Binding protein dependent ABC transporters

In bacteria and archaea the large subfamily of ABC transporters that mediate nutrient uptake have an additional component, a periplasmic or membrane anchored binding protein that binds nutrients with high affinity prior to translocation (Boos and Lucht, 1996). The liganded binding protein binds to TMDs and stimulates via the TMDs the ATPase activity of the ABC cassettes in the cytoplasm. By this the binding protein becomes tightly bound to the TMDs to ensure that the substrate is passed through the transporter which refers to the transition state of the transport cycle (Chen et al., 2001). In the TMDs of binding protein dependent ABC transporters a local sequence similarity, the EAA-motif (EAAAx₃Gx₉IxLP), has been found which is referred to as the L-loop (Locher et al., 2002). Among the best studied transporters of this class are the maltose transporter MalEFGK₂ fromE. coli and the histidine permease HisJQMP₂ from Salmonella typhimurium. Nevertheless, all attempts to determine the structure of the intact MalFGK₂ and HisQMP₂ transporters at atomic resolution failed until today.

Only their water soluble components have been structurally characterized HisJ (Oh et al., 1994), HisP (Hung et al., 1998), MalE (Spurlino et al., 1991), and MalK (Chen et al., 2003). Instead, the first structure of a binding protein dependent ABC transporter was that of Btu(CD)2 (see Figure 1.3C).

Figure 1.5: Binding proteins ProX from Archaeoglobus fulgidus in the open unliganded conformation (left) and the closed liganded conformation (right). The domains are colored blue and yellow and the ligand in red.

Binding proteins

Binding proteins bind their substrates with affinities in the low µM range. During evolution they have been optimized to bind a large variety of ligands like sugars, amino acids, vitamins, and so forth. Nevertheless, crystal structures of several binding proteins revealed a common blueprint. They consist of two globular domains or lobes which are flexibly linked to each other by one to three polypeptide switches between them, forming a hinge. The hinge region is able to undergo large conformational changes, allowing the two domains to move with respect to each other (Figure 1.5). The ligand binding site is located between the two domains in the hinge region. In the unliganded form several open states can be adopted by the binding protein (Bj¨orkman and Mowbray, 1998;

Magnusson et al., 2002). Ligand binding induces a large conformational change that moves both domains toward each other, engulfing the ligand between both domains.

The maltose transporter MalEFGK₂

Maltose and maltodextrins are a major carbon source for microorganisms. The maltose transporter is responsible for the transport of these sugars across the cytoplasmic mem-brane. It consists of the periplasmic binding protein MalE, the two homologous TMDs MalF and MalG, and two copies of the ABC ATPases MalK. Additionally to its sugar transport function MalEFGK₂ is also involved in regulatory processes. This regulatory function is mediated by MalK that is not only an energizing module within the trans-port complex. The C-terminal 2/5 of MalK fold into a distinct barrel-like structure that is present only in a subset of all bacterial and archaeal ABC transporters. When no substrate is transported by MalFGK2, MalT, the transcriptional activator of most of

1.3 ATP binding cassette transporters

Figure 1.6: Maltose transporter from Thermococcus litoralis Structurally character-ized components are shown as molecular model TMBP in green and the MalK dimer in blue (ATPase domain) and yellow (regulatory domain). The membrane spanning domains MalF and MalG are schematically drawn in red and orange

the mal genes, is bound to the C-terminal part of MalK andmal gene expression cannot occur. Furthermore, the dephosphorylated form of EIIA^Glcbinds to the C-terminal part of MalK as well. This inhibits non-PTS (phosphotransferases systems) sugar uptake systems like the maltose transporter as long as glucose is being phosphorylated during PTS transport, which leads in a series of phosphotransfer reactions to the dephospho-rylation of EIIA^Glc (Boos and Shuman, 1998). Both binding sites are located in two distinct areas in the barrel-like structure and do not interfere with each other (B¨ohm et al., 2002).

A very homologous system has been found in the hyperthermophilic archaeon Thermo-coccus litoralis (Xavier et al., 1996). This has some unique properties: it recognizes maltose and trehalose with equal affinities, but no maltodextrins, it exhibits high affin-ity transport at 85 ^◦C, the optimal growth temperature of T. litoralis, and the binding protein is anchored in the cytoplasmic membrane. In our laboratory the structures of the ATP binding cassette MalK and the trehalose/maltose binding protein TMBP have been already structurally determined (Diederichs et al., 2000; Diez et al., 2001) (see Figure 1.6). Part of the present thesis is aimed at the crystallization and structural characterization of the transport complex MalFGK₂. Crystals were obtained which, however, did not diffract beyond 5 ˚A and no high resolution structure could be deter-mined so far (Chapter 2). Currently, we are working on new constructs for the expression of MalFGK₂ from Thermococcus litoralis as well asThermus thermophilus inE. coli in order to obtain more suitable crystals for structural analyses.

A homologue of MalEFGK₂ has also been found in the Gram-positive bacterium Alicy-clobacillus acidocaldarius. This thermoacidophilic organism grows best at pH 3.6 and 57 ^◦C. All its extracellular proteins, like the lipid anchored binding protein MalE, are

mation is available for mesophilic as well as for thermophilic MalE homologues (Sharff et al., 1992; Evdokimov et al., 2001; Diez et al., 2001), MalE from A. acidocaldarius is an excellent candidate to study acidostability. The structural analysis of MalE and its adaptation towards acidostability are discussed in Chapter 3.

The compatible solute transporter ProU

Microorganisms lack systems for active water transport. They adjust their cellular wa-ter content and turgor by controlling the pool of osmolytically active substances in the cytoplasm. As a first response to an osmotic upshift, bacterial cells transport potassium ions into the cytoplasm. Since ions influence protein stability and function, this is only a transient solution to cope with high osmolarity. After a while, the ions are replaced by the synthesis or accumulation of compounds known as compatible solutes. Compatible solutes are defined as compounds that can be accumulated up to molar concentrations without disturbing cellular functions. According to the preferential exclusion model, compatible solutes are excluded from the immediate hydration shell of proteins due to unfavorable interactions. The resulting non-uniform distribution of these compounds within the cytoplasm forces proteins to occupy a smaller volume by reducing the amount of hydration water. These effects are not only compatible with cellular functions but also stabilize the tertiary structures of proteins.

An important system for the uptake of compatible solutes is the osmoregulated binding protein dependent ABC transporter ProU from E. coli. It consists of two copies of the ABC ATPase ProV, two TMDs ProW and the periplasmic binding protein ProX. ProU shows a rather broad substrate specificity but has a clear preference for N,N,N-trimethyl glycine (glycine betaine) and N,N-dimethyl-L-proline (proline betaine) (Gouesbet et al., 1994; Haardt et al., 1995). As deduced from genome sequence, a homologous system has been found in the hyperthermophilic archaeonArchaeoglobus fulgidus which consists of two distinct TMD copies ProW1 and ProW2, two copies of ProV and the lipid anchored binding protein ProX. This shows the same ligand preferences as ProU fromE. coli.

Both systems require a binding protein, ProX, that is able to overcome the usual ex-clusion of the ligand within its binding site. Crystallization and structural analysis of both ProX proteins has been achieved in the thesis presented here. The structural pre-requisites for high affinity binding of glycine betaine and proline betaine is discussed in Chapter 4 for ProX from E. coli and in Chapter 5 for ProX fromA. fulgidus.