Unfolding events in the water-soluble monomeric Cry1Ab toxin during transition to oligomeric pre-pore and membrane-inserted pore channel

The insecticidal crystal (Cry) proteins produced by Bacillus thuringiensis undergo several conformational changes from crystal inclusion protoxins to membrane-inserted channels in the midgut epithelial cells of the target insect. Here we analyzed the stability of the different forms of Cry1Ab toxin, monomeric toxin, pre-pore complex, and membrane-inserted channel, after urea and thermal denaturation by monitoring intrinsic tryptophan fluorescence of the protein and 1-anilinonaphthalene-8-sulfonic acid binding to partially unfolded proteins. Our results showed that flexibility of the monomeric toxin was dramatically enhanced upon oligomerization and was even further increased by insertion of the pre-pore into the membrane as shown by the lower concentration of chaotropic agents needed to achieve unfolding of the oligomeric species. The flexibility of the toxin structures is further increased by alkaline pH. We found that the monomer-monomer interaction in the pre-pore is highly stable because urea promotes oligomer denaturation without disassembly. Partial unfolding and limited proteolysis studies demonstrated that domains II and III were less stable and unfold first, followed by unfolding of the most stable domain I, and also that domain I is involved in monomer-monomer interaction. The thermal-induced unfolding and analysis of energy transfer from Trp residues to bound 1-anilinonaphthalene-8-sulfonic acid dye showed that in the membrane-inserted pore domains II and III are particularly sensitive to heat denaturation, in contrast to domain I, suggesting that only domain I may be inserted into the membrane. Finally, the insertion into the membrane of the oligomeric pre-pore structure was not affected by pH. However, a looser conformation of the membrane-inserted domain I induced by neutral or alkaline pH correlates with active channel formation. Our studies suggest for the first time that a more flexible conformation of Cry toxin could be necessary for membrane insertion, and this flexible structure is induced by toxin oligomerization. Finally the alkaline pH found in the midgut lumen of lepidopteran insects could increase the flexibility of membrane-inserted domain I necessary for pore formation.     

Structure of colicin I receptor bound to the R-domain of colicin Ia: implications for protein import

Colicin Ia is a 69 kDa protein that kills susceptible Escherichia coli cells by binding to a specific receptor in the outer membrane, colicin I receptor (70 kDa), and subsequently translocating its channel forming domain across the periplasmic space, where it inserts into the inner membrane and forms a voltage-dependent ion channel. We determined crystal structures of colicin I receptor alone and in complex with the receptor binding domain of colicin Ia. The receptor undergoes large and unusual conformational changes upon colicin binding, opening at the cell surface and positioning the receptor binding domain of colicin Ia directly above it. We modelled the interaction with full-length colicin Ia to show that the channel forming domain is initially positioned 150 A above the cell surface. Functional data using full-length colicin Ia show that colicin I receptor is necessary for cell surface binding, and suggest that the receptor participates in translocation of colicin Ia across the outer membrane.     

Acid destabilization of the solution conformation of Bcl-xL does not drive its pH-dependent insertion into membranes

Regulation of programmed cell death by Bcl-xL is dependent on both its solution and integral membrane conformations. A conformational change from solution to membrane is also important in this regulation. This conformational change shows a pH-dependence similar to the translocation domain of diphtheria toxin, where an acid-induced molten globule conformation in the absence of lipid vesicles mediates the change from solution to membrane conformations. By contrast, Bcl-xL deltaTM in the absence of lipid vesicles exhibits no gross conformational changes upon acidification as observed by near- and far-UV circular dichroism spectropolarimetry. Additionally, no significant local conformational changes upon acidification were observed by heteronuclear NMR spectroscopy of Bcl-xL deltaTM. Under conditions that favor the solution conformation (pH 7.4), the free energy of folding for Bcl-xL deltaTM (deltaG(o)) was determined to be 15.8 kcal x mol(-1). Surprisingly, under conditions that favor a membrane conformation (pH 4.9), deltaG(o) was 14.6 kcal x mol(-1). These results differ from those obtained with many other membrane-insertable proteins where acid-induced destabilization is important. Therefore, other contributions must be necessary to destabilize the solution conformation Bcl-xL and favor the membrane conformation at pH 4.9. Such contributions might include the presence of a negatively charged membrane or an electrostatic potential across the membrane. Thus, for proteins that adopt both solution and membrane conformations, an obligatory molten globule intermediate may not be necessary. The absence of a molten globule intermediate might have evolved to protect Bcl-xL from intracellular proteases as it undergoes this conformational change essential for its activity.     

Influence of membrane fluidity on the assembly of Staphylococcus aureus alpha-toxin, a channel-forming protein, in liposome membrane.

By use of multilamellar phosphatidylcholine (PC) liposomes of different acyl composition and cholesterol content as model membranes, we studied whether or not membrane fluidity affects the assembly process of Staphylococcus aureus alpha-toxin. Under conditions using fluid and solid membranes, we assayed accessibility (or hemolytic activity) of liposome-bound alpha-toxin to rabbit erythrocytes added, hexamerization of membrane-bound toxin using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nondenaturating conditions, and susceptibility of liposome-bound toxin to trypsin digestion. Our data indicated 1) that alpha-toxin bound to PC membrane as a hemolytically active monomer (or reversibly bound state); 2) that when the membrane was fluidized either by phase transition of PC or by inclusion of cholesterol over 20 mol %, the hemolytically active monomer of the toxin was irreversibly converted to nonhemolytic monomer (and/or unstable oligomer) in a first-order kinetics with a t1/2 of about 1 min, and thereafter hexamerization of the toxin gradually proceeded in the following 60-90 min; 3) that alpha-toxin might have different topology and/or conformation in PC membrane, depending on the presence or absence of cholesterol in the PC membrane; and 4) that coexistence of unsaturated acyl chain-carrying PC and cholesterol was a prerequisite for efficient hexamerization of alpha-toxin in membrane. Thus, increase in membrane fluidity promoted the assembly process of S. aureus alpha-toxin.     

A rivet model for channel formation by aerolysin-like pore-forming toxins

The bacterial toxin aerolysin kills cells by forming heptameric channels, of unknown structure, in the plasma membrane. Using disulfide trapping and cysteine scanning mutagenesis coupled to thiol-specific labeling on lipid bilayers, we identify a loop that lines the channel. This loop has an alternating pattern of charged and uncharged residues, suggesting that the transmembrane region has a beta-barrel configuration, as observed for Staphylococcal alpha-toxin. Surprisingly, we found that the turn of the beta-hairpin is composed of a stretch of five hydrophobic residues. We show that this hydrophobic turn drives membrane insertion of the developing channel and propose that, once the lipid bilayer has been crossed, it folds back parallel to the plane of the membrane in a rivet-like fashion. This rivet-like conformation was modeled and sequence alignments suggest that such channel riveting may operate for many other pore-forming toxins.     

Integration of the Serratia marcescens haemolysin into human erythrocyte membranes

The haemolytic activity of Serratia marcescens is determined by two proteins, ShlA and ShlB. ShlA integrates into the erythrocyte membrane and causes osmotic lysis through channel formation. The conformation of ShlA and its interaction with erythrocyte membranes were studied by determining the cleavage of ShlA by added trypsin. Our results suggest that the conformation of inactive ShlA (from an ShlB- strain) differs from the active ShlA, and that in a hydrophobic environment (detergent or membrane) active ShlA assumes a conformation distinct from that in buffer. Only active haemolysin adsorbed to erythrocytes. ShlA was firmly integrated into the erythrocyte membrane since it was only released under conditions which also dissolved the integral erythrocyte membrane proteins. Moreover, ShlA integrated into 'ghosts' remained there and was not haemolytic when incubated with erythrocytes. From the trypsin cleavage pattern obtained with haemolysin and C-terminally truncated, but still active, haemolysin derivatives integrated into erythrocytes, and sealed and unsealed erythrocyte 'ghosts', we conclude that ShlA is preferentially cleaved by trypsin at a few sites but only from the inside of the erythrocyte. Haemolysin in the erythrocyte membrane forms a water-filled channel and is resistant to trypsin and other proteases.     

The SecYEG preprotein translocation channel is a conformationally dynamic and dimeric structure

Escherichia coli preprotein translocase comprises a membrane-embedded trimeric complex of SecY, SecE and SecG. Previous studies have shown that this complex forms ring-like assemblies, which are thought to represent the preprotein translocation channel across the membrane. We have analyzed the functional state and the quaternary structure of the SecYEG translocase by employing cross-linking and blue native gel electrophoresis. The results show that the SecYEG monomer is a highly dynamic structure, spontaneously and reversibly associating into dimers. SecG-dependent tetramers and higher order SecYEG multimers can also exist in the membrane, but these structures form at high SecYEG concentration or upon overproduction of the complex only. The translocation process does not affect the oligomeric state of the translocase and arrested preproteins can be trapped with SecYEG or SecYE dimers. Dissociation of the dimer into a monomer by detergent induces release of the trapped preprotein. These results provide direct evidence that preproteins cross the bacterial membrane, associated with a translocation channel formed by a dimer of SecYEG.     

Secondary structure and assembly mechanism of an oligomeric channel protein

The alpha-toxin of Staphylococcus aureus is secreted as a water-soluble, monomeric polypeptide (Mr 33 182) that can assemble into an oligomeric membrane channel. By chemical cross-linking, we have confirmed that the major form of the channel is a hexamer. The circular dichroism spectrum of this hexamer in detergent revealed that it contains a high proportion of beta-sheet that we deduce must lie within the lipid bilayer when the protein is associated with membranes. The circular dichroism spectrum of the monomeric toxin in the presence or absence of detergent was closely similar to the spectrum of the hexamer, suggesting that the secondary structure of the polypeptide is little changed on assembly. Results of experiments involving limited proteolysis of the monomer and hexamer are consistent with the idea that assembly involves the movement of two rigid domains about a hinge located near the midpoint of the polypeptide chain. The hydrophilic monomer is thereby converted to an amphipathic rod that becomes a subunit of the hexamer.     

Conformational state of the SecYEG-bound SecA probed by single tryptophan fluorescence spectroscopy

The SecYEG complex is a membrane-embedded channel that permits the passage of precursor proteins (preproteins) across the inner membrane of Escherichia coli. SecA is a molecular motor that associates with the SecYEG pore and drives the stepwise translocation of preproteins across the membrane through multiple cycles of ATP binding and hydrolysis. We have investigated the conformational state of soluble and SecYEG-bound SecA using single tryptophan mutants of SecA. The fluorescence spectral properties of the single tryptophans of SecA and their accessibility to the quencher acrylamide demonstrate that SecA undergoes a conformational change that results in a more compact structure upon binding of ATP and binding to the SecYEG pore. In addition, SecYEG-bound SecA undergoes ATP-dependent conformational changes that are not observed for soluble SecA. These data support a model in which binding to the SecYEG channel has a major impact on the SecA conformation.     

Localization and environment of tryptophans in soluble and membrane-bound states of a pore-forming toxin from Staphylococcus aureus

The location and environment of tryptophans in the soluble and membrane-bound forms of Staphylococcus aureus alpha-toxin were monitored using intrinsic tryptophan fluorescence. Fluorescence quenching of the toxin monomer in solution indicated varying degrees of tryptophan burial within the protein interior. N-Bromosuccinimide readily abolished 80% of the fluorescence in solution. The residual fluorescence of the modified toxin showed a blue-shifted emission maximum, a longer fluorescence lifetime as compared to the unmodified and membrane-bound alpha-toxin, and a 5- to 6-nm red edge excitation shift, all indicating a restricted tryptophan environment and deeply buried tryptophans. In the membrane-bound form, the fluorescence of alpha-toxin was quenched by iodide, indicating a conformational change leading to exposure of some tryptophans. A shorter average lifetime of tryptophans in the membrane-bound alpha-toxin as compared to the native toxin supported the conclusions based on iodide quenching of the membrane-bound toxin. Fluorescence quenching of membrane-bound alpha-toxin using brominated and spin-labeled fatty acids showed no quenching of fluorescence using brominated lipids. However, significant quenching was observed using 5- and 12-doxyl stearic acids. An average depth calculation using the parallax method indicated that the doxyl-quenchable tryptophans are located at an average depth of 10 A from the center of the bilayer close to the membrane interface. This was found to be in striking agreement with the recently described structure of the membrane-bound form of alpha-toxin.     

Reversible transition between the surface trimer and membrane-inserted monomer of annexin 12

Under mildly acidic conditions, annexin 12 (ANX) inserts into lipid membranes to form a transbilayer pore [Langen, R., et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 14060]. In this study, we have addressed the question of the oligomeric state of ANX in this transbilayer conformation by means of Forster-type resonance energy transfer (FRET). Two single-cysteine mutants (K132C and N244C) were labeled with either Alexa-532 (donor) or Alexa-647 (acceptor). The labels were positioned at the sites thought to be on the cis side of the known transmembrane regions [Ladokhin, A. S., et al. (2002) Biochemistry 41, 13617]. If the pore were comprised of an annexin oligomer, efficient energy transfer should be observed. Fluorescence excitation spectra of several mixtures of donor- and acceptor-labeled ANX were recorded under various conditions. Spectroscopic hallmarks of oligomerization-related FRET were established by following a well-documented transition of ANX from the soluble monomer to surface trimer upon addition of calcium at neutral pH. These hallmarks, however, were not detected for the membrane-inserted form of ANX at pH 4.5, suggesting that the transbilayer form is a monomer. This implies that the pore is formed by several transmembrane regions of the same ANX molecule. FRET and other fluorescence experiments demonstrate that the transitions between the surface trimer and membrane-inserted monomer are reversible. This reversibility, in combination with the absence of oligomerization in the water-soluble and inserted state, makes ANX a good experimental model for thermodynamic studies of folding and stability of membrane proteins.     

Structural and functional analysis of the pre-pore and membrane-inserted pore of Cry1Ab toxin

Bacillus thuringiensis produces insecticidal Cry proteins that are active against different insect species. The primary action of Cry toxins is to lyse midgut epithelial cells in the target insect by forming lytic pores on the apical membrane. After interaction with cadherin receptor, Cry proteins undergo conformational changes from a monomeric structure to a pre-pore-oligomeric form that is able to interact with a second GPI-anchored aminopeptidase-N receptor and then insert into lipid membranes. Here, we review the recent advances in the understanding of the structural changes presented by Cry1Ab toxin upon membrane insertion. Based on analysis of the Trp fluorescence of pure monomeric and oligomeric Cry1Ab structures in solution and in membrane-bound state we reported that oligomerization caused 27% reduction of Trp exposed to the solvent. After membrane insertion there is another conformational change that allows an additional rearrangement of the Trp residues resulting in a total protection of these residues from exposure to the solvent. The oligomeric structure is membrane insertion competent since more than 96% of the Cry1Ab oligomer inserts into the membrane as a function of lipid:protein ratio, in contrast to the monomer of which only 5-10%, inserts into the membrane. Finally, analysis of the stability of monomeric, pre-pore and pore structures of Cry1Ab toxin after urea and thermal denaturation suggested that a more flexible conformation could be necessary for membrane insertion and this flexible structure is obtained by toxin oligomerization and by alkaline pH. Domain I is involved in the intermolecular interaction within the oligomeric Cry1Ab and this domain is inserted into the membrane in the membrane-inserted state.     

In vitro synthesis and oligomerization of the mechanosensitive channel of large conductance, MscL, into a functional ion channel

Elucidation of high-resolution structures of integral membrane proteins is drastically lagging behind that of cytoplasmic proteins. In vitro synthesis and insertion of membrane proteins into synthetic membranes could circumvent bottlenecks associated with the overexpression of membrane proteins, producing sufficient membrane-inserted, correctly folded protein for structural studies. Using the mechanosensitive channel of large conductance, MscL, as a model protein we show that in vitro synthesized MscL inserts into YidC-containing proteoliposomes and oligomerizes to form a homopentamer. Using planar membrane bilayers, we show that MscL forms functional ion channels capable of ion transport. These data demonstrate that membrane insertion of MscL is YidC mediated, whereas subsequent oligomerization into a functional homopentamer is a spontaneous event.     

Bcl-2 changes conformation to inhibit Bax oligomerization

Bcl-2 inhibits apoptosis by regulating the release of cytochrome c and other proteins from mitochondria. Oligomerization of Bax promotes cell death by permeabilizing the outer mitochondrial membrane. In transfected cells and isolated mitochondria, Bcl-2, but not the inactive point mutants Bcl-2-G145A and Bcl-2-V159D, undergoes a conformation change in the mitochondrial membrane in response to apoptotic agonists such as tBid and Bax. A mutant Bcl-2 with two cysteines introduced at positions predicted to result in a disulfide bond that would inhibit the mobility of alpha5-alpha6 helices (Bcl-2-S105C/E152C) was only active in a reducing environment. Thus, Bcl-2 must change the conformation to inhibit tBid-induced oligomerization of integral membrane Bax monomers and small oligomers. The conformationally changed Bcl-2 sequesters the integral membrane form of Bax. If Bax is in excess, apoptosis resumes as Bcl-2 is consumed by the conformational change and in complexes with Bax. Thus, Bcl-2 functions as an inhibitor of mitochondrial permeabilization by changing conformation in the mitochondrial membrane to bind membrane-inserted Bax monomers and prevent productive oligomerization of Bax.     

Tryptophan spectroscopy studies and black lipid bilayer analysis indicate that the oligomeric structure of Cry1Ab toxin from Bacillus thuringiensis is the membrane-insertion intermediate

During intoxication, the Cry protoxins must change from insoluble crystals into membrane-inserted toxins, which form ionic pores. Binding of Cry1A toxins to the cadherin receptor promotes the formation of a 250 kDa oligomer. In this work, we analyzed for the first time the structural changes presented by Cry1Ab toxin upon membrane insertion. Trp fluorescence of pure monomeric and oligomeric structures in solution and in a membrane-bound state was analyzed. Cry1Ab has nine Trp residues, seven of them in pore-forming domain I. Trp quenching analysis with iodide indicated that oligomerization caused a 27% reduction in the level of Trp exposed to the solvent. Most of the oligomeric structure (96%) inserts into the membrane as a function of the lipid:protein ratio, in contrast to the monomer (10%). Additionally, the membrane-associated oligomer presented a blue shift of 5 nm in lambda(max) of the emission spectrum, indicating a more hydrophobic environment for some Trp residues. In agreement with this, iodide was unable to quench the Trp of the membrane-bound oligomer, suggesting that a significant part of the protein may be buried in the membrane. Quenching analysis using brominated and spin-labeled phospholipids in the vesicles indicates that most of the Trp residues are located close to the membrane-water interface. Finally, ionic currents in black lipid bilayers revealed that the oligomeric structure has kinetics different from those of the monomer, producing stable channels with a high probability of being open in contrast to the monomer that exhibited unstable opening patterns. These data show that the oligomer, in contrast to the monomer, is able to interact efficiently with phospholipid membranes forming stable pores.     

The Influence of Natural Lipid Asymmetry upon the Conformation of a Membrane-inserted Protein (Perfringolysin O)

Eukaryotic membrane proteins generally reside in membrane bilayers that have lipid asymmetry. However, in vitro studies of the impact of lipids upon membrane proteins are generally carried out in model membrane vesicles that lack lipid asymmetry. Our recently developed method to prepare lipid vesicles with asymmetry similar to that in plasma membranes and with controlled amounts of cholesterol was used to investigate the influence of lipid composition and lipid asymmetry upon the conformational behavior of the pore-forming, cholesterol-dependent cytolysin perfringolysin O (PFO). PFO conformational behavior in asymmetric vesicles was found to be distinct both from that in symmetric vesicles with the same lipid composition as the asymmetric vesicles and from that in vesicles containing either only the inner leaflet lipids from the asymmetric vesicles or only the outer leaflet lipids from the asymmetric vesicles. The presence of phosphatidylcholine in the outer leaflet increased the cholesterol concentration required to induce PFO binding, whereas phosphatidylethanolamine and phosphatidylserine in the inner leaflet of asymmetric vesicles stabilized the formation of a novel deeply inserted conformation that does not form pores, even though it contains transmembrane segments. This conformation may represent an important intermediate stage in PFO pore formation. These studies show that lipid asymmetry can strongly influence the behavior of membrane-inserted proteins.     

Formation of an unfolding intermediate state of soluble chloride intracellular channel protein CLIC1 at acidic pH

CLIC proteins function as anion channels when their structures convert from a soluble form to an integral membrane form. While very little is known about the mechanism of the conversion process, channel formation and activity are highly pH-dependent. In this study, the structural properties and conformational stability of CLIC1 were determined as a function of pH in the absence of membranes to improve our understanding of how its conformation changes when the protein encounters the acidic environment at the surface of a membrane. Although the global conformation and size of CLIC1 are not significantly altered by pH in the range of 5.5-8.2, equilibrium unfolding studies reveal that the protein molecule becomes destabilized at low pH, resulting in the formation of a highly populated intermediate with a solvent-exposed hydrophobic surface. Unlike the intermediates formed by many soluble pore-forming proteins for their insertion into membranes, the CLIC1 intermediate is not a molten globule. Acid-induced destabilization and partial unfolding of CLIC1 involve helix alpha1 which is the major structural element of the transmembrane region. We propose that the acidic environment encountered by CLICs at the surface of membranes primes the transmembrane region in the N-domain, thereby lowering the energy barrier for the conversion of soluble CLICs to their membrane-inserted forms.     

Conformational Itinerary of Pseudomonas aeruginosa 1,6-Anhydro-N-acetylmuramic Acid Kinase during Its Catalytic Cycle

Anhydro-sugar kinases are unique from other sugar kinases in that they must cleave the 1,6-anhydro ring of their sugar substrate to phosphorylate it using ATP. Here we show that the peptidoglycan recycling enzyme 1,6-anhydro-N-acetylmuramic acid kinase (AnmK) from Pseudomonas aeruginosa undergoes large conformational changes during its catalytic cycle, with its two domains rotating apart by up to 32° around two hinge regions to expose an active site cleft into which the substrates 1,6-anhydroMurNAc and ATP can bind. X-ray structures of the open state bound to a nonhydrolyzable ATP analog (AMPPCP) and 1,6-anhydroMurNAc provide detailed insight into a ternary complex that forms preceding an operative Michaelis complex. Structural analysis of the hinge regions demonstrates a role for nucleotide binding and possible cross-talk between the bound ligands to modulate the opening and closing of AnmK. Although AnmK was found to exhibit similar binding affinities for ATP, ADP, and AMPPCP according to fluorescence spectroscopy, small angle x-ray scattering analyses revealed that AnmK adopts an open conformation in solution in the absence of ligand and that it remains in this open state after binding AMPPCP, as we had observed for our crystal structure of this complex. In contrast, the enzyme favored a closed conformation when bound to ADP in solution, consistent with a previous crystal structure of this complex. Together, our findings show that the open conformation of AnmK facilitates binding of both the sugar and nucleotide substrates and that large structural rearrangements must occur upon closure of the enzyme to correctly align the substrates and residues of the enzyme for catalysis.     

Topography of the hydrophilic helices of membrane-inserted diphtheria toxin T domain: TH1-TH3 as a hydrophilic tether

After low pH-triggered membrane insertion, the T domain of diphtheria toxin helps translocate the catalytic domain of the toxin across membranes. In this study, the hydrophilic N-terminal helices of the T domain (TH1-TH3) were studied. The conformation triggered by exposure to low pH and changes in topography upon membrane insertion were studied. These experiments involved bimane or BODIPY labeling of single Cys introduced at various positions, followed by the measurement of bimane emission wavelength, bimane exposure to fluorescence quenchers, and antibody binding to BODIPY groups. Upon exposure of the T domain in solution to low pH, it was found that the hydrophobic face of TH1, which is buried in the native state at neutral pH, became exposed to solution. When the T domain was added externally to lipid vesicles at low pH, the hydrophobic face of TH1 became buried within the lipid bilayer. Helices TH2 and TH3 also inserted into the bilayer after exposure to low pH. However, in contrast to helices TH5-TH9, overall TH1-TH3 insertion was shallow and there was no significant change in TH1-TH3 insertion depth when the T domain switched from the shallowly inserting (P) to deeply inserting (TM) conformation. Binding of streptavidin to biotinylated Cys residues was used to investigate whether solution-exposed residues of membrane-inserted T domain were exposed on the external or internal surface of the bilayer. These experiments showed that when the T domain is externally added to vesicles, the entire TH1-TH3 segment remains on the cis (outer) side of the bilayer. The results of this study suggest that membrane-inserted TH1-TH3 form autonomous segments that neither deeply penetrate the bilayer nor interact tightly with the translocation-promoting structure formed by the hydrophobic TH5-TH9 subdomain. Instead, TH1-TH3 may aid translocation by acting as an A-chain-attached flexible tether.     

Structure of the monomeric 8-kDa dynein light chain and mechanism of the domain-swapped dimer assembly

The 8-kDa light chain of dynein (DLC8) is ubiquitously expressed in various cell types. Other than serving as a light chain of the dynein complexes, this highly conserved protein has been shown to bind a larger number of proteins with diverse biological functions. DLC8 forms a homodimer via three-dimensional domain swapping of an internal beta-strand (the beta2-strand) at neutral pH. The protein undergoes non-reversible dimer-to-monomer dissociation when the pH value of the protein solution decreases. The three-dimensional structure of the DLC8 monomer determined by NMR spectroscopy at pH 3.0 showed that the protein is well folded. The major conformational change accompanied by dimer dissociation is in the beta2-strand of the protein, which undergoes transition from a beta-strand to a nascent alpha-helix. The monomer form of DLC8 is not capable of binding to target proteins. Insertion of two flexible amino acid residues in the tight beta1/beta2-loop dramatically stabilized the monomer conformation of the protein. NMR studies showed that the mutation altered the conformation as well as the three-dimensional domain swapping-mediated assembly of the DLC8 dimer. The mutant DLC8 was unable to bind to its targets even at physiological pH. The three-dimensional structure of the mutant protein in its monomeric form provides the structural basis of the mutation-induced stabilization of the monomer conformation. Based on the experimental data, we conclude that the formation of the beta2-strand swapping-mediated dimer is mandatory for the structure and function of DLC8. We further note that the DLC8 dimer represents a novel mode of three-dimensional domain swapping.