Closure of the cytoplasmic gate formed by TM5 and TM11 during transport in the oxalate/formate exchanger from Oxalobacter formigenes.

OxlT, the oxalate/formate exchanger of Oxalobacter formigenes, is a member of the major facilitator superfamily of transporters. In the present work, substrate (oxalate) was found to enhance the reactivity of the cysteine mutant S336C on the cytoplasmic end of helix 11 to methanethiosulfonate ethyl carboxylate. In addition, S336C is found to spontaneously cross-link to S143C in TM5 in either native or reconstituted membranes under conditions that support transport. Continuous wave EPR measurements are consistent with this result and indicate that positions 143 and 336 are in close proximity in the presence of substrate. These two residues are localized within helix interacting GxxxG-like motifs (G140LASG144 and S336DIFG340) at the cytoplasmic poles of TM5 and TM11. Pulse EPR measurements were used to determine distances and distance distributions across the cytoplasmic or periplasmic ends of OxlT and were compared with the predictions of an inside-open homology model. The data indicate that a significant population of transporter is in an outside-open configuration in the presence of substrate; however, each end of the transporter exhibits significant conformational heterogeneity, where both inside-open and outside-open configurations are present. These data indicate that TM5 and TM11, which form part of the transport pathway, transiently close during transport and that there is a conformational equilibrium between inside-open and outside-open states of OxlT in the presence of substrate.

Gout-causing Q141K mutation in ABCG2 leads to instability of the nucleotide-binding domain and can be corrected with small molecules.

The multidrug ATP-binding cassette, subfamily G, 2 (ABCG2) transporter was recently identified as an important human urate transporter, and a common mutation, a Gln to Lys substitution at position 141 (Q141K), was shown to cause hyperuricemia and gout. The nature of the Q141K defect, however, remains undefined. Here we explore the Q141K ABCG2 mutation using a comparative approach, contrasting it with another disease-causing mutation in an ABC transporter, the deletion of Phe-508 (ΔF508) in the cystic fibrosis transmembrane conductance regulator (CFTR). We found, much like in ΔF508 CFTR, that the Q141K mutation leads to instability in the nucleotide-binding domain (NBD), a defect that translates to significantly decreased protein expression. However, unlike the CFTR mutant, the Q141K mutation does not interfere with the nucleotide-binding domain/intracellular loop interactions. This investigation has also led to the identification of critical residues involved in the protein-protein interactions necessary for the dimerization of ABCG2: Lys-473 (K473) and Phe-142 (F142). Finally, we have demonstrated the utility of using small molecules to correct the Q141K defect in expression and function as a possible therapeutic approach for hyperuricemia and gout.

Nanochannel-based single molecule recycling.

We present a method for measuring the fluorescence from a single molecule hundreds of times without surface immobilization. The approach is based on the use of electroosmosis to repeatedly drive a single target molecule in a fused silica nanochannel through a stationary laser focus. Single molecule fluorescence detected during the transit time through the laser focus is used to repeatedly reverse the electrical potential controlling the flow direction. Our method does not rely on continuous observation and therefore is less susceptible to fluorescence blinking than existing fluorescence-based trapping schemes. The variation in the turnaround times can be used to measure the diffusion coefficient on a single molecule level. We demonstrate the ability to recycle both proteins and DNA in nanochannels and show that the procedure can be combined with single-pair Förster energy transfer. Nanochannel-based single molecule recycling holds promise for studying conformational dynamics on the same single molecule in solution and without surface tethering.

Oligomeric state of the oxalate transporter, OxlT.

OxlT, the oxalate transporter of Oxalobacter formigenes, was studied to determine its oligomeric state in solution and in the membrane. Three independent approaches were used. First, we used triple-detector (SEC-LS) size exclusion chromatography to analyze purified OxlT in detergent/lipid micelles. These measurements evaluate protein mass in a manner independent of contributions from detergent and lipid; such work shows an average OxlT mass near 47 kDa for detergent-solubilized material, consistent with that expected for monomeric OxlT (46 kDa). A disulfide-linked OxlT mutant was used to verify that it was possible detect dimers under these conditions. A second approach used amino-reactive cross-linkers of varying spacer lengths to study OxlT in detergent/lipid micelles and in natural or artificial membranes, followed by analysis via sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These tests, performed under conditions where the presence of dimers can be documented for either of two known dimeric transporters (AdiC or TetL), indicate that OxlT exists as a monomer in the membrane and retains this status upon detergent solubilization. In a final test, we showed that reconstitution of OxlT into lipid vesicles at variable protein/lipid ratios has no effect on the specific activity of subsequent oxalate transport, as the OxlT content varies between 0.027 and 5.4 OxlT monomers/proteoliposome. We conclude that OxlT is a functional monomer in the membrane and in detergent/lipid micelles.

Structural and functional importance of transmembrane domain 3 (TM3) in the aspartate:alanine antiporter AspT: topology and function of the residues of TM3 and oligomerization of AspT.

AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, a membrane protein of 543 amino acids with 10 putative transmembrane (TM) helices, is the prototype of the aspartate:alanine exchanger (AAE) family of transporters. Because TM3 (isoleucine 64 to methionine 85) has many amino acid residues that are conserved among members of the AAE family and because TM3 contains two charged residues and four polar residues, it is thought to be located near (or to form part of) the substrate translocation pathway that includes the binding site for the substrates. To elucidate the role of TM3 in the transport process, we carried out cysteine-scanning mutagenesis. The substitutions of tyrosine 75 and serine 84 had the strongest inhibitory effects on transport (initial rates of l-aspartate transport were below 15% of the rate for cysteine-less AspT). Considerable but less-marked effects were observed upon the replacement of methionine 70, phenylalanine 71, glycine 74, arginine 76, serine 83, and methionine 85 (initial rates between 15% and 30% of the rate for cysteine-less AspT). Introduced cysteine residues at the cytoplasmic half of TM3 could be labeled with Oregon green maleimide (OGM), whereas cysteines close to the periplasmic half (residues 64 to 75) were not labeled. These results suggest that TM3 has a hydrophobic core on the periplasmic half and that hydrophilic residues on the cytoplasmic half of TM3 participate in the formation of an aqueous cavity in membranes. Furthermore, the presence of l-aspartate protected the cysteine introduced at glycine 62 against a reaction with OGM. In contrast, l-aspartate stimulated the reactivity of the cysteine introduced at proline 79 with OGM. These results demonstrate that TM3 undergoes l-aspartate-induced conformational alterations. In addition, nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses and a glutaraldehyde cross-linking assay suggest that functional AspT forms homo-oligomers as a functional unit.

Ins and outs of major facilitator superfamily antiporters.

The major facilitator superfamily (MFS) represents the largest group of secondary active membrane transporters, and its members transport a diverse range of substrates. Recent work shows that MFS antiporters, and perhaps all members of the MFS, share the same three-dimensional structure, consisting of two domains that surround a substrate translocation pore. The advent of crystal structures of three MFS antiporters sheds light on their fundamental mechanism; they operate via a single binding site, alternating-access mechanism that involves a rocker-switch type movement of the two halves of the protein. 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. In this review, we attempt to give an account of a set of mechanistic principles that characterize all MFS antiporters.

Cysteine scanning mutagenesis of TM5 reveals conformational changes in OxlT, the oxalate transporter of Oxalobacter formigenes.

We constructed a single-cysteine panel encompassing TM5 of the oxalate transporter, OxlT. The 25 positions encompassed by TM5 were largely tolerant of mutagenesis, and functional product was recovered for 21 of the derived variants. For these derivatives, thiol-directed MTS-linked agents (MTSEA, MTSCE, and MTSES) were used as probes of transporter function, yielding 11 mutants that responded to probe treatment, as indicated by effects on oxalate transport. Further study identified three biochemical phenotypes among these responders. Group 1 included seven mutants, exemplified by G151C, displaying substrate protection against probe inhibition. Group 2 was comprised of a single mutant, P156C, which had unexpected behavior. In this case, we observed increased activity if weak acid/base or neutral probes were used, while exposure to probes introducing a fixed charge led to decreased function. In both instances, the presence of substrate prevented the observed response. Group 3 contained three mutants (e.g., S143C) in which probe sensitivity was increased by the presence of substrate. The finding of substrate-protectable probe modification in groups 1 and 2 suggests that TM5 lies on the permeation pathway, as do its structural counterparts, TM2, TM8, and TM11. In addition, we speculate that substrate binding facilitates TM5 conformational changes that allow new regions to become accessible to MTS-linked probes (group 3). These biochemical data are consistent with the recently developed OxlT homology model.

ABC transporters: how small machines do a big job.

Transporters from the ATP-binding cassette (ABC) superfamily operate in all organisms, from bacteria to humans, to pump substances across biological membranes. Recent high-resolution views of ABC transporters in different conformational states provide clues as to how ATP might be used to drive the structural reorganizations that accompany membrane transport. Importantly, it now appears that a putative translocation pathway running through the center of the transporter might be gated alternately, either at the inside or the outside of the cytoplasmic membrane, coupling substrate translocation to a cycle of ATP-dependent conformational changes. ATP binding and ATP hydrolysis have distinct roles in this cycle: binding favors the outward-facing orientation, whereas hydrolysis returns the transporter to an inward-facing conformation.

Kinetic evidence is consistent with the rocker-switch mechanism of membrane transport by GlpT.

Secondary active transport of substrate across the cell membrane is crucial to many cellular and physiological processes. The crystal structure of one member of the secondary active transporter family, the sn-glycerol-3-phosphate (G3P) transporter (GlpT) of the inner membrane of Escherichia coli, suggests a mechanism for substrate translocation across the membrane that involves a rocker-switch-type movement of the protein. This rocker-switch mechanism makes two specific predictions with respect to kinetic behavior: the transport rate increases with the temperature, whereas the binding affinity of the transporter to a substrate is temperature-independent. In this work, we directly tested these two predictions by transport kinetics and substrate-binding experiments, integrating the data on this single system into a coherent set of observations. The transport kinetics of the physiologically relevant G3P-phosphate antiport reaction were characterized at different temperatures using both E. coli whole cells and GlpT reconstituted into proteoliposomes. Substrate-binding affinity of the transporter was measured using tryptophan fluorescence quenching in detergent solution. Indeed, the substrate transport velocity of GlpT increased dramatically with temperature. In contrast, neither the apparent Michaelis constant (Km) nor the apparent substrate-binding dissociation constant (Kd) showed temperature dependence. Moreover, GlpT-catalyzed G3P translocation exhibited a completely linear Arrhenius function with an activation energy of 35.2 kJ mol-1 for the transporter reconstituted into proteoliposomes, suggesting that the substrate-loaded transporter is delicately poised between the inward- and outward-facing conformations. When these results are taken together, they are in agreement with a rocker-switch mechanism for GlpT.

Topology of AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, determined by site-directed fluorescence labeling.

The gram-positive lactic acid bacterium Tetragenococcus halophilus catalyzes the decarboxylation of L-aspartate (Asp) with release of L-alanine (Ala) and CO(2). The decarboxylation reaction consists of two steps: electrogenic exchange of Asp for Ala catalyzed by an aspartate:alanine antiporter (AspT) and intracellular decarboxylation of the transported Asp catalyzed by an L-aspartate-beta-decarboxylase (AspD). AspT belongs to the newly classified aspartate:alanine exchanger family (transporter classification no. 2.A.81) of transporters. In this study, we were interested in the relationship between the structure and function of AspT and thus analyzed the topology by means of the substituted-cysteine accessibility method using the impermeant, fluorescent, thiol-specific probe Oregon Green 488 maleimide (OGM) and the impermeant, nonfluorescent, thiol-specific probe [2-(trimethylammonium)ethyl]methanethiosulfonate bromide. We generated 23 single-cysteine variants from a six-histidine-tagged cysteineless AspT template. A cysteine position was assigned an external location if the corresponding single-cysteine variant reacted with OGM added to intact cells, and a position was assigned an internal location if OGM labeling required cell lysis. The topology analyses revealed that AspT has a unique topology; the protein has 10 transmembrane helices (TMs), a large hydrophilic cytoplasmic loop (about 180 amino acids) between TM5 and TM6, N and C termini that face the periplasm, and a positively charged residue (arginine 76) within TM3. Moreover, the three-dimensional structure constructed by means of the full automatic modeling system indicates that the large hydrophilic cytoplasmic loop of AspT possesses a TrkA_C domain and a TrkA_C-like domain and that the three-dimensional structures of these domains are similar to each other even though their amino acid sequences show low similarity.

Analysis of substrate-binding elements in OxlT, the oxalate:formate antiporter of Oxalobacter formigenes.

An OxlT homology model suggests R272 and K355 in transmembrane helices 8 and 11, respectively, are critical to OxlT-mediated transport. We offer positive evidence supporting this idea by studying OxlT function after cysteine residues were separately introduced at these positions. Without further treatment, both mutant proteins had a null phenotype when they were reconstituted into proteoliposomes. By contrast, significant recovery of function occurred when proteoliposomes were treated with MTSEA (methanethiosulfonate ethylamine), a thiol-specific reagent that implants a positively charged amino group. In each case, there was a 2-fold increase in the Michaelis constant (K(M)) for oxalate self-exchange (from 80 to 160 microM), along with a 5-fold (K355C) or 100-fold (R272C) reduction in V(max) compared to that of the cysteine-less parental protein. Analysis by MALDI-TOF confirmed that MTSEA introduced the desired modification. We also examined substrate selectivity for the treated derivatives. While oxalate remained the preferred substrate, there was a shift in preference among other substrates so that the normal rank order (oxalate > malonate > formate) was altered to favor smaller substrates (oxalate > formate > malonate). This shift is consistent with the idea that the substrate-binding site is reduced in size via introduction of the SCH(2)CH(2)NH(3)(+) adduct, which generates a side chain that is approximately 1.85 A longer than that of lysine or arginine. These findings lead us to conclude that R272 and K355 are essential components of the OxlT substrate-binding site.

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

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.

Altered oxyanion selectivity in mutants of UhpT, the Pi-linked sugar phosphate carrier of Escherichia coli.

In Escherichia coli, the UhpT transporter catalyzes the electroneutral accumulation of sugar 6-phosphate by exchange with internal inorganic phosphate (Pi). The substrate specificity of UhpT is regulated at least in part by constituents of an Asp388-Lys391 intrahelical salt bridge, and mutations that remove one but not both of these residues alter UhpT preference for organophosphate substrates. Using site-directed mutagenesis, we examined the role played by these two positions in the selection of the oxyanion countersubstrate. We show that derivatives having aliphatic or polar residues at positions 388 and 391 are gain-of-function mutants capable of transporting SO4 as well as Pi. These oxyanions share similar structures but differ significantly in the presence of a proton(s) on Pi. Our findings therefore lead us to suggest that the Asp388-Lys391 ion pair acts normally as a filter that prevents substrates lacking a proton that can be donated from occupying the UhpT active site.

Impact of the deltaF508 mutation in first nucleotide-binding domain of human cystic fibrosis transmembrane conductance regulator on domain folding and structure.

Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator (CFTR), commonly the deletion of residue Phe-508 (DeltaF508) in the first nucleotide-binding domain (NBD1), which results in a severe reduction in the population of functional channels at the epithelial cell surface. Previous studies employing incomplete NBD1 domains have attributed this to aberrant folding of DeltaF508 NBD1. We report structural and biophysical studies on complete human NBD1 domains, which fail to demonstrate significant changes of in vitro stability or folding kinetics in the presence or absence of the DeltaF508 mutation. Crystal structures show minimal changes in protein conformation but substantial changes in local surface topography at the site of the mutation, which is located in the region of NBD1 believed to interact with the first membrane spanning domain of CFTR. These results raise the possibility that the primary effect of DeltaF508 is a disruption of proper interdomain interactions at this site in CFTR rather than interference with the folding of NBD1. Interestingly, increases in the stability of NBD1 constructs are observed upon introduction of second-site mutations that suppress the trafficking defect caused by the DeltaF508 mutation, suggesting that these suppressors might function indirectly by improving the folding efficiency of NBD1 in the context of the full-length protein. The human NBD1 structures also solidify the understanding of CFTR regulation by showing that its two protein segments that can be phosphorylated both adopt multiple conformations that modulate access to the ATPase active site and functional interdomain interfaces.

Characterization of the adenosinetriphosphatase and transport activities of purified cystic fibrosis transmembrane conductance regulator.

The cystic fibrosis transmembrane conductance regulator (CFTR) functions in vivo as a cAMP-activated chloride channel. A member of the ATP-binding cassette superfamily of membrane transporters, CFTR contains two transmembrane domains (TMDs), two nucleotide-binding domains (NBDs), and a regulatory (R) domain. It is presumed that CFTR couples ATP hydrolysis to channel gating, and as a first step in addressing this issue directly, we have established conditions for purification of biochemical quantities of human CFTR expressed in Sf9 insect cells. Use of an 8-azido[alpha-(32)P]ATP-binding and vanadate-trapping assay allowed us to devise conditions to preserve CFTR function during purification of a C-terminal His(10)-tagged variant after solubilization with lysophosphatidylglycerol (1%) and diheptanoylphosphatidylcholine (0.3%) in the presence of excess phospholipid. Study of purified and reconstituted CFTR showed that it binds nucleotide with an efficiency comparable to that of P-glycoprotein and that it hydrolyzes ATP at rates sufficient to account for presumed in vivo activity [V(max) of 58 +/- 5 nmol min(-1) (mg of protein)(-1), K(M)(MgATP) of 0.15 mM]. In further work, we found that neither nucleotide binding nor ATPase activity was altered by phosphorylation (using protein kinase A) or dephosphorylation (with protein phosphatase 2B); we also observed inhibition (approximately 40%) of ATP hydrolysis by reduced glutathione but not by DTT. To evaluate CFTR function as an anion channel, we introduced an in vitro macroscopic assay based on the equilibrium exchange of proteoliposome-entrapped radioactive tracers. This revealed a CFTR-dependent transport of (125)I that could be inhibited by known chloride channel blockers; no significant CFTR-dependent transport of [alpha-(32)P]ATP was observed. We conclude that heterologous expression of CFTR in Sf9 cells can support manufacture and purification of fully functional CFTR. This should aid in further biochemical characterization of this important molecule.

Structure of nucleotide-binding domain 1 of the cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a chloride channel. Nucleotide-binding domain 1 (NBD1), one of two ABC domains in CFTR, also contains sites for the predominant CF-causing mutation and, potentially, for regulatory phosphorylation. We have determined crystal structures for mouse NBD1 in unliganded, ADP- and ATP-bound states, with and without phosphorylation. This NBD1 differs from typical ABC domains in having added regulatory segments, a foreshortened subdomain interconnection, and an unusual nucleotide conformation. Moreover, isolated NBD1 has undetectable ATPase activity and its structure is essentially the same independent of ligand state. Phe508, which is commonly deleted in CF, is exposed at a putative NBD1-transmembrane interface. Our results are consistent with a CFTR mechanism, whereby channel gating occurs through ATP binding in an NBD1-NBD2 nucleotide sandwich that forms upon displacement of NBD1 regulatory segments.

Functional characterization of cysteine residues in GlpT, the glycerol 3-phosphate transporter of Escherichia coli.

In Escherichia coli, the GlpT transporter, a member of the major facilitator superfamily, moves external glycerol 3-phosphate (G3P) into the cytoplasm in exchange for cytoplasmic phosphate. Study of intact cells showed that both GlpT and HisGlpT, a variant with an N-terminal six-histidine tag, are inhibited (50% inhibitory concentration approximately 35 microM) by the hydrophilic thiol-specific agent p-mercurichlorobenzosulfonate (PCMBS) in a substrate-protectable fashion; by contrast, two other thiol-directed probes, N-maleimidylpropionylbiocytin (MPB) and [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET), have no effect. Use of variants in which the HisGlpT native cysteines are replaced individually by serine or glycine implicates Cys-176, on transmembrane helix 5 (TM5), as the major target for PCMBS. The inhibitor sensitivity of purified and reconstituted HisGlpT or its cysteine substitution derivatives was found to be consistent with the findings with intact cells, except that a partial response to PCMBS was found for the C176G mutant, suggesting the presence of a mixed population of both right-side-out (RSO) (resistant) and inside-out (ISO) (sensitive) orientations after reconstitution. To clarify this issue, we studied a derivative (P290C) in which the RSO molecules can be blocked independently due to an MPB-responsive cysteine in an extracellular loop. In this derivative, comparisons of variants with (P290C) and without (P290C/C176G) Cys-176 indicated that this residue shows substrate-protectable inhibition by PCMBS in the ISO orientation in proteoliposomes. Since PCMBS gains access to Cys-176 from both periplasmic and cytoplasmic surfaces of the protein (in intact cells and in a reconstituted ISO orientation, respectively) and since access is unavailable when the substrate is present, we propose that Cys-176 is located on the transport pathway and that TM5 has a role in lining this pathway.

Structural model for 12-helix transporters belonging to the major facilitator superfamily.

The major facilitator superfamily includes a large collection of evolutionarily related proteins that have been implicated in the transport of a variety of solutes and metabolites across the membranes of organisms ranging from bacteria to humans. We have recently reported the three-dimensional structure, at 6.5 A resolution, of the oxalate transporter, OxlT, a representative member of this superfamily. In the oxalate-bound state, 12 helices surround a central cavity to form a remarkably symmetrical structure that displays a well-defined pseudo twofold axis perpendicular to the plane of the membrane as well as two less pronounced, mutually perpendicular pseudo twofold axes in the plane of the membrane. Here, we combined this structural information with sequence information from other members of this protein family to arrive at models for the arrangement of helices in this superfamily of transport proteins. Our analysis narrows down the number of helix arrangements from about a billion starting possibilities to a single probable model for the relative spatial arrangement for the 12 helices, consistent both with our structural findings and with the majority of previous biochemical studies on members of this superfamily.

Three-dimensional structure of a bacterial oxalate transporter.

The major facilitator superfamily (MFS) represents one of the largest classes of evolutionarily related membrane transporter proteins. Here we present the three-dimensional structure at 6.5 A resolution of a bacterial member of this superfamily, OxlT. The structure, derived from an electron crystallographic analysis of two-dimensional crystals, reveals that the 12 helices in the OxlT molecule are arranged around a central cavity, which is widest at the center of the membrane. The helices divide naturally into three groups: a peripheral set comprising helices 3, 6, 9 and 12; a second set comprising helices 2, 5, 8 and 11 that faces the central substrate transport pathway across most of the length of the membrane; and a third set comprising helices 1, 4, 7 and 10 that participate in the pathway either on the cytoplasmic side (4 and 10) or on the periplasmic side (1 and 7). Overall, the architecture of the protein is remarkably symmetric, providing a compelling molecular explanation for the ability of such transporters to carry out bi-directional substrate transport.

Pyridoxal 5-phosphate inhibition of substrate selectivity mutants of UhpT, the sugar 6-phosphate carrier of Escherichia coli.

In the sugar phosphate transporter UhpT, gain-of-function derivatives that prefer phosphoenolpyruvate (PEP) as substrate have an uncompensated lysine residue on transmembrane segment 11. We show here that these variants are also highly susceptible to substrate-protectable inhibition by covalent modification of lysine with pyridoxal 5-phosphate. The chemical requirements of this interaction provide evidence that the gain-of-function phenotype results from the pairing of the uncompensated lysines in these mutants with the anionic carboxyl group of PEP.