Double Cys Mutants Research Articles

Probing the Dynamics of AcrB Through Disulfide Bond Formation.

The resistant-nodulation-division (RND) superfamily member tripartite AcrA-AcrB-TolC efflux pump is a major contributor to the multidrug resistance in Escherichia coli. AcrB is the inner membrane protein of the efflux complex and is responsible for the recognition and binding of compounds before their transportation out of the cell. Understanding the dynamics of AcrB during functional rotation in the process of drug efflux is the focus of this study. For this purpose, we introduced six inter-subunit disulfide bonds into the periplasmic domain of AcrB using site-directed mutagenesis to study the importance of the relative flexibility at the inter-subunit interface. Western blot analysis revealed the formation of disulfide bond-linked AcrB oligomers, which were reduced into monomers under reducing conditions. The impact of mutation and formation of disulfide bond on efflux were evaluated via comparison of the minimum inhibitory concentration (MIC) of an acrB knockout strain expressing different mutants. The double Cys mutants tested led to equal or higher susceptibility to AcrB substrates compared to their corresponding single mutants. To determine if the reduction of activity in a double mutant is due to restriction on conformational changes by the disulfide bond formation, ethidium bromide accumulation assays were conducted utilizing dithiothreitol (DTT) as the reducing agent. In two cases, the activities of the double Cys mutants were partially restored by DTT reduction, confirming the importance of relative movement in the respective location for function. These findings provide new insights into the dynamics of the AcrAB-TolC efflux pump in E. coli.

Engineered occluded apo-intermediate of LacY

The lactose permease of Escherichia coli (LacY) utilizes an alternating access symport mechanism with multiple conformational intermediates, but only inward (cytoplasmic)- or outward (periplasmic)-open structures have been characterized by X-ray crystallography. It is demonstrated here with sugar-binding studies that cross-linking paired-Cys replacements across the closed cytoplasmic cavity stabilize an occluded conformer with an inaccessible sugar-binding site. In addition, a nanobody (Nb) that stabilizes a periplasmic-open conformer with an easily accessible sugar-binding site in WT LacY fails to cause the cytoplasmic cross-linked mutants to become accessible to galactoside, showing that the periplasmic cavity is closed. These results are consistent with tight association of the periplasmic ends in two pairs of helices containing clusters of small residues in the packing interface between N- and C-terminal six-helix bundles of the symporter. However, after reduction of the disulfide bond, the Nb markedly increases the rate of galactoside binding, indicating unrestricted access to the Nb epitope and the galactoside-binding site from the periplasm. The findings indicate that the cross-linked cytoplasmic double-Cys mutants resemble an occluded apo-intermediate in the transport cycle.

Bioresponsive interfaces composed of calmodulin and poly(ethylene glycol): Toggling the interfacial film thickness by protein-ligand binding

Responsive interfaces are often realized by polymer films that change their structure and properties upon changing the pH-value, ionic strength or temperature. Here, we present a bioresponsive interfacial structure that is based on a protein, calmodulin (CaM), which undergoes a huge conformational change upon ligand binding. At first, we characterize the conformational functionality of a double Cys mutant of CaM by small-angle X-ray scattering (SAXS) and Fourier transform infrared (FTIR) spectroscopy. The CaM mutant is then used to cross-link poly(ethylene glycol) (PEG) chains, which are bound covalently to a supporting planar Si surface. These films are characterized by X-ray reflectometry (XR) in a humidity chamber providing full hydration. It is well known that Ca2+-saturated holo-CaM binds trifluoperazine (TFP) and changes its conformation from an open, dumbbell-shaped to a closed, globular one in solution. At the interface, we observe an increase of the PEG-CaM film thickness, when TFP is binding and inducing the closed conformation, whereas the removal of Ca2+-ions and a concomitant release of TFP is associated with a decrease of the film thickness. This toggling of the film thickness is largely reversible. In this way, a structural change of the interface is achieved via protein functionality which has the advantage of being selective for ligand molecules without changing the environmental conditions in a harsh way via physico-chemical parameters.

Structural and functional alterations associated with deutan N94K and R330Q mutations of green cone opsin

Deuteranopia is an X-linked congenital dichromatic condition in which single point mutations in green cone opsin lead to defective non-functional cone photoreceptor cells. Green cone opsin belongs to the G protein-coupled receptor superfamily and consists of a seven transmembrane helical apoprotein covalently bound to 11-cis-retinal, by means of a protonated Schiff base linkage, in its inactive dark state. Several point mutations in green cone opsin have been reported to cause deuteranopia, but the structural details underlying the molecular mechanisms behind the malfunction of mutated opsins have not been clearly established. Here, deutan N94K and R330Q mutants were studied by introducing these substitutions into the native green cone opsin gene by site-directed mutagenesis. The mutant proteins were purified and analyzed using UV-vis spectroscopy and transducin activation assay. We find that the N94K mutant binds the retinal chromophore by means of an unprotonated Schiff base linkage in contrast to previous studies that reported no chromophore regeneration. The other mutant studied, R330Q, showed impaired functionality as measured by its reduced transducin activation ability when compared to wild-type green cone opsin. A double Cys mutant that could form a stabilizing disulfide bond was used in an attempt to address the instability of the green opsin mutants. Our results suggest the presence of key intramolecular networks which may be disrupted in deuteranopia, and these findings could help in finding therapeutic solutions for treating color blindness. Furthermore, our results can also have implications for the study of other visual pigments and other rhodopsin-like G protein-coupled receptors.

An Asymmetric Conformational Change in LacY.

Galactoside/H+ symport by the lactose permease of Escherichia coli (LacY) involves reciprocal opening and closing of periplasmic and cytoplasmic cavities so that sugar- and H+-binding sites become alternatively accessible to either side of the membrane. After reconstitution into proteoliposomes, LacY with the periplasmic cavity sealed by cross-linking paired-Cys residues does not bind sugar from the periplasmic side. However, reduction of the S-S bond restores opening of the periplasmic cavity and galactoside binding. Furthermore, nanobodies that stabilize the double-Cys mutant in a periplasmic-open conformation and allow free access of galactoside to the binding site do so only after reduction of the S-S bond. In contrast, when cross-linked LacY is solubilized in detergent, galactoside binding is observed, indicating that the cytoplasmic cavity is patent. Sugar binding from the cytoplasmic side exhibits nonlinear stopped-flow kinetics, and analysis reveals a two-step process in which a conformational change precedes binding. Because the cytoplasmic cavity is spontaneously closing and opening in the symporter with a sealed periplasmic cavity, it is apparent that an asymmetrical conformational transition controls access of sugar to the binding site.

Disulfide mapping the voltage-sensing mechanism of a voltage-dependent potassium channel.

Voltage-dependent potassium (Kv) channels allow for the selective permeability of potassium ions in a membrane potential dependent manner, playing crucial roles in neurotransmission and muscle contraction. Kv channel is a tetramer, in which each subunit possesses a voltage-sensing domain (VSD) and a pore domain (PD). Although several lines of evidence indicated that membrane depolarization is sensed as the movement of helix S4 of the VSD, the detailed voltage-sensing mechanism remained elusive, due to the difficulty of structural analyses at resting potential. In this study, we conducted a comprehensive disulfide locking analysis of the VSD using 36 double Cys mutants, in order to identify the proximal residue pairs of the VSD in the presence or absence of a membrane potential. An intramolecular SS-bond was formed between 6 Cys pairs under both polarized and depolarized environment, and one pair only under depolarized environment. The multiple conformations captured by the SS-bond can be divided by two states, up and down, where S4 lies on the extracellular and intracellular sides of the membrane, respectively, with axial rotation of 180°. The transition between these two states is caused by the S4 translocation of 12 Å, enabling allosteric regulation of the gating at the PD.

Serine suppresses the motor function of a periplasmic PomB mutation in the Vibrio flagella stator.

The flagellar motor of Vibrio alginolyticus is made of two parts: a stator consisting of proteins PomA and PomB, and a rotor whose main component is FliG. The interaction between FliG and PomA generates torque for flagellar rotation. Based on cross-linking experiments of double-Cys mutants of PomB, we previously proposed that a conformational change in the periplasmic region of PomB caused stator activation. Double-Cys mutants lost their motility due to an intramolecular disulfide bridge. In this study, we found that the addition of serine, a chemotactic attractant, to a PomB(L160C/I186C) mutant restored motility without cleaving the disulfide bridge. We speculate that serine changed the rotor (FliG) conformation, affecting rotational direction. Combined with the counterclockwise (CCW)-biased mutation FliG(G214S), motility of PomB(L160C/I186C) was restored without the addition of serine. Likewise, motility was restored without serine in Che(-) mutants, in either a CCW-locked or clockwise (CW)-locked strain. In contrast, in a ΔcheY (CCW-locked) strain, Vibrio (L160C/I186C) required serine to be rescued. We speculate that CheY affects stator conformation and motility restoration by serine is independent on the chemotaxis signaling pathway.

Abstract 644: Impact of conjugation site on pharmacokinetics and off-target toxicity of site-specific antibody drug conjugates

Abstract Site-specific conjugation of toxic payload to antibody yields homogeneous ADCs resulting in improved therapeutic index over conventional conjugation. To understand the role of conjugation site on pharmacokinetics, off-target toxicity, cysteine(cys) was engineered into a humanized anti-IL13Rα2 antibody, hAB08, at various sites within the IgG1/k constant domains. Single cys mutants L443C and Q347C with DAR 2, and double cys mutants K392C+L443C, L443C/kK183C and Q347C/kK183C with DAR 4 were conjugated using auristatin 0101 via maleimidocapronic - valine-citruline-p-aminobenzyloxycarbonyl (vc, cleavable linker). All cys mutants and their conjugates maintained their binding properties compared to parental hAb08. Cysteine substitution sites were selected based on conjugation efficiency and in vitro stability. In vivo efficacy was evaluated in mouse xenograph tumor models. Pharmacokinetics (PK) and off target toxicity (TOX) were assessed in both mouse and rat in vivo studies. Our data demonstrate that that all site specific conjugates show improved in vitro stability, comparable or better in vivo efficacy in mice xenograph models and in vivo stability (ratio of ADC-AUC/Ab-AUC) compared to conventional conjugates. However PK and off-target toxicity of antibody drug conjugates had been significantly impacted by conjugation site and animal species. L443 mutants show higher ADC-AUC, lower clearance and longer half life than Q347 mutants in mice while a reverse TK behavior was observed in rat. All site specific conjugates show higher dose tolerance and less off-target toxicity relative to conventional conjugates in rat. This off-target toxicity improvement depends on sites of conjugation. The L443 mutants exhibited a better off-target TOX profile relative to Q347 mutants. Taken together, site-specific conjugation improved in vivo efficacy and off-target toxicity relative to conventionally conjugated ADC. PK differences and off-target toxicity improvements depend on conjugation sites and animal species. Note: This abstract was not presented at the meeting. Citation Format: Dangshe Ma, Fang Jin, Frank Barletta, George Hu, Nathan Tumey, Haige Zhang, Tao He, Eric Sousa, Manoj Charti, Kiran Khadke, Judy Lucas, Darren Ferguson, Christoper Brown, Weijun Ma, Scott Gatto, William Brady, Edmund Graziani, Hans-Peter Gerber, Puja Sapra, Lioudmila Tchistikova. Impact of conjugation site on pharmacokinetics and off-target toxicity of site-specific antibody drug conjugates. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 644. doi:10.1158/1538-7445.AM2015-644

Apoptosis inducing, conformationally constrained, dimeric peptide analogs of KLA with submicromolar cell penetrating abilities.

The apoptosis inducing KLA peptide, (KLAKLAK)2, possesses an ability to disrupt mitochondrial membranes. However, this peptide has a poor eukaryotic cell penetrating potential and, as a result, it requires the assistance of other cell penetrating peptides for effective translocation in micromolar concentrations. In an effort to improve the cell penetrating potential of KLA, we have created a library in which pairs of residues on its hydrophobic face are replaced by Cys. The double Cys mutants were then transformed to bundle dimers by oxidatively generating two intermolecular disulfide bonds. We envisioned that once transported into cells, the disulfide bonds would undergo reductive cleavage to generate the monomeric peptides. The results of these studies showed that one of the mutant peptides, dimer B, has a high cell penetrating ability that corresponds to 100% of fluorescence positive cells at 250 nM. Even though dimer B induces disruption of the mitochondrial potential and cytochrome c release followed by caspase activation at submicromolar concentrations, it displays an LD50 of 1.6 μM under serum conditions using HeLa cells. Taken together, the results demonstrate that the strategy involving formation of bundle dimeric peptides is viable for the design of apoptosis inducing KLA peptide that translocate into cells at submicromolar concentrations.

Conformational change in the periplamic region of the flagellar stator coupled with the assembly around the rotor.

The torque of the bacterial flagellum is generated by the rotor-stator interaction coupled with the ion flow through the channel in the stator. Anchoring the stator unit to the peptidoglycan layer with proper orientation around the rotor is believed to be essential for smooth rotation of the flagellar motor. The stator unit of the sodium-driven flagellar motor of Vibrio is composed of PomA and PomB, and is thought to be fixed to the peptidoglycan layer and the T-ring by the C-terminal periplasmic region of PomB. Here, we report the crystal structure of a C-terminal fragment of PomB (PomBC) at 2.0-Å resolution, and the structure suggests a conformational change in the N-terminal region of PomBC for anchoring the stator. On the basis of the structure, we designed double-Cys replaced mutants of PomB for in vivo disulfide cross-linking experiments and examined their motility. The motility can be controlled reproducibly by reducing reagent. The results of these experiments suggest that the N-terminal disordered region (121-153) and following the N-terminal two-thirds of α1(154-164) in PomBC changes its conformation to form a functional stator around the rotor. The cross-linking did not affect the localization of the stator nor the ion conductivity, suggesting that the conformational change occurs in the final step of the stator assembly around the rotor.

A Step Forward in Understanding the Mechanism of VDAC Voltage-Gating

The voltage-dependent anion channel (VDAC) governs the exchange of ions and metabolites between the mitochondria and the rest of the cell. In its open state VDAC exhibits high conductance and selectivity for anions that facilitates the passage of ADP, ATP, and other metabolites. At increased voltages (>30mV) VDAC switches to lower conducting states, termed as “closed” states. Closed states are cation-selective and impermeable for ATP. The voltage-induced transition from the open to closed states is referred to as voltage-gating. Although it is well established that VDAC voltage-gating involves large structural rearrangements, the precise molecular mechanism of this process is still under debate. We investigated VDAC voltage-gating by systematically titrating VDAC charge residues and by using thermodynamic and kinetic approaches to study opening and closing of the channel. All the models proposed so far agree that N-terminal region plays a key role in VDAC voltage-gating. According to the original idea, the N-terminal region is a part of a mobile voltage sensor domain, which slides in and out of the channel lumen in response to the applied voltage. The alternative models consider independent movement of the N-terminal region upon gating. In order to test the role of VDAC N-terminal region in voltage-gating, we engineered a double Cys mutant of murine VDAC1 that cross-links the α-helix to the β-strand 11 of the pore wall. The cross-linked VDAC1 reconstituted into planar lipid membranes exhibited typical voltage gating, which suggests that the N-terminal α-helix is located inside the pore of VDAC in the open state and remains associated with the pore wall during voltage gating. Our findings support a model where β-barrel is not rigid but undergoes a conformational change that leads to a partial constriction upon transition to the closed states.

Structural Dynamics of Actin-Myosin Bound and Unbound States of Cardiac Myosin Binding Protein-C Detected by Dipolar EPR

We have used site-directed spin labeling and pulsed dipolar electron-electron paramagnetic resonance (DEER) to resolve the structure and dynamics of flexible and disordered regions of myosin binding protein-C (MyBP-C)'s cardiac isoform, with implications for the pathophysiology of hypertrophic cardiomyopathy (HCM). N-terminal domains of cMyBP-C contain binding domains for several interaction partners in the myofilament, including myosin heavy chain subfragment 2 (S2) and actin. We engineered pairs of labeling sites in protein fragments of mouse cMyBP-C to measure with high resolution distance and disorder between (1) domains C0 and C1, flanking the flexible Pro/Ala-rich linker, and between (2) domains C1 and C2, flanking the partially disordered phosphorylation motif, using DEER. Changes in distance and disorder were assessed for double-Cys mutant cMyBP-C's free in solution and when bound to myosin S2 or actin, with or without cMyBP-C phosphorylation by protein kinase A (PKA). Understanding conformational transitions in the flexible and dynamic portions of cMyBP-C upon actin-myosin binding and phosphorylation provide new molecular insight into defining its modulatory role in muscle force development. (NIH-F32 to BAC; NIH-R01 to DDT)

Affixing N-terminal α-Helix to the Wall of the Voltage-dependent Anion Channel Does Not Prevent Its Voltage Gating

The voltage-dependent anion channel (VDAC) governs the free exchange of ions and metabolites between the mitochondria and the rest of the cell. The three-dimensional structure of VDAC1 reveals a channel formed by 19 β-strands and an N-terminal α-helix located near the midpoint of the pore. The position of this α-helix causes a narrowing of the cavity, but ample space for metabolite passage remains. The participation of the N-terminus of VDAC1 in the voltage-gating process has been well established, but the molecular mechanism continues to be debated; however, the majority of models entail large conformational changes of this N-terminal segment. Here we report that the pore-lining N-terminal α-helix does not undergo independent structural rearrangements during channel gating. We engineered a double Cys mutant in murine VDAC1 that cross-links the α-helix to the wall of the β-barrel pore and reconstituted the modified protein into planar lipid bilayers. The modified murine VDAC1 exhibited typical voltage gating. These results suggest that the N-terminal α-helix is located inside the pore of VDAC in the open state and remains associated with β-strand 11 of the pore wall during voltage gating.

Structural Dynamics in Chloroplast Signal Recognition Particle (cpSRP) Proteins Studied with Single Molecule Fluorescence

The interdomain structural dynamics of cpSRP43 protein interacting with cpSRP54 protein is studied by single molecule FRET, FCS, and ensemble fluorescence spectroscopy in order to understand the protein-protein interactions that help to transport LHCP into the thylakoid membrane for photosynthesis. Several double Cys and single Cys mutants of cpSRP43 and cpSRP54 proteins are cloned, expressed, purified and then specifically labeled with fluorescent dyes. The single molecule FRET and FCS equilibrium binding results suggest that the fluorescence labeling does not affect the cpSRP43-cpSRP54 interaction. Furthermore, we find that cpSRP43 proteins are very heterogeneous in structure and that cpSRP43 proteins undergo complex interdomain structural dynamics when interacting with cpSRP54 protein.

Beta Subunits Bridge Two Alpha Subunits Within the BK Channel Tetramer

BK potassium channels contain four pore-forming alpha subunits and four modulatory beta subunits. Transmembrane (TM) helices S1-S6 of BK alpha are homologous to S1-S6 of other V-gated potassium channels; however, BK alpha contains a unique seventh TM helix, S0, N terminal to S1. The beta subunits contain two TM helices, TM1 and TM2. From the extent of endogenous disulfide crosslinking between Cys substituted for the four residues just flanking the extracellular ends of alpha S0-S6 and of beta1 TM1 and TM2, we previously inferred that the flank of S0 was closest to the four-residue loop between S3 and S4 and also contacted the flanks of S1 and S2. Furthermore, the flank of beta1 TM1 was closest to the flanks of S1 and S2, and the flank of TM2 was closest to the flank of S0. We have now extended this analysis to the membrane doman. We find that Cys in the first helical turn of S0 within the membrane forms disulfides with Cys subsituted in the first helical turns of S3 and S4 but not with similarly located Cys in S1 or S2. Thus, in the membrane, S0 is next to S3 and S4 but not to S1 and S2, although the flank of S0 reaches the flanks of S1 and S2. Furthermore, co-expression of the double-Cys mutant of alpha, W23C in the first helical turn of S0 and F144C in the S2 flank, and the double-Cys mutant of beta1, Y42C in the TM1 flank and L157C in the first helical turn of TM2, resulted in the crosslinking of two alphas through one beta1, S0 to TM2 and TM1 to S2. Thus, TM1 and TM2 of each beta subunit lie between the voltage-sensing domains (S0-S4) of adjacent alpha subunits.

Structural dynamics of the myosin relay helix by time-resolved EPR and FRET

We have used two complementary time-resolved spectroscopic techniques, dipolar electron-electron resonance and fluorescence resonance energy transfer to determine conformational changes in a single structural element of the myosin motor domain, the relay helix, before and after the recovery stroke. Two double-Cys mutants were labeled with optical probes or spin labels, and interprobe distances were determined. Both methods resolved two distinct structural states of myosin, corresponding to straight and bent conformations of the relay helix. The bent state was occupied only upon nucleotide addition, indicating that relay helix, like the entire myosin head, bends in the recovery stroke. However, saturation of myosin with nucleotide, producing a single biochemical state, did not produce a single structural state. Both straight and bent structural states of the relay helix were occupied when either ATP (ADP.BeF(x)) or ADP.P(i) (ADP.AlF(4)) analogs were bound at the active site. A greater population was found in the bent structural state when the posthydrolysis analog ADP.AlF(4) was bound. We conclude that the bending of the relay helix in the recovery stroke does not require ATP hydrolysis but is favored by it. A narrower interprobe distance distribution shows ordering of the relay helix, despite its bending, during the recovery stroke, providing further insight into the dynamics of this energy-transducing structural transition.

Conformational Flexibility of the GM2 Activator Protein Loop Regions Investigated By Site Directed Spin Labeling EPR Spectroscopy

The GM2 activator protein (GM2AP) is an essential component in the degradation pathway of neuronal gangliosides. GM2AP is a required accessory protein for the hydrolytic conversion of GM2 to GM3 by a water soluble hydrolase. The X-ray structure of GM2AP reveals a β-cup topology with multiple conformations of the protein within the unit cell. Because the crystal structures show different conformations of the putative membrane binding loops, we have utilized site-directed spin labeling to investigate conformational flexibility of these loops for protein in solution and bound with GM2 ligand. As such, a series of single and double CYS mutants (still with original 8 CYS in 4 disulfide bridges) have been generated and spin labeled with MTSL. EPR spectra of spin labeled GM2AP were collected with and without GM2 ligand, and no significant changes in the EPR lineshape were seen. EPR spectra were simulated for spin labels located in the loop regions and reveal multiple component fits, while those in the backside of the β-cup beta strands have single component fits. For certain sites in the mobile loops, spectra were acquired as a function of temperature. From these lineshape simulations, the activation energy for the conformational change has been determined. The SDSL EPR results indicate that the multiple conformations observed in the crystallographic unit cell are populated in solution and represent conformational flexibility of the protein; which is not necessarily altered by binding to lipid ligands. Additionally, spin labeled protein was analyzed by mass spectrometry to confirm proper formation of the four disulfide linkages and addition of only one spin label at the mutant cysteine.

Identification and Role of the Homodimerization Interface of the Glycosylphosphatidylinositol-anchored Membrane Type 6 Matrix Metalloproteinase (MMP25)

The membrane type (MT) 6 matrix metalloproteinase (MMP) (MMP25) is a glycosylphosphatidylinositol-anchored matrix metalloproteinase (MMP) that is highly expressed in leukocytes and in some cancer tissues. We previously showed that natural MT6-MMP is expressed on the cell surface as a major reduction-sensitive form of M(r) 120, likely representing enzyme homodimers held by disulfide bridges. Among the membrane type-MMPs, the stem region of MT6-MMP contains three cysteine residues at positions 530, 532, and 534 which may contribute to dimerization. A systematic site-directed mutagenesis study of the Cys residues in the stem region shows that Cys(532) is involved in MT6-MMP dimerization by forming an intermolecular disulfide bond. The mutagenesis data also suggest that Cys(530) and Cys(534) form an intramolecular disulfide bond. The experimental observations on cysteines were also investigated by computational studies of the stem peptide, which validate these proposals. Dimerization is not essential for transport of MT6-MMP to the cell surface, partitioning into lipid rafts or cleavage of alpha-1-proteinase inhibitor. However, monomeric forms of MT6-MMP exhibited enhanced autolysis and metalloprotease-dependent degradation. Collectively, these studies establish the stem region of MT6-MMP as the dimerization interface, an event whose outcome imparts protease stability to the protein.

Opening and closing of the periplasmic gate in lactose permease

X-ray crystal structures of lactose permease (LacY) reveal pseudosymmetrically arranged N- and C-terminal six-transmembrane helix bundles surrounding a deep internal cavity open on the cytoplasmic side and completely closed on the periplasmic side. The residues essential for sugar recognition and H(+) translocation are located at the apex of the cavity and are inaccessible from the outside. On the periplasmic side, helices I/II and VII from the N- and C- six helix bundles, respectively, participate in sealing the cavity from the outside. Three paired double-Cys mutants-Ile-40 --> Cys/Asn-245 --> Cys, Thr-45 --> Cys/Asn-245 --> Cys, and Ile-32 --> Cys/Asn-245 --> Cys-located in the interface between helices I/II and VII on the periplasmic side of LacY were constructed. After cross-linking with homobifunctional reagents less than approximately 15 A in length, all three mutants lose the ability to catalyze lactose transport. Strikingly, however, full or partial activity is observed when cross-linking is mediated by flexible reagents greater than approximately 15 A in length. The results provide direct support for the argument that transport via LacY involves opening and closing of a large periplasmic cavity.

Ligand-Specific Changes in M3 Muscarinic Acetylcholine Receptor Structure Detected by a Disulfide Scanning Strategy

G protein-coupled receptor (GPCR) function can be modulated by different classes of ligands including full and inverse agonists. At present, little is known about the conformational changes that agonist ligands induce in their target GPCRs. In this study, we employed an in situ disulfide cross-linking strategy to monitor ligand-induced structural changes in a series of cysteine (Cys)-substituted mutant M 3 muscarinic acetylcholine receptors. One of our goals was to study whether the cytoplasmic end of transmembrane domain V (TM V), a region known to be critically involved in receptor/G protein coupling, undergoes a major conformational change, similar to the adjacent region of TM VI. Another goal was to determine and compare the disulfide cross-linking patterns observed after treatment of the different mutant receptors with full versus inverse muscarinic agonists. Specifically, we generated 20 double Cys mutant M 3 receptors harboring one Cys substitution within the cytoplasmic end of TM V (L249-I253) and a second one within the cytoplasmic end of TM VI (A489-L492). These receptors were transiently expressed in COS-7 cells and subsequently characterized in pharmacological and disulfide cross-linking studies. Our cross-linking data, in conjunction with a three-dimensional model of the M 3 muscarinic receptor, indicate that M 3 receptor activation does not trigger major structural disturbances within the cytoplasmic segment of TM V, in contrast to the pronounced structural changes predicted to occur at the cytoplasmic end of TM VI. We also demonstrated that full and inverse muscarinic agonists had distinct effects on the efficiency of disulfide bond formation in specific double Cys mutant M 3 receptors. The present study provides novel information about the dynamic changes that accompany M 3 receptor activation and how the receptor conformations induced (or stabilized) by full versus inverse muscarinic agonists differ from each other at the molecular level. Because all class I GPCRs are predicted to share a similar transmembrane topology, the conclusions drawn from the present study should be of broad general relevance.