Cys Accessibility Research Articles

The cofactors and domains of a staphylococcal capsule-producing enzyme preserve its structure, stability, shape and dimerization ability.

CapF, a staphylococcal capsule-producing enzyme, binds Zn2+ ion and NADPH using its C-terminal domain (CTD) and N-terminal domain (NTD), respectively. To elucidate the roles of cofactors and domains, we have systematically investigated the related recombinant proteins, rCapF, rCTD, recombinant NTD (rNTD) and the Zn2+-free rCapF/rCTD, Apo-rCapF/Apo-rCTD. The results show that the secondary structure, tertiary structure, shape and surface hydrophobicity of Apo-rCapF and Apo-rCTD are different from those of rCapF and rCTD. The removal of Zn2+ made rCapF thermo-sensitive, whereas both rCTD and Apo-rCTD are thermo-resistant proteins. Further, Apo-rCapF and rCapF existed as the dimers, whereas rCTD and Apo-rCTD formed a mixture of dimers and tetramers in the aqueous solution. Zn2+ maintained the structure of NTD as well. The NADPH binding activity and Cys accessibility of rNTD, rCapF and Apo-rCapF were significantly different from each other. The binding of NADPH to the above three proteins freely occurred, liberated heat at 25°C and increased their diameters. In addition, the structure, stability, shape and oligomerization ability of rNTD, rCTD and rCapF little resembled each other. Collectively, the domains and cofactors of CapF contribute to preserving its conformation, stability, shape and dimerization ability.

Topological Dissection of the Membrane Transport Protein Mhp1 Derived from Cysteine Accessibility and Mass Spectrometry.

Cys accessibility and quantitative intact mass spectrometry (MS) analyses have been devised to study the topological transitions of Mhp1, the membrane protein for sodium-linked transport of hydantoins from Microbacterium liquefaciens. Mhp1 has been crystallized in three forms (outward-facing open, outward-facing occluded with substrate bound, and inward-facing open). We show that one natural cysteine residue, Cys327, out of three, has an enhanced solvent accessibility in the inward-facing (relative to the outward-facing) form. Reaction of the purified protein, in detergent, with the thiol-reactive N-ethylmalemide (NEM), results in modification of Cys327, suggesting that Mhp1 adopts predominantly inward-facing conformations. Addition of either sodium ions or the substrate 5-benzyl-l-hydantoin (L-BH) does not shift this conformational equilibrium, but systematic co-addition of the two results in an attenuation of labeling, indicating a shift toward outward-facing conformations that can be interpreted using conventional enzyme kinetic analyses. Such measurements can afford the Km for each ligand as well as the stoichiometry of ion–substrate-coupled conformational changes. Mutations that perturb the substrate binding site either result in the protein being unable to adopt outward-facing conformations or in a global destabilization of structure. The methodology combines covalent labeling, mass spectrometry, and kinetic analyses in a straightforward workflow applicable to a range of systems, enabling the interrogation of changes in a protein’s conformation required for function at varied concentrations of substrates, and the consequences of mutations on these conformational transitions.

Core Transmembrane Domain 6 Plays a Pivotal Role in the Transport Cycle of the Sodium/Proline Symporter PutP

Crystal structures of transporters with a LeuT-type structural fold assign core transmembrane domain 6 (TM6′) a central role in substrate binding and translocation. Here, the function of TM6′ in the sodium/proline symporter PutP, a member of the solute/sodium symporter family, was investigated. A complete scan of TM6′ identified eight amino acids as particularly important for PutP function. Of these residues, Tyr-248, His-253, and Arg-257 impact sodium binding, whereas Arg-257 and Ala-260 may participate in interactions leading to closure of the inner gate. Furthermore, the previous suggestion of an involvement of Trp-244, Tyr-248, and Pro-252 in proline binding is further supported. In addition, substitution of Gly-245, Gly-247, and Gly-250 affects the amount of PutP in the membrane. A Cys accessibility analysis suggests an involvement of the inner half of TM6′ in the formation of a hydrophilic pathway that is open to the inside in the absence of ligands and closed in the presence of sodium and proline. In conclusion, the results demonstrate that TM6′ plays a central role in substrate binding and release on the inner side of the membrane also in PutP and extend the knowledge on functionally relevant amino acids in transporters with a LeuT-type structural fold.

A role for loop G in the β1 strand in GABAA receptor activation.

The role of the β1 strand in GABAA receptor function is unclear. It lies anti-parallel to the β2 strand, which is known to participate in receptor activation. Molecular dynamics simulation revealed solvent accessible residues within the β1 strand of the GABAA β3 homopentamer that might be amenable to analysis using the substituted Cys accessibility method. Cys substitutions from Asp43 to Thr47 in the GABAA α1 subunit showed that D43C and T47C reduced the apparent potency of GABA. F45C caused a biphasic GABA concentration-response relationship and increased spontaneous gating. Cys43 and Cys47 were accessible to 2-aminoethyl methanethiosulphonate (MTSEA) modification, whereas Cys45 was not. Both GABA and the allosteric agonist propofol reduced MTSEA modification of Cys43 and Cys47. By contrast, modification of Cys64 in the β2 strand loop D was impeded by GABA but unaffected by propofol. These data reveal movement of β1 strand loop G residues during agonist activation of the GABAA receptor. The GABAA receptor α subunit β1 strand runs anti-parallel to the β2 strand, which contains loop D, known to participate in receptor activation and agonist binding. However, a role for the β1 strand has yet to be established. We used molecular dynamics simulation to quantify the solvent accessible surface area (SASA) of β1 strand residues in the GABAA β3 homopentamer structure. Residues in the complementary interface equivalent to those between Asp43 and Thr47 in the α1 subunit have an alternating pattern of high and low SASA consistent with a β strand structure. We investigated the functional role of these β1 strand residues in the α1 subunit by individually replacing them with Cys residues. D43C and T47C substitutions reduced the apparent potency of GABA at α1β2γ2 receptors by 50-fold and eight-fold, respectively, whereas the F45C substitution caused a biphasic GABA concentration-response relationship and increased spontaneous gating. Receptors with D43C or T47C substitutions were sensitive to 2-aminoethyl methanethiosulphonate (MTSEA) modification. However, GABA-evoked currents mediated by α1(F45C)β2γ2 receptors were unaffected by MTSEA, suggesting that this residue is inaccessible. Both GABA and the allosteric agonist propofol reduced MTSEA modification of α1(D43C)β2γ2 and α1(T47C)β2γ2 receptors, indicating movement of the β1 strand even during allosteric activation. This is in contrast to α1(F64C)β2γ2 receptors, where only GABA, but not propofol, reduced MTSEA modification. These findings provide the first functional evidence for movement of the β1 strand during gating of the receptor and identify residues that are critical for maintaining GABAA receptor function.

Transmembrane signaling in the sensor kinase DcuS of Escherichia coli: A long-range piston-type displacement of transmembrane helix 2

The C4-dicarboxylate sensor kinase DcuS is membrane integral because of the transmembrane (TM) helices TM1 and TM2. Fumarate-induced movement of the helices was probed in vivo by Cys accessibility scanning at the membrane-water interfaces after activation of DcuS by fumarate at the periplasmic binding site. TM1 was inserted with amino acid residues 21-41 in the membrane in both the fumarate-activated (ON) and inactive (OFF) states. In contrast, TM2 was inserted with residues 181-201 in the OFF state and residues 185-205 in the ON state. Replacement of Trp 185 by an Arg residue caused displacement of TM2 toward the outside of the membrane and a concomitant induction of the ON state. Results from Cys cross-linking of TM2/TM2' in the DcuS homodimer excluded rotation; thus, data from accessibility changes of TM2 upon activation, either by ligand binding or by mutation of TM2, and cross-linking of TM2 and the connected region in the periplasm suggest a piston-type shift of TM2 by four residues to the periplasm upon activation (or fumarate binding). This mode of function is supported by the suggestion from energetic calculations of two preferred positions for TM2 insertion in the membrane. The shift of TM2 by four residues (or 4-6 Å) toward the periplasm upon activation is complementary to the periplasmic displacement of 3-4 Å of the C-terminal part of the periplasmic ligand-binding domain upon ligand occupancy in the citrate-binding domain in the homologous CitA sensor kinase.

Abstract 782: Transmembrane 2-3 loop domain of the proton coupled folate transporter (PCFT) forms a novel “reentrant loop” structure and is functionally important

Abstract The proton coupled folate transporter (PCFT) is a proton-folate symporter with a twelve transmembrane domain (TMD) structure. Human PCFT (hPCFT) is highly expressed in many solid tumors and has an acidic pH optimum, suggesting its high activity in the acidic tumor microenvironment. Our recent studies have focused on exploiting PCFT for targeting solid tumors with novel cytotoxic antifolates. Predicted topology models suggest that the loop domain between TMDs 2 and 3 resides in the cytosol. However, mutations in TMD2-3 loop residues Asp109 or Arg113 result in loss of PCFT activity, suggesting an important role in transport activity. Additionally, TMD2 may itself form a part of the substrate binding domain, suggesting functional importance for TMD2 residues. Using site-directed mutagenesis, we replaced each of the seven cysteine (cys) residues of hPCFT with serine, creating functional “cys-less” PCFT for use in substituted cys accessibility methods (SCAM). We then used site-directed mutagenesis to create 34 single cys mutants spanning TMD2 and the TMD 2-3 loop region (positions 85-118) in a cys-less PCFT background. These mutants were transfected into R1-11 HeLa cells, which express no endogenous folate transporters. We measured [3H]methotrexate uptake at pH 5.5 as a readout of PCFT activity and confirmed PCFT protein expression by western blotting. All 34 mutants were detected on westerns and 29 mutants were active, while W107C, D109C, G112C, R113C, and R114C were inactive. Using functionally active mutants, we performed pull-down assays with 2-aminoethyl methanethiosulfonate-biotin (MTSEA-biotin) and steptavidin beads, in order to determine the aqueous accessibility of each mutant. We found that mutants G93C, F94W, G105C, A106C, S110C, and L116C reacted with MTSEA-biotin, suggesting that these residues are aqueous-accessible. For G93C, F94C, and L116C, MTSEA-biotin labeling was inhibited by pemetrexed pretreatment, and MTSEA-biotin treatment inhibited transport activity of G93C and F94C. Our results suggest that the TMD 2-3 loop domain is aqueous accessible and must form a novel functionally important “reentrant loop” structure. Additionally, residues located in TMD2 (Gly93 and Phe94) appear to play a key role in substrate binding to PCFT. Better understanding of functional and structural determinants of PCFT membrane transport will be critical to the development of novel PCFT-selective antifolate drugs for targeting solid tumors. Citation Format: Michael R. Wilson, Zhanjun Hou, Larry H. Matherly. Transmembrane 2-3 loop domain of the proton coupled folate transporter (PCFT) forms a novel “reentrant loop” structure and is functionally important. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 782. doi:10.1158/1538-7445.AM2014-782

Role of the fourth transmembrane domain in proton-coupled folate transporter function as assessed by the substituted cysteine accessibility method

The proton-coupled folate transporter (PCFT, SLC46A1) mediates folate transport across the apical brush-border membrane of the proximal small intestine and the basolateral membrane of choroid plexus ependymal cells. Two loss-of-function mutations in PCFT, which are the basis for hereditary folate malabsorption, have been identified within the fourth transmembrane domain (TMD4) in subjects with this disorder. We have employed the substituted Cys accessibility method (SCAM) to study the accessibilities of all residues in TMD4 and their roles in folate substrate binding to the carrier. When residues 146-167 were replaced by Cys, all except R148C were expressed at the cell surface. Modification of five of these substituted Cys residues (positions 147, 152, 157, 158, and 161) by methanethiosulfonate (MTS) reagents led to reduction of PCFT function. All five residues could be labeled with N-biotinylaminoethyl-MTS, and this could be blocked by the high-affinity PCFT substrate pemetrexed. Pemetrexed also protected PCFT mutant function from inhibitory modification of the substituted Cys at positions 157, 158, and 161 by a MTS. The findings indicate that these five residues in TMD4 are accessible to the aqueous translocation pathway, play a role in folate substrate binding, and are likely located within or near the folate binding pocket. A homology model of PCFT places three of these residues, Phe¹⁵⁷, Gly¹⁵⁸, and Leu¹⁶¹, within a breakpoint in the midportion of TMD4, a region that likely participates in alterations in the PCFT conformational state during carrier cycling.

Structure of the M2 Transmembrane Segment of GLIC, a Prokaryotic Cys Loop Receptor Homologue from Gloeobacter violaceus, Probed by Substituted Cysteine Accessibility

GLIC is a homopentameric proton-gated, prokaryotic homologue of the Cys loop receptor family of neurotransmitter-gated ion channels. Recently, crystal structures of GLIC hypothesized to represent an open channel state were published. To explore the channel structure in functional GLIC channels, we tested the ability of p-chloromercuribenzenesulfonate to react with 30 individual cysteine substitution mutants in and flanking the M2 channel-lining segment in the closed state (pH 7.5) and in a submaximally activated state (pH 5.0). Nine mutants did not tolerate cysteine substitution and were not functional. From positions 10' to 27', p-chloromercuribenzenesulfonate significantly modified the currents at pH 7.5 and 5.0 in all mutants except H234C (11'), I235C (12'), V241C (18'), T243C (20'), L245C (22'), and Y250C (27'), which were not functional, except for 12'. Currents for P246C (23') and K247C (24') were only significantly altered at pH 5.0. The reaction rates were all >1000 m(-1) s(-1). The reactive residues were more accessible in the activated than in the resting state. We infer that M2 is tightly associated with the adjacent transmembrane helices at the intracellular end but is more loosely packed from 10' to the extracellular end than the x-ray structures suggest. We infer that the charge selectivity filter is in the cytoplasmic half of the channel. We also show that below pH 5.0, GLIC desensitizes on a time scale of minutes and infer that the crystal structures may represent a desensitized state.

Site-Directed Alkylation Studies with LacY Provide Evidence for the Alternating Access Model of Transport

In total, 59 single Cys-replacement mutants in helix VII and helix X of the lactose permease of Escherichia coli were subjected to site-directed fluorescence labeling in right-side-out membrane vesicles to complete the testing of Cys accessibility or reactivity. For both helices, accessibility/reactivity is relatively low at the level of the sugar-binding site where the helices are tightly packed. However, labeling of Cys substitutions in helix VII with tetramethylrhodamine-5-maleimide decreases from the middle toward the cytoplasmic end and increases toward the periplasmic end. Helix X is labeled mainly on the side facing the central hydrophilic cavity with relatively small or no changes in the presence of ligand. In contrast, sugar binding causes a significant increase in accessibility/reactivity at the periplasmic end of helix VII. When considered with similar findings from N-ethylmaleimide alkylation studies, the results confirm and extend support for the alternating access model.

Structure-Function Studies of the SLC17 Transporter Sialin Identify Crucial Residues and Substrate-induced Conformational Changes

Salla disease and infantile sialic acid storage disorder are human diseases caused by loss of function of sialin, a lysosomal transporter that mediates H(+)-coupled symport of acidic sugars N-acetylneuraminic acid and glucuronic acid out of lysosomes. Along with the closely related vesicular glutamate transporters, sialin belongs to the SLC17 transporter family. Despite their critical role in health and disease, these proteins remain poorly understood both structurally and mechanistically. Here, we use substituted cysteine accessibility screening and radiotracer flux assays to evaluate experimentally a computationally generated three-dimensional structure model of sialin. According to this model, sialin consists of 12 transmembrane helices (TMs) with an overall architecture similar to that of the distantly related glycerol 3-phosphate transporter GlpT. We show that TM4 in sialin lines a large aqueous cavity that forms a part of the substrate permeation pathway and demonstrate substrate-induced alterations in accessibility of substituted cysteine residues in TM4. In addition, we demonstrate that one mutant, F179C, has a dramatically different effect on the apparent affinity and transport rate for N-acetylneuraminic acid and glucuronic acid, suggesting that it may be directly involved in substrate recognition and/or translocation. These findings offer a basis for further defining the transport mechanism of sialin and other SLC17 family members.

Transmembrane Domain 6 of the Human Serotonin Transporter Contributes to an Aqueously Accessible Binding Pocket for Serotonin and the Psychostimulant 3,4-Methylene Dioxymethamphetamine

The plasma membrane serotonin (5-HT) transporter (SERT, SLC6A4) clears 5-HT after release at nerve termini and is targeted by both antidepressant medications and psychostimulants (e.g. MDMA, cocaine). Homology modeling of human SERT (hSERT), based on high resolution structures of the microbial SLC6 family member LeuT(Aa), along with biochemical studies of wild type and mutant transporters, predicts transmembrane (TM) domains 1, 3, 6, and 8 comprise the 5-HT-binding pocket. We utilized the substituted cysteine accessibility method along with surface and site-specific biotinylation to probe TM6 for aqueous accessibility and differential interactions with 5-HT and psychostimulants. Our results are consistent with TM6 being composed of an aqueous-accessible, alpha-helical extracellular domain (TM6a) that is separated by a central, unwound section from a cytoplasmically localized domain (TM6b) with limited aqueous accessibility. The substitution G338C appears to lock hSERT in an outward-facing conformation that, although accessible to aminoethylmethanethiosulfonate-biotin, 5-HT, and citalopram, is incapable of inward 5-HT transport. Transport of 5-HT by G338C can be partially restored by the TM1 mutation Y95F. With regard to methanethiosulfonate (MTS) inactivation of uptake, TM6a Cys mutants demonstrate Na(+)-dependent [2-(trimethylammonium)ethyl]-MTS sensitivity. Studies with the centrally located substitution S336C reveal features of a common binding pocket for 5-HT and 3,4-methylenedioxymethamphetamine (MDMA). Interestingly, the substitution I333C reveals an MDMA-induced conformation not observed with 5-HT. In the context of prior studies on TM1, our findings document shared and unique features of TM6 contributing to hSERT aqueous accessibility, ligand recognition, and conformational dynamics.

Evidence for a Second, High Affinity Gβγ Binding Site on Gαi1(GDP) Subunits

It is well known that Galpha(i1)(GDP) binds strongly to Gbetagamma subunits to form the Galpha(i1)(GDP)-Gbetagamma heterotrimer, and that activation to Galpha(i1)(GTP) results in conformational changes that reduces its affinity for Gbetagamma subunits. Previous studies of G protein subunit interactions have used stoichiometric amounts of the proteins. Here, we have found that Galpha(i1)(GDP) can bind a second Gbetagamma subunit with an affinity only 10-fold weaker than the primary site and close to the affinity between activated Galpha(i1) and Gbetagamma subunits. Also, we find that phospholipase Cbeta2, an effector of Gbetagamma, does not compete with the second binding site implying that effectors can be bound to the Galpha(i1)(GDP)-(Gbetagamma)(2) complex. Biophysical measurements and molecular docking studies suggest that this second site is distant from the primary one. A synthetic peptide having a sequence identical to the putative second binding site on Galpha(i1) competes with binding of the second Gbetagamma subunit. Injection of this peptide into cultured cells expressing eYFP-Galpha(i1)(GDP) and eCFP-Gbetagamma reduces the overall association of the subunits suggesting this site is operative in cells. We propose that this second binding site serves to promote and stabilize G protein subunit interactions in the presence of competing cellular proteins.

Spatial Arrangement of the β-Glucoside Transporter fromEscherichia coli

The Escherichia coli BglF protein, a sugar permease of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), catalyzes concomitant transport and phosphorylation of beta-glucosides across the cytoplasmic membrane. Despite intensive studies of PTS permeases, the mechanism that couples sugar translocation to phosphorylation and the nature of the translocation apparatus are poorly understood. Like many PTS permeases, BglF consists of a transmembrane domain, which in addition to transmembrane helices (TMs) contains a big cytoplasmic loop and two hydrophilic domains, one containing a conserved cysteine that phosphorylates the incoming sugar. We previously reported that the big hydrophilic loop, which connects TM VI to TM VII, contains regions that alternate between facing-in and facing-out states and speculated that it is involved in creating the sugar translocation channel. In the current study we used [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET), a membrane-impermeative thiol-specific reagent, to identify sites that are involved in sugar transport. These sites map to the regions that border the big loop. Using cross-linking reagents that penetrate the cell, we could demonstrate spatial proximity between positions at the center of the big loop and the phosphorylation site, suggesting that the two regions come together to execute sugar phosphotransfer. Additionally, positions on opposite ends of the big loop were found to be spatially close. Cys accessibility analyses suggested that the sugar induces a change in this region. Taken together, our results demonstrate that the big loop participates in creating the sugar pathway and explain the observed coupling between translocation of PTS sugars from the periplasm to the cytoplasm and their phosphorylation.

Agrobacterium VirB10 domain requirements for type IV secretion and T pilus biogenesis

Agrobacterium tumefaciens VirB10 couples inner membrane (IM) ATP energy consumption to substrate transfer through the VirB/D4 type IV secretion (T4S) channel and also mediates biogenesis of the virB-encoded T pilus. Here, we determined the functional importance of VirB10 domains denoted as the: (i) N-terminal cytoplasmic region, (ii) transmembrane (TM) alpha-helix, (iii) proline-rich region (PRR) and (iv) C-terminal beta-barrel domain. Mutations conferring a transfer- and pilus-minus (Tra(-), Pil(-)) phenotype included PRR deletion and beta-barrel substitution mutations that prevented VirB10 interaction with the outer membrane (OM) VirB7-VirB9 channel complex. Mutations permissive for substrate transfer but blocking pilus production (Tra(+), Pil(-)) included a cytoplasmic domain deletion and TM domain insertion mutations. Another class of Tra(+) mutations also selectively disrupted pilus biogenesis but caused release of pilin monomers to the milieu; these mutations included deletions of alpha-helical projections extending from the beta-barrel domain. Our findings, together with results of Cys accessibility studies, indicate that VirB10 stably integrates into the IM, extends via its PRR across the periplasm, and interacts via its beta-barrel domain with the VirB7-VirB9 channel complex. The data further support a model that distinct domains of VirB10 regulate formation of the secretion channel or the T pilus.

Hydrophobic Interactions as Key Determinants to the KCa3.1 Channel Closed Configuration: AN ANALYSIS OF KCa3.1 MUTANTS CONSTITUTIVELY ACTIVE IN ZERO Ca2+

In this study we present evidence that residue Val282 in the S6 transmembrane segment of the calcium-activated KCa3.1 channel constitutes a key determinant of channel gating. A Gly scan of the S6 transmembrane segment first revealed that the substitutions A279G and V282G cause the channel to become constitutively active in zero Ca2+. Constitutive activity was not observed when residues extending from Cys276 to Ala286, other than Ala279 and Val282, were substituted to Gly. The accessibility of Cys engineered at Val275 deep in the channel cavity was next investigated for the ion-conducting V275C/V282G mutant and closed V275C channel in zero Ca2+ using Ag+ as probe. These experiments demonstrated that internal Ag+ ions have free access to the channel cavity independently of the channel conducting state, arguing against an activation gate located at the S6 segment C-terminal end. Experiments were also conducted where Val282 was substituted by residues differing in size and/or hydrophobicity. We found a strong correlation between constitutive activity in zero Ca2+ and the hydrophobic energy for side chain burial. Single channel recordings showed finally that constitutive activation in zero Ca2+ is better explained by a model where the channel is locked in a low conducting state with a high open probability rather than resulting from a change in the open/closed energy balance that would favor channel openings to a full conducting state in the absence of Ca2+. We conclude that hydrophobic interactions involving Val282 constitute key determinants to KCa3.1 gating by modulating the ion conducting state of the selectivity filter through an effect on the S6 transmembrane segment.

Clustering and synaptic targeting of PICK1 requires direct interaction between the PDZ domain and lipid membranes

Protein interacting with c kinase 1 (PICK1) regulates the trafficking of receptors and ion-channels such as AMPA receptors. Traditionally, the PICK1 PDZ domain is regarded as an adaptor capable of binding to receptors trafficked by PICK1, and the lipid-binding BAR domain functions to tether PICK1 directly to membranes. Here, we show that the PICK1 PDZ domain can directly interact with lipid membranes. The PDZ domain and lipid membrane interaction is mediated by both a polybasic amino-acid cluster and a conserved 'Cys-Pro-Cys' motif located away from the peptide ligand-binding groove. Disruption of the PDZ and lipid membrane interaction totally abolished synaptic targeting of PICK1. Although mutation of the CPC motif did not affect the interaction between PICK1 and AMPA receptors, the mutant PICK1 was unable to cluster the GluR2 subunit of the receptor. In neurons, PICK1 containing the same mutation displayed dramatically compromised capacity in the trafficking of AMPA receptors. Taken together, our findings not only uncovered the novel lipid membrane-binding property of the PICK1 PDZ domain, but also provided direct evidence supporting the functional relevance of the PDZ-lipid interaction.

Constitutively Active Homo-oligomeric Angiotensin II Type 2 Receptor Induces Cell Signaling Independent of Receptor Conformation and Ligand Stimulation

Members of the G-protein-coupled receptor superfamily (GPCRs) undergo homo- and/or hetero-oligomerization to induce cell signaling. Although some of these show constitutive activation, it is not clear how such GPCRs undergo homo-oligomerization with transmembrane helix movement. We previously reported that angiotensin II (Ang II) type 2 (AT(2)) receptor, a GPCR, showed constitutive activation and induced apoptosis independent of its ligand, Ang II. In the present study, we analyzed the translocation and oligomerization of the AT(2) receptor with transmembrane movement when the receptor induces cell signaling. Constitutively active homo-oligomerization, which was due to disulfide bonding between Cys(35) in one AT(2) receptor and Cys(290) in another AT(2) receptor, was localized in the cell membrane without Ang II stimulation and induced apoptosis without changes in receptor conformation. These results provide the direct evidence that the constitutively active homo-oligomeric GPCRs by intermolecular interaction in two extracellular loops is translocated to the cell membrane and induces cell signaling independent of receptor conformation and ligand stimulation.

TM2-TM7 Interaction in Coupling Movement of Transmembrane Helices to Activation of the Angiotensin II Type-1 Receptor

Agonist-induced rigid body motion of transmembrane (TM) helices has been established as a unifying mechanism in the activation of the G protein-coupled receptors. In attempts to measure specific conformational transitions during the activation of the type 1 receptor for angiotensin II (AT(1)), we found a decrease in accessibility of Cys(76) in the second TM helix, suggesting that the orientation of TM2 is altered (Miura, S., and Karnik, S. S. (2002) J. Biol. Chem. 277, 24299-24305). Now we provide evidence that the TM2 helical movement plays a role in regulating the activated state of the AT(1) receptor, and this role may involve an interaction between TM2 and TM7. Alanine substitution of native Cys(296) in TM7 leads to increased accessibility of Cys(289) and diminished response to bound agonist. Both effects of the C296A mutation are suppressed when combined with F77A and N111G mutants. The TM7 conformation and the sensitivity of Cys(289) altered by C296A mutation are suppressed by the F77A mutation in TM2 to salvage function. We show that the F77A mutant alters orientation of both TM2 and TM7 but does not induce constitutive activity in suppressing the C296A mutant effects. Thus, interaction of TM2 and TM7 is important for transmembrane signal transduction in the AT(1) receptor.

β2 Adrenergic Receptor Activation: MODULATION OF THE PROLINE KINK IN TRANSMEMBRANE 6 BY A ROTAMER TOGGLE SWITCH

In many rhodopsin-like G-protein-coupled receptors, agonist binding to a cluster of aromatic residues in TM6 may promote receptor activation by altering the configuration of the TM6 Pro-kink and by the subsequent movement of the cytoplasmic end of TM6 away from TM3. We hypothesized that the highly conserved Cys(6.47), in the vicinity of the conserved Pro(6.50), modulates the configuration of the aromatic cluster and the TM6 Pro-kink through specific interactions in its different rotamer configurations. In the beta(2) adrenergic receptor, mutation of Cys(6.47) to Thr, which in an alpha-helix has a different rotamer distribution from Cys and Ser, produced a constitutively active receptor, whereas the Ser mutant was similar to wild-type receptor. Use of the biased Monte Carlo technique of Conformational Memories showed that the rotamer changes among Cys/Ser/Thr(6.47), Trp(6.48), and Phe(6.52) are highly correlated, representing a rotamer "toggle switch" that may modulate the TM6 Pro-kink. Differential modulation of the accessibility of Cys(6.47) and an engineered Cys(6.52) in wild type and a constitutively active background provides experimental support for the association of this rotamer switch with receptor activation.

Mapping the agonist binding site of the nicotinic acetylcholine receptor by cysteine scanning mutagenesis: antagonist footprint and secondary structure prediction.

To further define the surface of the Torpedo californica nicotinic acetylcholine receptor (nAChR) contributing to the agonist binding site structure, we used the substituted Cys accessibility method to identify novel residues and determined the "footprint" of residues protected from modification by the reversible competitive antagonist d-tubocurarine (dTC). nAChRs containing single Cys substitutions within regions of the alpha- or gamma-subunit primary structure known to contribute to the agonist binding site were expressed in Xenopus laevis oocytes. Cys substitutions in binding site segments A (alphaTyr-93 and alphaAsn-94), C (alphaTyr-198), and D (gammaGlu-57) had been shown previously to be accessible for modification. We now introduced cysteines from alphaAsp-195 to alphaIle-201 and from gammaAla-106 to gammaAsp-113 and identified positions accessible for modification in segments C (alphaAsp-195, alphaThr-196, alphaPro-197, alphaAsp-200, and alphaIle-201) and E (gammaAsn-107 and gammaLeu-109). dTC protected against alkylation in segments D (gammaGlu-57) and E (gammaLeu-109) but not in segment A (alphaTyr-93 and alphaAsn-94). In segment C, dTC protection experiments revealed a pattern in which every other residue (alpha196, alpha198, and alpha200, but not alpha197 or alpha201) was protected from alkylation. This pattern of protection provides evidence that bound dTC is near amino acids in segments C, D, and E but not in segment A, and identifies a beta-strand surface within segment C contributing to the binding site. These results are discussed in terms of a homology model, based on the molluscan acetylcholine binding protein crystal structure, of the T. californica nAChR agonist binding site.