2.A.15 The Betaine/Carnitine/Choline Transporter (BCCT) Family
Proteins of the BCCT family are found in Gram-negative and Gram-positive bacteria and archaea. Their common functional feature is that they all transport molecules with a quaternary ammonium group [R-N (CH3)3]. The BCCT family proteins vary in length between 481 and 706 amino acyl residues and possess 12 putative transmembrane α-helical spanners (TMSs). The x-ray structures (see next paragraph) reveal two 5 TMS repeats with the total number of TMSs being 10. These porters catalyze bidirectional uniport or are energized by pmf-driven or smf-driven proton or sodium ion symport, respectively, or else by substrate:substrate antiport. Some of these permeases exhibit osmosensory and osmoregulatory properties inherent to their polypeptide chains. The BCCT family has been reviewed (Ziegler et al. 2010).
Schulze et al. (2010) reported the structures of the sodium-independent carnitine/butyrobetaine antiporter CaiT from Proteus mirabilis (PmCaiT) at 2.3 Å and from E. coli (EcCaiT) at 3.5 Å resolution. Most members of the BCCT family are Na+- or H+-dependent, whereas EcCaiT is Na+- and H+-independent. The three-dimensional architecture of CaiT resembles that of the Na+-dependent transporters LeuT and BetP, but in CaiT, a methionine sulphur takes the place of the Na+ to coordinate the substrate in the central transport site, accounting for Na+ independence. Both CaiT structures show the fully open, inward-facing conformation, and thus complete the set of functional states that describe the alternating access mechanism. EcCaiT contains two bound butyrobetaine substrate molecules, one in the central transport site, the other in an extracellular binding pocket. In the structure of PmCaiT, a tryptophan side chain occupies the transport site, and access to the extracellular site is blocked. Binding of both substrates to CaiT reconstituted into proteoliposomes is cooperative, with Hill coefficients of up to 1.7, indicating that the extracellular site is regulatory. Schulze et al. (2010) proposed a mechanism whereby the occupied regulatory site increases the binding affinity of the transport site and initiates substrate translocation.
Most secondary-active transporters transport their substrates using an electrochemical ion gradient, but the carnitine transporter (CaiT) is an ion-independent, l-carnitine/gamma-butyrobetaine antiporter. Crystal structures of CaiT from E. coli and Proteus mirabilis revealed the inverted five-transmembrane-helix repeat similar to that in the amino acid/Na+ symporter, LeuT. Kalayil et al.(2013) showed that mutations of arginine 262 (R262) made CaiT Na+-dependent with increased transport activity in the presence of a membrane potential, in agreement with substrate/Na+ cotransport. R262 also plays a role in substrate binding by stabilizing the partly unwound TM1' helix.
Modeling CaiT from P. mirabilis in the outward-open and closed states on the corresponding structures of the related symporter BetP revealed alternating orientations of the buried R262 side chain, which mimic sodium binding and unbinding in the Na+-coupled substrate symporters. A similar mechanism may be operative in other Na+/H+-independent transporters, in which a positively
charged amino acid replaces the cotransported cation. The oscillation of the R262 side chain in CaiT indicates how a positive charge triggers the change between outward-open and inward-open conformations (Kalayil et al., 2013).
The generalized transport reactions catalyzed by members of the BCCT family are:
Substrate (out) + nH+ (out) → Substrate (in) + nH+ (in)
Substrate (out) + Na+ (out) → Substrate (in) + Na+ (in)
Substrate1 (out) + Substrate2 (in) → Substrate1 (in) + Substrate2 (out)
Substrate (out) ⇌ Substrate (in)
Substrate = a quaternary amine
Glycine betaine:Na+ symporter (also transports dimethylsulfonioacetate and dimethylsulfoniopropionate)
OpuD of Bacillus subtilis
Glycine betaine transporter, BetP. BetP is a transporter with three different functions: betaine transport, osmosensing, and osmoregulation (Krämer and Morbach 2004). The x-ray structure is known (3PO3; 2WIT; Ressl et al., 2009). Regulatory crosstalk in the trimeric BetP has been reported (Gärtner et al., 2011). An extracellular K+ -dependent interaction site modulates betaine-binding (Ge et al., 2011). The porter is trimeric and exhibits structural asymmetry (Tsai et al., 2011). The C-terminal domain is involved in osmosensing and is trimeric like wild-type BetP. The two Na+ binding sites are between TMSs 1 and 8 in the first and second 5 TMS repeats, and between the equivalent TMSs 6 and 3 in the second and first repeats, respectively (Khafizov et al. 2012). interdependent binding of betaine and two sodium ions is observed during the coupling process. All three sites undergo progressive reshaping and dehydration during the alternating-access cycle, with the most optimal coordination of all substrates found in the closed state (Perez et al. 2014). BetP is active and regulated only when negatively charged lipids such as phosphatidyl glycerol are present, and the mechanism has been discussed (Güler et al. 2016). The K+-sensing C-terminal domain results in K+-dependent cooperative betaine-binding (Ge et al. 2011). BetP is a homotrimer lacking exact 3-fold symmetry. The observed differences may be due to crystal packing, or they may reflect different functional states of the transporter, related to osmosensing and osmoregulation (Ziegler et al. 2004). Intracellular K+ alters the conformation of the disordered C- and N-terminal domains to allosterically reconfigure TMSs 3, 8 and 10 to enhance betaine interactions. A map of the betaine binding site, at near single amino acid resolution, revealed a critical extrahelical H-bond mediated by TMS3 with betaine (Tantirimudalige et al. 2022). Both the N- and C-terminal (45 aas) segments participate in autoregulation, transducing changes in K+ concentrations as well as lipid bilayer properties to the integral membrane part of the protein. The C-terminal segment has short helical elements and an orientation that confines interactions (Leone et al. 2022).
BetP of Corynebacterium glutamicum (P54582)
The glycine betaine transporter, BetH, of 505 aas and 12 TMSs (Lu et al. 2005).
BetH of Halobacillus trueperi
Ectosine/glycine betaine/proline:Na+ symporter
EctP of Corynebacterium glutamicum
Low affinity (0.9 mM), high efficiency, choline/glycine betaine:H+ symporter, BetT (Chen and Beattie, 2007)
BetT of Pseudomonas syringae (Q4ZLW8)
The high-affinity, proton- or sodium-driven, secondary symporter, BetT. The cytoplasmic C-terminal domain of plays a role in the regulation of BetT activity; C-terminal truncations cause BetT to be permanently locked in a low-transport-activity mode. (Tøndervik and Strøm 2007).
BetT of E. coli (P0ABC9)
Glycine-betaine/proline-betaine:Na+ symporter, BetS; BetT, OpuD (Kappes et al. 1996; Boscari et al. 2002; Ziegler et al. 2010).
BetS of Sinorhizobium meliloti (Q92WM0)
The glycine betaine, dimethylsulfoniopropionate:Na+ symporter (Ziegler et al., 2010).
Dddt of Psyohrobacter sp. J466 (D0U567)
The ectoine/glycine:Na+ symporter, LcoP (Ziegler et al., 2010).
LcoP of Corynebacterium glutamicum (Q8NN75)
The ectoine/hydroxyectoine:Na+ symporter, EctT (Ziegler et al., 2010).
EctT of Virgibacillus pantothenticus (Q93AK1)
High affinity glycine betaine uptake system
Glycine betaine transporter of Acinetobacter baylyi (Q6F754)
Carnitine:γ-butyrobetaine antiporter. The x-ray structure is known at 3.5 Å resolution (Schulze et al., 2010). The structure reveals a homotrimer where each protomer has 12 TMSs with 4 L-carnitine molecules outlining the pathway. There is a central binding site and another in the intracellular vestibule (Tang et al. 2010).
CaiT of E. coli (P31553)
The L-carnitine:γ-butyrobetaine antiporter, CaiT. The x-ray structure is known at 2.3 Å resolution (Schulze et al., 2010).
CaiT of Proteus mirabilis (B4EY22)
Uncharacterized transporter, YeaV, sometimes called CaiT, of 481 or 536 aas with 10 - 12 TMSs.
YeaV of Escherichia coli