Increase In Apparent Affinity Research Articles

Staphylococcal peroxidase inhibitor (SPIN): Investigation of the inhibitory N-terminal domain via a stabilizing disulfide insertion

Staphylococcus aureus secretes an array of small proteins that inhibit key enzyme-catalyzed reactions necessary for proper function of the human innate immune system. Among these, the Staphylococcal Peroxidase Inhibitor, SPIN, blocks the activity of myeloperoxidase (MPO) and thereby disrupts the HOCl-generating system of neutrophils. Previous studies on S. aureus SPIN have shown that it relies on a C-terminal α-helical bundle domain to mediate initial binding to MPO, but requires a disordered N-terminal region to fold into a β-hairpin conformation to inhibit MPO activity. To further investigate the structure/function relationship of SPIN, we introduced two cysteine residues into its N-terminal region to trap SPIN in its MPO-bound conformation and characterized the modified protein, which we refer to here as SPIN-CYS. Although control experiments confirmed the presence of the disulfide bond in SPIN-CYS, solution structure determination revealed that the N-terminal region of SPIN-CYS adopted a physically constrained series of lariat-like structures rather than a well-defined β-hairpin. Nevertheless, SPIN-CYS exhibited a gain in inhibitory potency against human MPO when compared to wild-type SPIN. This gain of function persisted even in the presence of deleterious mutations within the C-terminal α-helical bundle domain. Surface plasmon resonance studies showed that the gain in potency arose through an increase in apparent affinity of SPIN-CYS for MPO, which was driven primarily by an increased association rate with MPO when compared to wild-type SPIN. Together, this work provides new information on the coupled binding and folding events required to manifest biological activity of this unusual MPO inhibitor.

Human Cardiac Myosin-Binding Protein C N-Terminal Domains Cooperatively Impact Actin Structural Dynamics

Cardiac myosin-binding protein C (cMyBP-C) is a thick filament-associated protein that modulates contractility by influencing interactions between myosin and actin. Despite being a leading cause of familial hypertrophic cardiomyopathy, the mechanism by which cMyBP-C modulates contractility is not well understood. N-terminal domains of cMyBP-C (C0 through C2) have been shown to interact with the actin-thin filament; however, the cMyBP-C regions involved in the interaction and how cMyBP-C influences actin's dynamic structure remain unclear. Here, we have used cosedimentation assays for measuring binding kinetics and time-resolved phosphorescence anisotropy (TPA) for measuring actin rotational dynamics. We examined the effects of actin interactions with human fragments C0-C1 and C0-C2 of cMyBP-C. We also evaluated roles of cMyBP-C phosphorylation sites known to influence contractility. We find that C0-C2 binds actin with ∼4-fold increase in apparent affinity compared to C0-C1. TPA of labeled-actin showed marked differences in the potency of effects between C0-C2, C0-C1, and phosphorylated cMyBP-C fragments with respect to decreasing the amplitude of torsional motions, increasing the rate of monomer rotation, and increasing relative resilience of the filament. A Hill equation was used to determine the level of cooperativity of the TPA effects for each fragment. Our results demonstrate that C0-C2 exhibits stronger cooperativity in propagating its effects on amplitude, rate, and resilience to more distant actin monomers than C0-C1. Assuming a thin filament cooperative unit of ∼7 actin monomers (length of tropomyosin), our TPA analyses suggest that one molecule of C0-C2 bound to actin propagated effects 2-3 cooperative units (14-21 monomers), whereas C0-C1 effects were confined to one unit, and phosphorylation reduced cooperativity. These findings provide new insight into the molecular basis for cMyBP-C effects on actin structure and function. This work was supported by an NIH R00HL122397 to B.A.C.

Structure/function studies of Diverse Forms of ADPGlucose Pyrophosphorylase

ADPGlucose Pyrophosphorylase (ADPG PPase) is an allosterically regulated enzyme that serves as the rate‐limiting step of starch synthesis in plants and glycogen synthesis in bacteria. Because starch is a source of renewable and biodegradable carbon, ADPG PPase is an attractive target for protein engineering to increase biomass yield. The microbial versions of this enzyme are quite diverse in their regulatory and physical properties in accord with adaptation to different environments; some of these properties would be useful to incorporate into crops to enhance starch production.The enzyme from Deinococcus radiodurans (D. rad), coded for by the glgC gene, is an extremophile resistant to radiation and harsh growth conditions. Using standard assays with magnesium (Mg) as a co‐factor, the purified D. rad enzyme was found to have relatively low apparent affinity for the substrate ATP and Mg with S0.5 values of 8.4 mM and 24 mM, respectively. However, this organism is known to have a high intracellular manganese (Mn) concentration, which correlates with ionizing radiation and desiccation resistance. Because stored glycogen could act as an energy and carbon source during cell recovery, it was hypothesized that this ADPG PPase may be more responsive to Mn. Use of Mn in the assay revealed a ~9.3 fold increase in apparent affinity for ATP, a ~5 fold increase in apparent affinity for Mn (versus Mg), and a two‐fold increase in Vmax which supports the hypothesis. Complete kinetic characterization of the wild‐type (WT) and selected altered enzymes is underway to probe the putative metal binding site(s). Several crystallization conditions were found to yield promising results and further crystallization trials are in progress.In contrast, the enzyme from the thermophile Thermotoga maritima (T.ma) consists of two subunits, coded for by the glgC and glgD genes. Previous studies indicated that the purified WT T.ma glgD alone had no activity but was found to stimulate the relatively low activity of the glgC subunit. The complex could also be activated by FBP while the glgC alone was insensitive to FBP. The apparent affinity for ATP and Mg increased ~2 and ~3 fold in the complex while the Vmax increased 4 fold. Previous results indicated that limited proteolysis of the C‐terminus of glgD resulted in lower activation of the complex. In order to probe this further, a 73 amino acid C‐terminal truncation was generated and the activity of the complexes with the WT and WT glgC/truncated glgD (altered complex) compared. The altered complex exhibited a lower apparent affinity for ATP than the WT complex as well as a 5‐fold reduction in Vmax, which still represented a ~3‐fold stimulation compared to the glgC alone. In the presence of the activator FBP, the altered complex was slightly inhibited by FBP in contrast to the 2‐fold activation of the WT complex. The results appear to support a role for the C‐terminus in allosteric activation by FBP and partial stimulation of the enzyme in the absence of FBP. A 3 Å crystal structure for the glgC subunit was recently solved which was in good agreement with a previously modeled structure based on the A. tumefaciens enzyme structure (PDB 3BRK). Complete kinetic and physical characterization of the altered enzyme complexes in the absence and presence of FBP, as well as crystallization of various complexes are underway.Support or Funding InformationSupported in part by NSF BIO MCB Grant 0448676 and BIO DBI.

Increasing Thyromimetic Potency through Halogen Substitution.

Sobetirome is one of the most studied thyroid hormone receptor β (TRβ)-selective thyromimetics in the field due to its excellent selectivity and potency. A small structural change-replacing the 3,5-dimethyl groups of sobetirome with either chlorine or bromine-produces significantly more potent compounds, both in vitro and in vivo. These halogenated compounds induce transactivation of a TRβ-mediated cell-based reporter with an EC50 value comparable to that of T3, access the central nervous system (CNS) at levels similar to their parent, and activate an endogenous TR-regulated gene in the brain with an EC50 value roughly five-fold lower than that of sobetirome. Previous studies suggest that this apparent increase in affinity can be explained by halogen bonding between the ligand and a backbone carbonyl group in the receptor. This makes the new analogues potential candidates for treating CNS disorders that may respond favorably to thyroid-hormone-stimulated pathways.

Structure‐Function Studies of Deinococcus radiodurans ADP Glucose Pyrophosphorylase: Role of Ser48 in Allosteric Regulation

Adenosine Diphosphate Glucose Pyrophosphorylase (ADPG PPase) is an allosterically regulated enzyme that functions as the rate‐limiting step of starch synthesis in plants and glycogen synthesis in bacteria. Because starch is a source of renewable and biodegradable carbon, ADPG PPase is an attractive target for protein engineering to increase biomass yield in crops. The microbial versions of this enzyme are quite diverse in their regulatory and physical properties; some of these properties would be useful to incorporate into transgenic crops to enhance starch production. While a number of ADPG PPases have been kinetically characterized, there is only one published x ray structure (PDB 3BRK) of an inhibited form. Very little is known about the enzyme from Deinococcus radiodurans (D. rad), an extremophile that is resistant to ionizing radiation and harsh growth conditions. When comparing the amino acid sequence of this enzyme to other characterized ADPG PPases, it was noted that position 48 differed with a serine substituted for alanine in a region known to be important for allostery. To probe the role of Ser‐48, the S48A enzyme was generated by site‐directed mutagenesis and the recombinant altered D. rad ADPG PPases successfully expressed in E. coli and purified via a scheme that includes anion exchange chromatography, size exclusion chromatography, and affinity chromatography. Initial studies on the S48A enzyme in the absence of activators have shown a dramatic 20‐fold increase in the apparent affinity for the substrate ATP, a 3‐fold increase in apparent affinity for the cofactor magnesium, and a 4‐fold increase in Vmax compared to wild‐type. Interestingly, in the presence of the activator FBP there was little change in the apparent binding affinity for substrates or Vmax compared to WT which displays a 14‐fold increase in Vmax and 12‐fold and 3‐fold increase in apparent affinity for ATP and magnesium, respectively. Similarly, in the presence of F6P, there were not a very large increase in Vmax or higher apparent affinity for ATP for the S48A enzyme, but there was a 9‐fold difference in Vmax and a 12‐fold and 2‐fold increase in apparent binding affinity for ATP and magnesium for WT, respectively. The WT enzyme also displayed higher apparent affinity for FBP and F6P compared to the S48A enzyme. The alanine substitution appears to result in an enzyme form that is partially activated but relatively insensitive to activators. Further kinetic characterization of S48A in the presence of sulfate and the inhibitor phosphate is in progress. With respect to physical characterization, two crystallization conditions for S48A, one including imidazole and the other with lithium sulfate and PEG, were found to yield preliminary results. Further crystallization trials are in progress as a first step in elucidating the three‐dimensional structure.Support or Funding InformationSupported in part by NSF and NSF BIO MCB grant #0448676.

Probing the Role of Ser‐48 in the Regulation of Deinococcus radiodurans ADP Glucose Pyrophosphorylase

ADPGlucose Pyrophosphorylase (ADPG PPase) is an allosterically regulated enzyme that serves as the rate-limiting step of starch synthesis in plants and glycogen synthesis in bacteria. Because starch is a source of renewable and biodegradable carbon, ADPG PPase is an attractive target for protein engineering to increase biomass yield. The microbial versions of this enzyme are quite diverse in their regulatory and physical properties; some of these properties would be useful to incorporate into crops to enhance starch production. Little is known about the enzyme from Deinococcus radiodurans (D. rad), an extremophile resistant to radiation and harsh growth conditions. When comparing the sequence of this enzyme to others, position 48 differed with a serine substituted for alanine in a region known to be important for allostery. To probe the role of S48, the S48A enzyme was generated by site-directed mutagenesis and the altered D. rad ADPG PPase successfully expressed and purified. Initial studies on the S48A enzyme in the absence of activators have shown a 20-fold increase in the apparent affinity for the substrate ATP and a 4-fold increase in Vmax compared to wild-type. Interestingly, in the presence of the activators FBP and F6P there were no change in the apparent binding affinity for substrates or Vmax compared to WT which displays a 5-fold increase in Vmax and 12-fold and 3-fold increase in apparent affinity for ATP and magnesium, respectively. The alanine substitution appears to result in an enzyme form that is partially activated but insensitive to activators. Complete kinetic analyses of the S48A enzyme are in progress. Supported in part by NSF Grant 0448676.

Structure‐function studies of the ADPglucose pyrophosphorylase from Thermotoga maritima: probing the role of a conserved aspartate residue in the glgD and glgC subunits (769.20)

ADPGlucose pyrophosphorylase (ADPG PPase, glgC gene) catalyzes the rate‐limiting step of glucan biosynthesis in plants and bacteria. Structure‐function studies of the diverse forms of the enzyme will allow for the engineering of a more active enzyme to produce more renewable carbon. In Thermotoga maritima (T.ma) there are two genes, glgC and glgD, which code for two subunits of this ADPG PPase. The recombinant His‐tagged subunits were successfully purified using a heat step and nickel chromatography; the wild type (WT) glgD alone had little to no activity, while the glgC protein displayed a specific activity of ~3 Units/mg with S0.5 values for ATP and magnesium (Mg) of 5.4 mM and 18.7 mM, respectively. However, the WT glgC/D complex was stimulated ~5‐fold compared to glgC alone and also activated ~2‐fold by FBP. Previous alignment studies and molecular modeling indicated that glgC D126 plays a role in catalysis so the D126A enzyme was generated to test this hypothesis. The glgC D126A protein displayed a modest increase in apparent affinity for substrates compared to WT but a 25‐fold decrease in Vmax indicating a role in optimal catalysis. Interestingly, the inactive glgD protein also harbors an aspartate (D125) at this position and the D125A glgD enzyme was generated for characterization. Surprisingly, the altered protein was found to have activity with S0.5 values for ATP, Mg, and G1P of 4.90 mM, 14.8 mM, and 39.2 µM, respectively, with a Vmax of 0.26 U/mg. In preliminary assays, we found that the glgD D125A/glgC complex displayed only a small stimulation compared to glgC alone while the glgD/glgC D126A complex displayed stimulation comparable to WT; a very large stimulation of activity was found when the glgD D125A/glgC D126A was combined, rising to the level of the glgD D125A/glgC2 complex. These aspartate residues may affect activity and stimulation of the subunits in a complex in a synergistic manner. Complete kinetic and physical characterization of the altered enzyme complexes is underway.Grant Funding Source: Supported by NSF award 0448676.

In Vivo Measurement of Hippocampal GABAA/cBZR Density with [18F]-Flumazenil PET for the Study of Disease Progression in an Animal Model of Temporal Lobe Epilepsy

PurposeImbalance of inhibitory GABAergic neurotransmission has been proposed to play a role in the pathogenesis of temporal lobe epilepsy (TLE). This study aimed to investigate whether [18F]-flumazenil ([18F]-FMZ) PET could be used to non-invasively characterise GABAA/central benzodiazepine receptor (GABAA/cBZR) density and affinity in vivo in the post-kainic acid status epilepticus (SE) model of TLE.MethodsDynamic [18F]-FMZ -PET scans using a multi-injection protocol were acquired in four male wistar rats for validation of the partial saturation model (PSM). SE was induced in eight male Wistar rats (10 weeks of age) by i.p. injection of kainic acid (7.5–25 mg/kg), while control rats (n = 7) received saline injections. Five weeks post-SE, an anatomic MRI scan was acquired and the following week an [18F]-FMZ PET scan (3.6–4.6 nmol). The PET data was co-registered to the MRI and regions of interest drawn on the MRI for selected structures. A PSM was used to derive receptor density and apparent affinity from the [18F]-FMZ PET data.Key FindingsThe PSM was found to adequately model [18F]-FMZ binding in vivo. There was a significant decrease in hippocampal receptor density in the SE group (p<0.01), accompanied by an increase in apparent affinity (p<0.05) compared to controls. No change in cortical receptor binding was observed. Hippocampal volume reduction and cell loss was only seen in a subset of animals. Histological assessment of hippocampal cell loss was significantly correlated with hippocampal volume measured by MRI (p<0.05), but did not correlate with [18F]-FMZ binding.SignificanceAlterations to hippocampal GABAA/cBZR density and affinity in the post-kainic acid SE model of TLE are detectable in vivo with [18F]-FMZ PET and a PSM. These changes are independent from hippocampal cell and volume loss. [18F]-FMZ PET is useful for investigating the role that changes GABAA/cBZR density and binding affinity play in the pathogenesis of TLE.

Dissecting Gating Rules of GIRK Channels: Role of PIP2 in Ethanol-Dependent Activation

G protein-gated inwardly rectifying potassium (GIRK) channels are implicated in alcohol abuse and addiction. We discovered a discrete alcohol-binding pocket in the channel mediating ethanol-dependent activation. Here, we investigated the role of G proteins and PIP2 in ethanol-dependent gating. We engineered GIRK2 with single, modifiable cysteine at L257 in alcohol pocket and found that alcohol-like methanthiosulfonate (MTS) reagents activate GIRK2-L257C, similar to ethanol-dependent activation. We assessed role of G proteins in alcohol activation by either increasing levels of Gβγ (+Gβ1γ2) or decreasing Gβγ through chelation with membrane bound Phosducin (+mPhos). Neither Gβ1γ2 nor mPhos altered the rate of MTS-HE-mediated GIRK2-L257C activation. For comparison, we examined GIRK2-L344C, a key site for Gβγ activation that is inhibited by MTS modification. In contrast to L257C, rate of MTS modification showed a clear dependence on Gβγ levels. These results suggest that alcohol activation of GIRK channel is independent of G proteins.To investigate the role of PIP2, we used voltage-sensitive phosphatase DR-VSP to transiently deplete PIP2 in the membrane. Activation of DR-VSP completely reversed MTS-HE activated current of GIRK2-L257C. Furthermore, MTS-HE treatment significantly slowed the rate of GIRK2-L257C current inhibition following DR-VSP activation, suggesting an increase in apparent affinity for PIP2 and GIRK2-L257C channels modified by MTS-HE. Lastly, we examined the role of PIP2 on alcohol-dependent activation of wild-type GIRK2. Addition of propanol significantly slowed the rate of wild-type GIRK2 current inhibition following PIP2 depletion. Taken together, these data demonstrate that alcohol-dependent activation of GIRK involves an increase in apparent affinity for PIP2, with little influence of Gβγ subunits. The fundamental dichotomy between alcohol and Gβγ arises from distinct gating mechanisms converging on PIP2-dependent opening, revealing novel pathways for antagonizing alcohol's effects on ion channels.

Signaling Active CD95 Receptor Molecules Trigger Co-translocation of Inactive CD95 Molecules into Lipid Rafts

The capability of soluble CD95L trimers to trigger CD95-associated signaling pathways is drastically increased by oligomerization. The latter can be achieved, for example, by antibodies recognizing a N-terminal epitope tag in recombinant CD95L variants or by genetic engineering-enforced formation of hexamers. Using highly sensitive and accurate binding studies with recombinant CD95L variants equipped with a Gaussia princeps luciferase reporter domain, we found that oligomerization of CD95L has no major effect on CD95 occupancy. This indicates that the higher activity of oligomerized CD95L trimers is not related to an avidity-related increase in apparent affinity and points instead to a crucial role of aggregation of initially formed trimeric CD95L-CD95 complexes in CD95 activation. Furthermore, binding of soluble CD95L trimers was found to be insufficient to increase the association of CD95 with the lipid raft-containing membrane fraction. However, when Gaussia princeps luciferase-CD95L trimers were used as tracers to "mark" inactive CD95 molecules, increased association of these inactive receptors was observed upon activation of the remaining CD95 molecules by help of highly active hexameric Fc-CD95L or membrane CD95L. Moreover, in cells expressing endogenous CD95 and chimeric CD40-CD95 receptors, triggering of CD95 signaling via endogenous CD95 resulted in co-translocation of CD40-CD95 to the lipid raft fraction, whereas vice versa activation of CD95-associated pathways with Fc-CD40L via CD40-CD95 resulted in co-translocation of endogenous CD95. In sum, this shows that signaling-active CD95 molecules not only enhance their own association with the lipid raft-containing membrane fraction but also those of inactive CD95 molecules.

Specific targeting of pancreatic β‐cells by simultaneous binding of GLP‐1 and α2‐adrenergic receptors with a multivalent ligand

Diabetes Mellitus is characterized by a decline in β‐cell mass and function. The lack of specificity of β‐cell targeting agents poses a challenge to observe changes in β‐cell mass and thus monitor disease progression. We propose β‐cell specificity can be enhanced by using multivalent ligands that simultaneously bind to multiple unique surface receptors that, as a combination, identify a cell type of interest. We synthesized a ligand composed of Yohimbine (Yhb) (α2 adrenergic receptor (α2AR) antagonist) linked to Glucagon‐like Peptide 1(GLP‐1). Receptors to both ligands are expressed on βTC3 cells at a ratio of ~ 1.5:1 (GLP‐1R/α2AR). Microscopy of a fluorophore tagged ligand indicated that binding was rapid with removal of ligand from the cell surface seen within 2–3 min. From population based competition assays, the binding affinity for monovalent GLP‐1 was ~10 nM, whereas GLP‐1/Yhb exhibited 2 distinct binding sites. The high affinity binding (9 pM) is correlated to MVL crosslinking the complementary receptors causing a decrease in ligand off rate, resulting in a 1000X increase in apparent affinity. The low affinity binding (225 nM) is consistent with weak binding of the GLP‐1 moiety in the MVL to the spare GLP‐1 receptors in βTC3 cells. Our findings suggest that GLP‐1/Yhb acts as a specific targeting agent to β‐cells as it exhibits enhanced binding to and retention in β‐cells compared to other cells in the imaging field.Supported by: Juvenile Diabetes Research Foundation, and the Arizona Biomedical Research Commission

Transmembrane AMPA receptor regulatory protein regulation of competitive antagonism: a problem of interpretation

Synaptic AMPA receptors are greatly influenced by a family of transmembrane AMPA receptor regulatory proteins (TARPs) which control trafficking, channel gating and pharmacology. The prototypical TARP, stargazin (or γ2), shifts the blocking ability of several AMPAR-selective compounds including the commonly used quinoxalinedione antagonists, CNQX and NBQX. Stargazin's effect on CNQX is particularly intriguing as it not only apparently lowers the potency of block, as with NBQX, but also renders it a partial agonist. Given this, agonist behaviour by CNQX has been speculated to account for its weaker blocking effect on AMPAR-TARP complexes. Here we show that this is not the case. The apparent effect of stargazin on CNQX antagonism can be almost entirely explained by an increase in the apparent affinity for l-glutamate (l-Glu), a full agonist and neurotransmitter at AMPAR synapses. Partial agonism at best plays a minor role but not through channel gating per se but rather because CNQX elicits AMPAR desensitization. Our study reveals that CNQX is best thought of as a non-competitive antagonist at glutamatergic synapses due to the predominance of non-equilibrium conditions. Consequently, CNQX primarily reports the proportion of AMPARs available for activation but may also impose additional block by receptor desensitization.

Verapamil Block of T-Type Calcium Channels

Verapamil is a prototypical phenylalkylamine (PAA), and it was the first calcium channel blocker to be used clinically. It tonically blocks L-type channels in the inner pore with micromolar affinity, and its affinity increases at depolarized membrane potentials. In T-type calcium channels, verapamil blocks with micromolar affinity and has modestly increased affinity at depolarized potentials. We found that a related PAA, 4-desmethoxyverapamil (D888), is comparable with verapamil both in affinity and in state-dependence. Permanently charged verapamil was more effective intracellularly than neutral verapamil. Charged PAAs were able to access their binding site from both inside and outside the cell. Furthermore, membrane-impermeant [2-(trimethylammonium)ethyl]methanethiosulfonate was able to access the inner pore from outside of the cell. We examined a homology model of the T-type calcium channel to look for possible routes of drug entry. Mutation of L1825W produced a channel that was blocked significantly more slowly by charged verapamil from the outside, with an increase in apparent affinity when the drug was applied from the inside. Data suggest that T-type channels have a back pathway through which charged drugs can access the inner pore of the channel without passing through the plasma membrane.

Identification of Residues in the Drug Translocation Pathway of the Human Multidrug Resistance P-glycoprotein by Arginine Mutagenesis

P-glycoprotein (P-gp, ATP-binding cassette B1) is a drug pump that extracts toxic drug substrates from the plasma membrane and catalyzes their ATP-dependent efflux. To map the residues in the drug translocation pathway, we performed arginine-scanning mutagenesis on all transmembrane (TM) segments (total = 237 residues) of a P-gp processing mutant (G251V) defective in folding (15% maturation efficiency) (glycosylation state used to monitor folding). The rationale was that arginines introduced into the drug-binding sites would mimic drug rescue and enhance maturation of wild-type or processing mutants of P-gp. It was found that 38 of the 89 mutants that matured had enhanced maturation. Enhancer mutations were found in 11 of the 12 TM segments with the largest number found in TMs 6 and 12 (seven in each), TMs that are critical for P-gp-drug substrate interactions. Modeling of the TM segments showed that the enhancer arginines were found on the hydrophilic face, whereas inhibitory arginines were located on a hydrophobic face that may be in contact with the lipid bilayer. It was found that many of the enhancer arginines caused large alterations in P-gp-drug interactions in ATPase assays. For example, mutants A302R (TM5), L339R (TM6), G872R (TM10), F942R (TM11), Q946R (TM11), V982R (TM12), and S993R (TM12) reduced the apparent affinity for verapamil by approximately 10-fold, whereas the F336R (TM6) and M986R (TM12) mutations caused at least a 10-fold increase in apparent affinity for rhodamine B. The results suggest that P-gp contains a large aqueous-filled drug translocation pathway with multiple drug-binding sites that can accommodate the bulky arginine side chains to promote folding of the protein.

The Crystal Structure of CHIR-AB1: A Primordial Avian Classical Fc Receptor

CHIR-AB1 is a newly identified avian immunoglobulin (Ig) receptor that includes both activating and inhibitory motifs and was therefore classified as a potentially bifunctional receptor. Recently, CHIR-AB1 was shown to bind the Fc region of chicken IgY and to induce calcium mobilization via association with the common γ-chain, a subunit that transmits signals upon ligation of many different immunoreceptors. Here we describe the 1.8-Å-resolution crystal structure of the CHIR-AB1 ectodomain. The receptor ectodomain consists of a single C2-type Ig domain resembling the Ig-like domains found in mammalian Fc receptors such as FcγRs and FcαRI. Unlike these receptors and other monomeric Ig superfamily members, CHIR-AB1 crystallized as a 2-fold symmetrical homodimer that bears no resemblance to variable or constant region dimers in an antibody. Analytical ultracentrifugation demonstrated that CHIR-AB1 exists as a mixture of monomers and dimers in solution, and equilibrium gel filtration revealed a 2:1 receptor/ligand binding stoichiometry. Measurement of the 1:1 CHIR-AB1/IgY interaction affinity indicates a relatively low affinity complex, but a 2:1 CHIR-AB1/IgY interaction allows an increase in apparent affinity due to avidity effects when the receptor is tethered to a surface. Taken together, these results add to the structural understanding of Fc receptors and their functional mechanisms.

In vitro evolution of an antibody fragment population to find high-affinity hapten binders

Recently, we constructed a focused antibody library tailored to interact with haptens. High functionality of this library was demonstrated, as specific binders could be retrieved to a range of different haptens. In the current study we have developed a mutagenesis and selection strategy in order to further fine-tune the hapten binding properties of these antibody fragments. Testosterone was chosen as model antigen for the investigation. A population, rather than a single clone, originating from this focused library and enriched for testosterone binders, was subjected to random mutagenesis and different phage display selection strategies of various stringencies. These included consecutively lowering the antigen concentration and having, or not having, soluble hapten present during the phage capture and elution steps. The different selection procedures resulted in a considerable increase in apparent affinities for several of the selected populations, from which the highest affinity antibody isolated had a K(D) of 2 nM, corresponding to an approximately 200-fold affinity improvement compared with the best clone of the starting population. Importantly, the polyclonal nature of the starting material allowed for the identification of novel unrelated variants that differed in fine-specificity, demonstrating that this approach is valuable for exploring different parts of structure space.

Regulation of ion channels and transporters by phosphatidylinositol 4,5‐bisphosphate

Regulation of ion channels and transporters by phosphatidylinositol 4,5‐bisphosphate

A Conserved Glutamate Residue in Transmembrane Helix 10 Influences Substrate Specificity of Rabbit OCT2 (SLC22A2)

OCT1 and OCT2 are involved in renal secretion of cationic drugs. Although they have similar selectivity for some substrates (e.g. tetraethylammonium (TEA)), they have distinct selectivities for others (e.g. cimetidine). We postulated that "homolog-specific residues," i.e. the 24 residues that are conserved in OCT1 orthologs as one amino acid and in OCT2 as a different one, influence homolog-specific selectivity and examined the influence on substrate binding of three of these conserved residues that are found in the C-terminal half of the rabbit orthologs of OCT1/2. The N353L and R403I substitutions (OCT2 to OCT1) did not significantly change the properties of OCT2. However, the E447Q replacement shifted substrate selectivity toward an OCT1-like phenotype. Substitution of glutamate with cationic amino acids (E447K and E447R) abolished transport activity, and the E447L mutant displayed markedly reduced transport of TEA and cimetidine while retaining transport of 1-methyl-4-phenylpyridinium. In a novel homology model of the three-dimensional structure of OCT2, Glu(447) was found in a putative docking region within a hydrophilic cleft of the protein. In addition, six residues identified in separate studies as exerting significant effects on OCT binding were also found within the putative cleft region. There was a significant correlation (r(2) = 0.82) between the IC(50) values for inhibition of TEA transport by 14 different compounds and their calculated K(D) values for binding to the model of rabbit OCT2. The results suggest that homology modeling offers an opportunity to direct future site-directed studies of OCT/substrate interaction.

Alterations in the α2 isoform of Na,K-ATPase associated with familial hemiplegic migraine type 2

A number of missense mutations in the Na,K-ATPase alpha2 catalytic subunit have been identified in familial hemiplegic migraine with aura. Two alleles (L764P and W887R) showed loss-of-function, whereas a third (T345A) is fully functional but with altered Na,K-ATPase kinetics. This study describes two additional mutants, R689Q and M731T, originally identified by Vanmolkot et al. [Vanmolkot, K. R., et al. (2003) Ann. Neurol. 54, 360-366], which we show here to also be functional and kinetically altered. Both mutants have reduced catalytic turnover and increased apparent affinity for extracellular K(+). For both R689Q and M731T, sensitivity to vanadate inhibition is decreased, suggesting that the steady-state E(1) <==> E(2) poise of the enzyme is shifted toward E(1). Whereas the K'(ATP) is not affected by the R689Q replacement, the M731T mutant has an increase in apparent affinity for ATP. Analysis of the structural changes effected by T345A, R689Q, and M731T mutations, based on homologous replacements in the known crystal structure of the sarcoplasmic reticulum Ca-ATPase, provides insights into the molecular bases for the kinetic alterations. It is suggested that the disease phenotype is the consequence of lowered molecular activity of the alpha2 pump isoform due to either decreased K(+) affinity (T345A) or catalytic turnover (R689Q and M731T), thus causing a delay in extracellular K(+) clearance and/or altered localized Ca(2+) handling/signaling secondary to reduced activity in colocalized Na(+)/Ca(2+) exchange.

Functional consequences of progressive cone dystrophy-associated mutations in the human cone photoreceptor cyclic nucleotide-gated channel CNGA3 subunit

Progressive cone dystrophies are a genetically heterogeneous group of disorders characterized by early deterioration of visual acuity and color vision, together with psychophysical and electrophysiological evidence of abnormal cone function and cone degeneration. Recently, three mutations in the gene encoding the CNGA3 subunit of cone photoreceptor cyclic nucleotide-gated (CNG) channels have been linked to progressive cone dystrophy in humans. To investigate the functional consequences of these mutations, we expressed mutant human CNGA3 subunits in Xenopus oocytes, alone or together with human CNGB3, and studied these channels using patch-clamp recording. Compared with wild-type channels, homomeric and heteromeric channels containing CNGA3-N471S or CNGA3-R563H subunits exhibited an increase in apparent affinity for cGMP and an increase in the relative agonist efficacy of cAMP compared with cGMP. In contrast, R277C subunits did not form functional homomeric or heteromeric channels. Cell surface expression levels, determined using confocal microscopy of green fluorescent protein-tagged subunits and patch-clamp recording, were significantly reduced for both R563H and R277C but unchanged for N471S. Overall, these results suggest that the plasma membrane localization and gating properties of cone CNG channels are altered by progressive cone dystrophy-associated mutations, providing evidence that supports the pathogenicity of these mutations.