acyl residues in length and possess either 12, 14, or occasionally, 24 transmembrane α-helical spanners (TMSs). The mechanistic principles applicable to all MFS carriers have been summarized by Law et al (2008), while Zhang et al. 2015 considered the interaction between protonation and the negative-inside membrane potential. Functional roles of the conserved sequence motifs were also discussed in the context of the 3D structures. Transporters can be modified posttranslationally by phsophorylation, ubiquitination, glycosylation and/or palmitoylation (Czuba et al. 2018). The mammalian proteins have been analyzed from phylogenetic standpoints, revealing three major groups with differing conserved residues (Jia et al. 2019). Proteins of different topologies were also identified. Hu et al. 2020 reviewed the main substrates and functions of SLCs that are expressed in the brain, with an emphasis on selected SLCs that are important physiologically, pathologically, and pharmacologically in the blood-brain barrier, astrocytes, and neurons. The glucose (hexose) transporters, GLUTs, TC# 2.A.1.1, have been reviewed (Holman 2020). Single-molecule FRET studies of membrane transport proteins has revealed their conformational flexibilities (Löw et al. 2021)..
The 24 TMS MFS permease, NarK, of Paracoccus pantotrophus has two 12 TMS domains, NarK1 and NarK2, both of which are required for normal nitrate uptake. NarK1 catalyzes NO3-:H+ symport, dependent on the pmf, while NarK2 catalyzes NO3-:NO2- antiport, independently of the pmf (Wood et al., 2002). Thus, the protein is a fusion protein of two homologous but distinct MFS permeases.
MFS permeases exhibit specificity for sugars, polyols, drugs, neurotransmitters, Krebs cycle metabolites, phosphorylated glycolytic intermediates, amino acids, peptides, osmolites, siderophores (efflux), iron-siderophores (uptake), nucleosides, organic anions, inorganic anions, etc. They are found ubiquitously in all three kingdoms of living organisms. Among the yeast MFS MDR transporters, the DHA2 family (TC 2.A.1.3) is more variable between species than the DHA1 family (TC 2.A.1.2) (Gbelska et al. 2006).
One member of the DHA2 family with 14 TMSs, the TetL Me2+ · tetracycline:H+ antiporter of B. subtilis (TC #2.A.1.3.16), which also exhibits monovalent ion antiport activity, can be converted to a monovalent cation (Na+, K+, H+) antiporter with no tetracycline transport activity by deletion of TMSs 7 and 8, the two central and extra TMSs (Jin et al., 2001). Genome analyses of MFS permeases have been published (Lorca et al., 2007).
A 6.5 Å resolution structure for the MFS permease, OxlT (TC #2.A.1.11.1) was obtained in early studies (Heymann et al., 2001; Hirai et al., 2002) which shows the positions of the transmembrane α-helices but does not allow assignment of the TMS # to these helices. Molecular modeling (Hirai et al., 2003) led to the suggestion that the 12 TMS protein arose from a 3 TMS element by two successive duplication events. The same suggestion resulted from sequence comparisons showing that the primordial 3 TMS element may have arisen from a VIC family (TC #1.A.1) 2 TMS channel-forming unit Hvorup & Saier, (2002).This conclusion has been extensively confirmed in several more recent studies.
The high-resolution 3-dimensional structures (3.3 and 3.5 Å resolution) of the glycerol-3-P:P antiporter (GlpT; TC #2.A.1.4.3) and the lactose:H+ symporter (LacY; TC #2.A.1.5.1), respectively (Huang et al., 2003 and Abramson et al., 2003, respectively; see also Locher et al., 2003; Guan et al., 2007) have been determined. These structures reveal the 2-fold symmetry expected, based on sequence similarity of the two halves. The substrate pathway is predicted to exist between the two halves of the permeases using an alternating access mechanism with a single substrate binding site (Huang et al., 2003). This mechanism is termed a 'rocker switch' type of movement.
As suggested above, MFS symporters and antiporters are believed to operate via a single binding site, alternating-access mechanism that involves a rocker-switch type movement of the two halves of the protein (Law et al., 2008). In the sn-glycerol-3-phosphate transporter (GlpT) from Escherichia coli, the substrate-binding site is formed by several charged residues and a histidine that can be protonated. Salt-bridge formation and breakage are involved in the conformational changes of the protein during transport (Law et al., 2008).
Vesicular glutamate transporters (VGLUTs [2.A.1.14.13 and 2.A.1.14.16]) are responsible for the vesicular storage of L-glutamate and play an essential role in glutamatergic signal transmission in the central nervous system. VGLUT2 facilitates L-glutamate uptake in a membrane potential (ΔΨ)-dependent fashion. Uptake exhibited an absolute requirement for ~4 mM Cl- and was sensitive to Evans blue, but was insensitive to D,L-aspartate. VGLUT2s with mutations in the transmembrane-located residues Arg184, His128, and Glu191 showed a dramatic loss in L-glutamate transport activity. VGLUT2 appears to possess two intrinsic transport machineries that are independent of each other: a ΔΨ-dependent L-glutamate uptake and a Na+-dependent Pi uptake (Juge et al., 2006).
Trichoderma spp. are avirulent, fungal, opportunistic plant symbionts present in nearly all climatic soils. These Trichoderma strains produce secondary metabolites that are potent bio-control agents against microbial pathogens and also can be plant growth promoters. The MFS includes a large proportion of efflux-pumps which are linked with membrane transport of these secondary metabolites in T. reesei (Chaudhary et al. 2016). Many of these export drugs such an anticancer agents and antibiotics. A comprehensive review of the classes of efflux pump inhibitors from various sources, highlighting their structure-activity relationships, which can be useful for medicinal chemists in the pursuit of novel efflux pump inhibitors has appeared (Durães et al. 2018).
The generalized transport reactions catalyzed by MFS porters are:
(1) Uniport: S (out) ⇌ S (in)
(2) Symport: S (out) + [H+ (or Na+)] (out) ⇌ S (in) + [H+ (or Na+)] (in)
(3) Antiport: S1 (out) + S2 (in) ⇌ S1 (in) + S2 (out) (S1 may be H+ or a solute)