Experimental tests of a homology model for OxlT, the oxalate transporter of Oxalobacter formigenes

Abstract

Using the x-ray structure of the glycerol 3-phosphate transporter (GlpT), we devised a model for the distantly related oxalate transporter, OxlT. The model accommodates all earlier biochemical information on OxlT, including the idea that Lys-355 lies on the permeation pathway, and predicts that Lys-355 and a second positive center, Arg-272, comprise the binding site for divalent oxalate. Study of R272K, R272A, and R272Q derivatives verifies that Arg-272 is essential, and comparisons with GlpT show that both anion transporters bind substrates within equivalent domains. In 22 single-cysteine variants in TM7 and TM8, topology as marked by accessibility to Oregon green maleimide is predicted by the model, with similar concordance for 52 positions probed earlier. The model also reconciles cross-linking of a cysteine pair placed near the periplasmic ends of TM2 and TM7, and retrospective study of TM2 and TM11 confirms that positions supporting disulfide trapping lie at a helical interface. Our work describes a pathway to the modeling of OxlT and other transporters in the major facilitator superfamily and outlines simple experimental tests to evaluate such proposals.anion-binding ͉ disulfide trapping ͉ major facilitator superfamily ͉ membrane protein ͉ permeation pathway O xlT, the oxalate͞formate antiporter of Oxalobacter formigenes (1, 2), belongs to the major facilitator superfamily (MFS), a large and diverse collection encompassing 30-40% of known transporters and permeases (www.biology.ucsd.edu͞ϳmsaier͞ transport). The main biochemical mechanisms associated with transporters (uniport, antiport, and symport) may be found within the MFS, whose individual members display a broad catalog of substrate specificity, including simple sugars and amino acids, intermediary metabolites, and even neurotransmitters (3). All members of the MFS share an architectural theme in which a central loop connects two groups of (typically) six transmembrane ␣-helices. Moreover, the superfamily as a whole is characterized by a short motif (GXXXDK͞R) at the cytoplasmic ends of TM2 and TM8 (3, 4), suggesting that these two six-helix clusters derived from a common ancestor; indeed, at times one finds a clear sequence homology between the N-and C-terminal domains (4, 5).Insight into the structure of MFS transporters is presently limited. Helix organization, symmetry, and connectivity were established first for OxlT, in work based on a low-resolution (6.5 Å) structure obtained by electron crystallography (6, 7). Subsequently, higher resolution (3.2-3.5 Å) was achieved by x-ray crystallography of two other MFS members from Escherichia coli, the H ϩ ͞lactose symporter (LacY) and the phosphate͞glycerol 3-phosphate antiporter (GlpT) (8, 9). These latter achievements have prompted several recent attempts to use LacY or GlpT as structural templates for models of other systems (10-13). With this in mind and to provide a detailed perspective to guide further work, we selected the GlpT structure as a template for derivation of a homology model of OxlT, ...

Analysis of Substrate-Binding Elements in OxlT, the Oxalate:Formate Antiporter of Oxalobacter formigenes

“…Early work showed that substitution at positions 272 or 355 abolished OxlT function unless the basic character of the original residue was preserved (15)(16)(17). But even like substitutions (e.g., R272K or K355R) yield substantially diminished catalytic activity ( Table 1), suggesting that side-chain architecture in this region is critical.…”

“…Examination of the OxlT homology model (17), as well as earlier biochemical work (11,15,16) suggests that two positively charged residues (R272 and K355) serve as ligand-binding elements for the anionic OxlT substrates. The work reported here supports this hypothesis, in part by showing that a requirement for positive charge at these positions reflects requirements for OxlT function, rather than biogenesis.…”

“…1B) was of some concern. On the one hand, since the modified target resembles lysine more closely than arginine, one might expect reduced activity, as for the R272K derivative (17). It was also feasible, however, that MTSEA modified only a small fraction of the target population.…”

“…In the OxlT homology model (17), R272 and K355 are positioned so that their side chains extend into the permeation pathway, where they are presumed to interact with the divalent substrate, oxalate. This hypothesis is consistent with the findings that among derivatives at these sites, only the R272K and K355R variants retain measurable function (11,17), although at relatively low levels (see below).…”

“…Since oxalate is a divalent anion at physiological pH (pH 7), it is of interest to ask if the OxlT hydrophobic sector has a second basic residue to facilitate substrate binding, and in this regard valuable insight comes from an OxlT homology model (17) based on the x-ray structure of GlpT (14). This model supports the idea that K355 is used in ligand binding and points to R272, in TM8, as a likely partner in this event; early work (17) shows that R272, too, is required for normal OxlT function.…”