Hemocyanin conformational changes associated with SDS-induced phenol oxidase activation

The enzymatic activity of phenoloxidase is assayed routinely in the presence of SDS. Similar assay conditions elicit phenoloxidase activity in another type 3 copper protein, namely hemocyanin, which normally functions as an oxygen carrier. The nature of the conformational changes induced in type 3 copper proteins by the denaturant SDS is unknown. This comparative study demonstrates that arthropod hemocyanins can be converted from being an oxygen carrier to a form which exhibits phenoloxidase activity by incubation with SDS, with accompanying changes in secondary and tertiary structure. Structural characterisation, using various biophysical methods, suggests that the micellar form of SDS is required to induce optimal conformational transitions in the protein which may result in opening a channel to the di-copper centre allowing bulky phenolic substrates access to the catalytic site.     

Substrate transport activation is mediated through second periplasmic loop of transmembrane protein MalF in maltose transport complex of Escherichia coli

In a recent study we described the second periplasmic loop P2 of the transmembrane protein MalF (MalF-P2) of the maltose ATP-binding cassette transporter (MalFGK(2)-E) as an important element in the recognition of substrate by the maltose-binding protein MalE. In this study, we focus on MalE and find that MalE undergoes a structural rearrangement after addition of MalF-P2. Analysis of residual dipolar couplings (RDCs) shows that binding of MalF-P2 induces a semiopen state of MalE in the presence and absence of maltose, whereas maltose is retained in the binding pocket. These data are in agreement with paramagnetic relaxation enhancement experiments. After addition of MalF-P2, an increased solvent accessibility for residues in the vicinity of the maltose-binding site of MalE is observed. MalF-P2 is thus not only responsible for substrate recognition, but also directly involved in activation of substrate transport. The observation that substrate-bound and substrate-free MalE in the presence of MalF-P2 adopts a similar semiopen state hints at the origin of the futile ATP hydrolysis of MalFGK(2)-E.     

ATP induces conformational changes of periplasmic loop regions of the maltose ATP-binding cassette transporter

We have studied cofactor-induced conformational changes of the maltose ATP-binding cassette transporter by employing limited proteolysis in detergent solution. The transport complex consists of one copy each of the transmembrane subunits, MalF and MalG, and of two copies of the nucleotide-binding subunit, MalK. Transport activity further requires the periplasmic maltose-binding protein, MalE. Binding of ATP to the MalK subunits increased the susceptibility of two tryptic cleavage sites in the periplasmic loops P2 of MalF and P1 of MalG, respectively. Lys(262) of MalF and Arg(73) of MalG were identified as probable cleavage sites, resulting in two N-terminal peptide fragments of 29 and 8 kDa, respectively. Trapping the complex in the transition state by vanadate further stabilized the fragments. In contrast, the tryptic cleavage profile of MalK remained largely unchanged. ATP-induced conformational changes of MalF-P2 and MalG-P1 were supported by fluorescence spectroscopy of complex variants labeled with 2-(4'-maleimidoanilino)naphthalene-6-sulfonic acid. Limited proteolysis was subsequently used as a tool to study the consequences of mutations on the transport cycle. The results suggest that complex variants exhibiting a binding protein-independent phenotype (MalF500) or containing a mutation that affects the "catalytic carboxylate" (MalKE159Q) reside in a transition state-like conformation. A similar conclusion was drawn for a complex containing a replacement of MalKQ140 in the signature sequence by leucine, whereas substitution of lysine for Gln(140) appears to lock the transport complex in the ground state. Together, our data provide the first evidence for conformational changes of the transmembrane subunits of an ATP-binding cassette import system upon binding of ATP.     

Analyzing conformational changes in the transport cycle of EmrE

The small multidrug resistance transporters represent a unique model system for studying the mechanism of secondary active transport and membrane protein evolution. However, this seemingly simple protein has been highly controversial. Recent studies have provided experimental evidence that EmrE exists as an asymmetric dimer that exchanges between identical inward-facing and outward-facing states. Re-examination of the published literature in light of these findings fills in many details of the microscopic steps in the transport cycle. Future work will need to examine how the symmetry observed in vitro affects EmrE function in the asymmetric environment of its native Escherichia coli membrane.     

Calmodulin conformational changes in the activation of protein kinases

The conformation of Ca2+/calmodulin changes from extended when free in solution to compact when bound in peptide complexes. The extent and kinetics of calmodulin compaction in association with Ca2+/calmodulin-dependent protein kinases (CaMKs), as well as target peptides, were investigated by fluorescence, resonance energy transfer and stopped-flow kinetics. Compaction of Ca2+/calmodulin labelled with resonance energy-transfer probes in association with target peptides was rapid (>350 s(-1)). With the target enzymes smooth-muscle myosin light-chain kinase, CaMKIV and CaMKII, the rates of calmodulin compaction were one-two orders of magnitude lower compared with those of the peptides and in the case of alphaCaMKII, ATP binding and Thr(286) auto-phosphorylation were required for calmodulin compaction. In the absence of nucleotides, Ca2+/calmodulin bound to alphaCaMKII in extended conformations, initially probably attached by one lobe only. Kinetic data suggest that in the activation process of Ca2+/calmodulin-dependent protein kinases, productive as well as unproductive complexes are formed. The formation of productive complexes with Ca2+/calmodulin thus may determine the rate of activation.     

Evidence for multiple pathways in the assembly of the Escherichia coli maltose transport complex

We used the maltose transport complex MalFGK2 of the Escherichia coli cytoplasmic membrane as a model for the study of the assembly of hetero-oligomeric membrane protein complexes. Analysis of other membrane protein complexes has led to a general model in which a unique, ordered pathway is followed from subunit monomers to a final oligomeric structure. In contrast, the studies reported here point to a fundamentally different mode for assembly of this transporter. Using co-immunoprecipitation and quantification of interacting partners, we found that all subunits of the maltose transport complex efficiently form heteromeric complexes in vivo. The pairwise complexes were stable over time, suggesting that they all represent assembly intermediates for the final MalFGK2 transporter. These results indicate that several paths can lead to assembly of this oligomer. We also characterized MalF and MalG mutants that caused reduced association between some or all of the subunits of the complex with this assay. The mutant analysis highlights some important motifs for subunit contacts and suggests that the promiscuous interactions between these Mal proteins contribute to the efficiency of complex assembly. The behaviors of the wild type and mutant proteins in the co-immunoprecipitations support a model of multiple assembly pathways for this complex.     

Ribosome conformational changes associated with protein S6 phosphorylation

The relative accessibility of rat liver ribosomal proteins to reductive methylation was examined using ribosomes with unphosphorylated, and extensively phosphorylated S6. Comparison of the results indicated that proteins S3, S4, S7, and S23/24 of the small subunit, and proteins L9, L10, L12, L18, L27, L34, and L36 are involved in a ribosomal conformational change.     

Vanadate-induced trapping of nucleotides by purified maltose transport complex requires ATP hydrolysis

The maltose transport system in Escherichia coli is a member of the ATP-binding cassette superfamily of transporters that is defined by the presence of two nucleotide-binding domains or subunits and two transmembrane regions. The bacterial import systems are unique in that they require a periplasmic substrate-binding protein to stimulate the ATPase activity of the transport complex and initiate the transport process. Upon stimulation by maltose-binding protein, the intact MalFGK(2) transport complex hydrolyzes ATP with positive cooperativity, suggesting that the two nucleotide-binding MalK subunits interact to couple ATP hydrolysis to transport. The ATPase activity of the intact transport complex is inhibited by vanadate. In this study, we investigated the mechanism of inhibition by vanadate and found that incubation of the transport complex with MgATP and vanadate results in the formation of a stably inhibited species containing tightly bound ADP that persists after free vanadate and nucleotide are removed from the solution. The inhibited species does not form in the absence of MgCl(2) or of maltose-binding protein, and ADP or another nonhydrolyzable analogue does not substitute for ATP. Taken together, these data conclusively show that ATP hydrolysis must precede the formation of the vanadate-inhibited species in this system and implicate a role for a high-energy, ADP-bound intermediate in the transport cycle. Transport complexes containing a mutation in a single MalK subunit are still inhibited by vanadate during steady-state hydrolysis; however, a stably inhibited species does not form. ATP hydrolysis is therefore necessary, but not sufficient, for vanadate-induced nucleotide trapping.     

Active-site conformational changes associated with hydride transfer in proton-translocating transhydrogenase

Transhydrogenase couples the redox (hydride-transfer) reaction between NAD(H) and NADP(H) to proton translocation across a membrane. The redox reaction is catalyzed at the interface between two components (dI and dIII) which protrude from the membrane. A complex formed from recombinant dI and dIII (the dI(2)dIII(1) complex) from Rhodospirillum rubrum transhydrogenase catalyzes fast single-turnover hydride transfer between bound nucleotides. In this report we describe three new crystal structures of the dI(2)dIII(1) complex in different nucleotide-bound forms. The structures reveal an asymmetry in nucleotide binding that complements results from solution studies and supports the notion that intact transhydrogenase functions by an alternating site mechanism. In one structure, the redox site is occupied by NADH (on dI) and NADPH (on dIII). The dihydronicotinamide rings take up positions which may approximate to the ground state for hydride transfer: the redox-active C4(N) atoms are separated by only 3.6 A, and the perceived reaction stereochemistry matches that observed experimentally. The NADH conformation is different in the two dI polypeptides of this form of the dI(2)dIII(1) complex. Comparisons between a number of X-ray structures show that a conformational change in the NADH is driven by relative movement of the two domains which comprise dI. It is suggested that an equivalent conformational change in the intact enzyme is important in gating the hydride-transfer reaction. The observed nucleotide conformational change in the dI(2)dIII(1) complex is accompanied by rearrangements in the orientation of local amino acid side chains which may be responsible for sealing the site from the solvent and polarizing hydride transfer.     

Anionic lipid-induced conformational changes in human phagocyte flavocytochrome b precede assembly and activation of the NADPH oxidase complex

Phagocyte NADPH oxidases generate superoxide at high rates in defense against infectious agents, a process regulated by second messenger anionic lipids using incompletely understood mechanisms. We reconstituted the catalytic core of the human neutrophil NADPH oxidase, flavocytochrome b (Cyt b) in 99% phosphatidylcholine vesicles in order to correlate anionic lipid-dependent conformational changes in membrane-bound Cyt b and oxidase activity. The anionic lipid 10:0 phosphatidic acid (10:0 PA) specifically induced conformational changes in Cyt b as measured by a combination of fluorescence resonance energy transfer methods and size exclusion chromatography. The fluorescence lifetime of a complex between Cyt b and Cascade Blue-derivatized anti-p22(phox) antibody (CCB-CS9), increased after exposure to 10:PA by ～50% of the change observed when the complex is dissociated, indicating a structural rearrangement of p22(phox) and/or the Cyt b heme prosthetic groups. Half of the quenching relaxation occurred at 10:0 PA concentrations permissive to less than 10% full NADPH oxidase activity, but saturated near the saturation in activity in a matched cell-free oxidase assay. We conclude that anionic lipids modulate the conformation of Cyt b in the membrane and suggest they may serve to modulate the structure of Cyt b as a control mechanism for the NADPH oxidase.     

Nanosecond pulse fluorometry of conformational change in phenylalanine hydroxylase associated with activation

Conformational change in rat liver phenylalanine hydroxylase associated with activation by phenylalanine or N-(1-anilinonaphth-4-yl)maleimide was investigated by measuring fluorescence spectra and fluorescence lifetimes of tryptophanyl residues as well as the probe fluorophore conjugated with SH groups of the hydroxylase. The fluorescence spectrum of tryptophan exhibited its maximum at 342 nm. It shifted by 8 nm toward longer wavelength accompanied by an increase in its intensity, by preincubation with 1 mM phenylalanine. The fluorescence intensity of tryptophan increased by 36% upon the activation. On the other hand, the binding of (6R)-L-erythro-tetrahydrobiopterin, a natural cofactor of the enzyme, induced a decrease in the fluorescence intensity by 79% without a shift of the maximum wavelength. The fluorescence lifetime of tryptophan of phenylalanine hydroxylase exhibited two components with lifetimes of 1.7 and 4.1 ns. The values of the lifetimes changed to 1.4 and 5.6 ns, respectively, upon the activation. It is considered that the change in the longer lifetime is correlated with the shift of the emission peak upon the activation. The values of both the lifetimes decreased to 0.64 and 3.6 ns upon the binding of (6R)-L-erythro-tetrahydrobiopterin, which is coincident with the decrease in the fluorescence intensity. Conjugation of N-(1-anilinonaphth-4-yl)maleimide with SH of phenylalanine hydroxylase brought about a decrease in both the fluorescence intensity and the value of the shorter lifetime of the tryptophanyl residues, while the longer lifetime remained unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorometric studies on conformational changes in tropomyosin associated with depolymerization.

Spectrofluorometric studies on the conformational changes in tropomyosin associated with depolymerization of the molecules were carried out using 1-anilino-8-naphthalene sulfonate (ANS). When ANS-probed tropomyosin was depolymerized to its monomer, the fluorescence intensity markedly increased, with a decrease in fluorescence polarization. On the other hand, the emission maxima of the ANS-tropomyosin complexes of both forms were the same. The temperature dependence of the polarization of the complexes at various KCl concentrations suggested that the segmental motion of a moiety containing the fluorophore was considerably activated by depolymerization of tropomyosin. In the polymerized and oligomeric forms, a thermal transition in the polarization was observed with a transition temperature of 30 degrees C. Titration curves of tropomyosin with ANS showed simple saturation kinetics with both monomer and polymer, and the apparent dissociation constants were estimated to be 9.93 X 10(-5) M (monomer) and 7.43 X 10(-5) M (polymer). On the other hand, the number of the ANS-binding sites increased from 0.5 to 2.0 per tropomyosin monomer on depolymerization of the molecules. Based on these results, the conformational state of tropomyosin in the polymerized form is discussed.     

Binding of the anticonvulsant drug lamotrigine and the neurotoxin batrachotoxin to voltage-gated sodium channels induces conformational changes associated with block and steady-state activation

Voltage-gated sodium channels are dynamic membrane proteins characterized by rapid conformational changes that switch the molecule between closed resting, activated, and inactivated states. Sodium channels are specifically blocked by the anticonvulsant drug lamotrigine, which preferentially binds to the channel pore in the inactivated open state. Batrachotoxin is a lipid-soluble alkaloid that causes steady-state activation and binds in the inner pore of the sodium channel with overlapping but distinct molecular determinants from those of lamotrigine. Using circular dichroism spectroscopy on purified voltage-gated sodium channels from Electrophorus electricus, the secondary structures associated with the mixture of states present at equilibrium in the absence of these ligands were compared with specific stabilized states in their presence. As the channel shifts to open states, there appears to be a significant change in secondary structure to a more alpha-helical conformation. The observed changes are consistent with increased order involving the S6 segments that form the pore, the domain III-IV linker, and the P-loops that form the outer pore and selectivity filter. A molecular model has been constructed for the sodium channel based on its homology with the pore-forming regions of bacterial potassium channels, and automated docking of the crystal structure of lamotrigine with this model produces a structure in which the close contacts of the drug are with the residues previously identified by mutational studies as forming the binding site for this drug.     

Photochemical intermediates of Arabidopsis phototropin 2 LOV domains associated with conformational changes

The photochemical reactions of Arabidopsis phototropin 2 light- oxygen-voltage domain 2 (LOV2) with the linker region (LOV2-linker), without the linker (LOV2), and LOV1 were studied using the time-resolved transient grating (TG) and transient lens (TrL) methods. Although the absorption spectra did not change after the formation of the adduct species, a small volume expansion process with a time constant of 9 ms was observed for LOV2. For the LOV2-linker, at 293 K, a volume contraction process with a time constant of 140 mus was observed in addition to a volume expansion process with 9 ms and the diffusion coefficient change with 2 ms. The reaction intermediate species were characterized on the basis of their thermodynamic properties, such as changes in enthalpy, thermal expansion, and heat capacity. For the first intermediate (S(390)), the values of these properties were similar to those of the ground state for both LOV2 and LOV2-linker. A relatively large thermal expansion volume (0.09 cm(3)mol(-1)K(-1)) and a positive heat capacity change (4.7 kJ mol(-1)K(-1)) were detected for the intermediates of LOV2-linker. These characteristic features were interpreted in terms of structural fluctuation and exposure of hydrophobic residues in the linker domain, respectively. The enthalpy change of S(390) of the LOV1 domain was significantly greater than changes for the LOV2 or LOV2-linker samples. Data from this study support a major conformational change of the linker region in the photochemical reaction of phototropin.     

Nucleotide-dependent conformational changes in the DnaA-like core of the origin recognition complex

Structural details of initiator proteins for DNA replication have provided clues to the molecular events in this process. EM reconstructions of the Drosophila melanogaster origin recognition complex (ORC) reveal nucleotide-dependent conformational changes in the core of the complex. All five AAA+ domains in ORC contain a conserved structural element that, in DnaA, promotes formation of a right-handed helix, indicating that helical AAA+ substructures may be a feature of all initiators. A DnaA helical pentamer can be docked into ORC, and the location of Orc5 uniquely positions this core. The results suggest that ATP-dependent conformational changes observed in ORC derive from reorientation of the AAA+ domains. By analogy to the DNA-wrapping activity of DnaA, we posit that ORC together with Cdc6 prepares origin DNA for helicase loading through mechanisms related to the established pathway of prokaryotes.     

Structural analysis of coxsackievirus A7 reveals conformational changes associated with uncoating

Coxsackievirus A7 (CAV7) is a rarely detected and poorly characterized serotype of the Enterovirus species Human enterovirus A (HEV-A) within the Picornaviridae family. The CAV7-USSR strain has caused polio-like epidemics and was originally thought to represent the fourth poliovirus type, but later evidence linked this strain to the CAV7-Parker prototype. Another isolate, CAV7-275/58, was also serologically similar to Parker but was noninfectious in a mouse model. Sequencing of the genomic region encoding the capsid proteins of the USSR and 275/58 strains and subsequent comparison with the corresponding amino acid sequences of the Parker strain revealed that the Parker and USSR strains are nearly identical, while the 275/58 strain is more distant. Using electron cryomicroscopy and three-dimensional image reconstruction, the structures of the CAV7-USSR virion and empty capsid were resolved to 8.2-Å and 6.1-Å resolutions, respectively. This is one of the first detailed structural analyses of the HEV-A species. Using homology modeling, reconstruction segmentation, and flexible fitting, we constructed a pseudoatomic T = 1 (pseudo T = 3) model incorporating the three major capsid proteins (VP1 to VP3), addressed the conformational changes of the capsid and its constituent viral proteins occurring during RNA release, and mapped the capsid proteins'' variable regions to the structure. During uncoating, VP4 and RNA are released analogously to poliovirus 1, the interfaces of VP2 and VP3 are rearranged, and VP1 rotates. Variable regions in the capsid proteins were predicted to map mainly to the surface of VP1 and are thus likely to affect the tropism and pathogenicity of CAV7.