Asymmetric Conformational Changes Research Articles

Asymmetric contribution of a selectivity filter gate in triggering inactivation of CaV1.3 channels.

Voltage-dependent and Ca2+-dependent inactivation (VDI and CDI, respectively) of CaV channels are two biologically consequential feedback mechanisms that fine-tune Ca2+ entry into neurons and cardiomyocytes. Although known to be initiated by distinct molecular events, how these processes obstruct conduction through the channel pore remains poorly defined. Here, focusing on ultrahighly conserved tryptophan residues in the interdomain interfaces near the selectivity filter of CaV1.3, we demonstrate a critical role for asymmetric conformational changes in mediating VDI and CDI. Specifically, mutagenesis of the domain III-IV interface, but not others, enhanced VDI. Molecular dynamics simulations demonstrate that mutations in distinct selectivity filter interfaces differentially impact conformational flexibility. Furthermore, mutations in distinct domains preferentially disrupt CDI mediated by the N- versus C-lobes of CaM, thus uncovering a scheme of structural bifurcation of CaM signaling. These findings highlight the fundamental importance of the asymmetric arrangement of the pseudotetrameric CaV pore domain for feedback inhibition.

Structure of the Anthrax Protective Antigen D425A Dominant Negative Mutant Reveals a Stalled Intermediate State of Pore Maturation

The tripartite protein complex produced by anthrax bacteria (Bacillus anthracis) is a member of the AB family of β-barrel pore-forming toxins. The protective antigen (PA) component forms an oligomeric prepore that assembles on the host cell surface and serves as a scaffold for binding of lethal and edema factors. Following endocytosis, the acidic environment of the late endosome triggers a pH-induced conformational rearrangement to promote maturation of the PA prepore to a functional, membrane spanning pore that facilitates delivery of lethal and edema factors to the cytosol of the infected host. Here, we show that the dominant-negative D425A mutant of PA stalls anthrax pore maturation in an intermediate state at acidic pH. Our 2.7 Å cryo-EM structure of the intermediate state reveals structural rearrangements that involve constriction of the oligomeric pore combined with an intramolecular dissociation of the pore-forming module. In addition to defining the early stages of anthrax pore maturation, the structure identifies asymmetric conformational changes in the oligomeric pore that are influenced by the precise configuration of adjacent protomers.

Structural Insights into Substrate Recognition and Processing by the 20S Proteasome.

Four decades of proteasome research have yielded extensive information on ubiquitin-dependent proteolysis. The archetype of proteasomes is a 20S barrel-shaped complex that does not rely on ubiquitin as a degradation signal but can degrade substrates with a considerable unstructured stretch. Since roughly half of all proteasomes in most eukaryotic cells are free 20S complexes, ubiquitin-independent protein degradation may coexist with ubiquitin-dependent degradation by the highly regulated 26S proteasome. This article reviews recent advances in our understanding of the biochemical and structural features that underlie the proteolytic mechanism of 20S proteasomes. The two outer α-rings of 20S proteasomes provide a number of potential docking sites for loosely folded polypeptides. The binding of a substrate can induce asymmetric conformational changes, trigger gate opening, and initiate its own degradation through a protease-driven translocation mechanism. Consequently, the substrate translocates through two additional narrow apertures augmented by the β-catalytic active sites. The overall pulling force through the two annuli results in a protease-like unfolding of the substrate and subsequent proteolysis in the catalytic chamber. Although both proteasomes contain identical β-catalytic active sites, the differential translocation mechanisms yield distinct peptide products. Nonoverlapping substrate repertoires and product outcomes rationalize cohabitation of both proteasome complexes in cells.

Exploring Subunit Communication of Malate Dehydrogenase through Interface Point‐ Mutations

Malate Dehydrogenase (MDH) catalyzes the oxidation/reduction of malate/oxaloacetate through coupling with NAD+/NADH conversion, a key area in cellular metabolism and has been shown to be regulated by citrate. MDH is a homo‐dimer and x‐ray structures show a flexible loop, which upon substrate or citrate binding induces a closed conformational in one subunit, while the other remains open. This implies an as of yet unknown means of communication between the two subunits to convey the open/closed loop response. Concurrent work in the Bell Lab has shown two mutations, S266A & L269A, located in the active site and interface regions display significant changes in kinetic parameters. To further probe these regions and assess individual residue contributions to subunit communication several additional point mutants (I88A, K261M/Q, S270A/C, T255E/V) located between the active site and the subunit interface have been constructed, expressed, purified and characterized. The I88A mutant protein has a 10,000 fold decrease in Vmax while that of the T255E mutant is decreased 1,000 fold. Km for NADH is significantly increased in the I88A mutant while that for T255E remains unchanged. This suggests that decreased affinity for NADH in the I88A mutant results from disruption of the subunit interface., I88 is thought to interact with L269 on the opposite chain, while T255 may be involved in oxaloacetate/malate/citrate binding at the active site. This data is consistent with predicted effects of interface and active site mutations and contributes to a detailed understanding of a predicted inter‐subunit relay system to induce asymmetric conformational changes and ligand binding in this homo‐dimeric enzyme.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Studying the recognition mechanism of TcaR and ssDNA using molecular dynamic simulations

The transcription regulator teicoplanin-associate locus regulator (TcaR) plays a vital role in interfering with ssDNA replication and resisting ssDNA phage invasion. Although recent studies demonstrated that TcaR had strong interaction with ssDNA, the dynamics and interaction mechanism of dimeric TcaR bound to ssDNA have not been rationalized at the atomic level. In our study, MD simulations combined with MM-GB/SA calculations were employed to study recognition mechanism between TcaR and ssDNA. The results illuminate that electrostatic interaction is the main driving force for the binding process. We put forward that six anchoring residues (Arg70, Arg71, Ser188, Gln191, Arg221 and Arg222) play a vital role in stabilizing the ssDNA by forming strong hydrogen bond and salt bridge interactions. TcaR undergoes the asymmetric conformational changes at the wHTH domain upon binding to ssDNA. This may be attributed to the changing of electrostatic potential, enhanced contacts and salt bridge interaction. The present study provides new insights into the recognition mechanism of TcaR bound to ssDNA, which could contribute to understanding about the multiple TcaR functions in staphylococci enrich our understanding of MarR family.

Autophagy in MCF-7 cancer cells induced by copper complexes.

Autophagy plays an important role in cancer cells. Targeting autophagy in cancer can provide new opportunities for drug development. In this study we tested four Schiff base Cu(II) complexes against human breast cancer cells (MCF-7) and human non-cancerous cells (HEK-293T). We have tested their cytotoxic effect by evaluating IC50 using MTT test. To detect morphological changes of the actin fibers we have used fluorescent microscopy. To determine the type of cell death we used electrophoretic analysis and western blot analysis (protein LC3). IC50 values of the complexes increased with time of their influence, indicating acquired resistance of MCF-7 to the complexes. Healthy cells HEK-293T were not sensitive to the Cu(II) complexes. Compared with the control cells (cells without Cu(II) complexes) which were without morphological changes of actin fibers, Cu(II) complexes induced condensation and asymmetric conformational changes in actin filaments. To examine the type of cell death induced by the Cu(II) complexes we treated MCF-7 cells with Cu(II) complexes (1, 10, 50 and 100μmol/L) during a 72h incubation period. By electrophoresis we have not detected any DNA fragmentation. To determine whether Cu(II) complexes induced autophagy or necrotic cell death we used the western blot analysis. MCF-7 cells influenced with tested Cu(II) complexes produced LC3 protein after their 72h incubation indicating autophagy in MCF-7 cancer cells. Tested Schiff base copper (II) complexes have antiproliferative activity against cancer cells but not against healthy cells. They have induced autophagy in the cancer cell line MCF-7.

Cofilin-induced unidirectional cooperative conformational changes in actin filaments revealed by high-speed atomic force microscopy.

High-speed atomic force microscopy was employed to observe structural changes in actin filaments induced by cofilin binding. Consistent with previous electron and fluorescence microscopic studies, cofilin formed clusters along actin filaments, where the filaments were 2-nm thicker and the helical pitch was ~25% shorter, compared to control filaments. Interestingly, the shortened helical pitch was propagated to the neighboring bare zone on the pointed-end side of the cluster, while the pitch on the barbed-end side was similar to the control. Thus, cofilin clusters induce distinctively asymmetric conformational changes in filaments. Consistent with the idea that cofilin favors actin structures with a shorter helical pitch, cofilin clusters grew unidirectionally toward the pointed-end of the filament. Severing was often observed near the boundaries between bare zones and clusters, but not necessarily at the boundaries.

Activation of the B Cell Receptor Leads to Increased Membrane Proximity of the Igα Cytoplasmic Domain

Binding of antigen to the B cell receptor (BCR) induces conformational changes in BCR's cytoplasmic domains that are concomitant with phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs). Recently, reversible folding of the CD3ε and ξ chain ITAMs into the plasma membrane has been suggested to regulate T cell receptor signaling. Here we show that the Igα and Igβ cytoplasmic domains of the BCR do not associate with plasma membrane in resting B cells. However, antigen binding and ITAM phosphorylation specifically increased membrane proximity of Igα, but not Igβ. Thus, BCR activation is accompanied by asymmetric conformational changes, possibly promoting the binding of Igα and Igβ to differently localized signaling complexes.

Molecular modeling studies give hint for the existence of a symmetric hβ2R-Gαβγ-homodimer

Several experimental studies suggest that GPCR dimers or oligomers may play an important role in signal transduction. In 2011 the crystal structure of a hβ2R-Gαβγ-complex was published and crystal structures of GPCR dimers are known. But until now, no crystal structure of a GPCR dimer including the Gαβγ-complex is available. In order to obtain detailed insights into interactions within hβ2R dimers including the Gαβγ-complex we performed a potential-energy-surface scan in order to identify favored asymmetric and symmetric hβ2R-Gαβγ-homodimers. This potential energy surface scan suggests, besides the existence of asymmetric dimers, the existence of a symmetric hβ2R-Gαβγ-homodimer with a TM I/VII-contact. A subsequent 20 ns MD simulation of the symmetric homodimer revealed large asymmetric conformational changes of both hβ2Rs, especially regarding TM VII and the interaction network between Asp(2.50), Val(7.44), Ser(7.46) and Tyr(7.43). Since similar conformational changes were not observed during the molecular dynamic simulation of the monomeric hβ2R-Gαβγ-complex, it may be suggested that the conformational changes in the symmetric homodimer are related to the presence of the second hβ2R-Gαβγ-complex. Due to the limitations of simulation time, conformational changes within a time scale of μs or ms may of course not be observed. However, the detected conformational changes, especially in TM VII, correspond to minima on the potential energy surface and thus, this study gives new insights into GPCR dimers on molecular level and furthermore, gives suggestions for site-directed mutagenesis studies.

A mechanical model for the role of the neck linker during kinesin stepping and gating

In this paper, considering the different elastic properties in the attached head and the free head, we propose a physical model, in which the free head undergoes a diffusive search in an entropic spring potential formed by undocking the neck linker, and there are asymmetric conformational changes in the attached head formed by docking the neck linker to support the load force and bias the diffusive search to the forward direction. By performing the thermodynamic analysis, we obtain the free energy difference between forward and backward binding sites. And using the Fokker-Planck equation with two absorbing boundaries, we obtain the dependence of the ratio of forward to backward steps on the backward force. Also, within the Michaelis-Menten model, we investigate the dependence of the velocity-load relationship on the effective length of the junction between the two heads. The results show that our model can provide a physical understanding for the processive movement of kinesin.

Structure–neurotoxicity relationships of amyloid β-protein oligomers

Amyloid beta-protein (Abeta) oligomers may be the proximate neurotoxins in Alzheimer's disease (AD). "Oligomer" is an ill-defined term because many kinds have been reported and they often exist in rapid equilibrium with monomers and higher-order assemblies. We report here results of studies in which specific oligomers have been stabilized structurally, fractionated in pure form, and then studied by using a combination of CD spectroscopy, Thioflavin T fluorescence, EM, atomic force microscopy (AFM), and neurotoxicity assays. Abeta monomers were largely unstructured, but oligomers exhibited order-dependent increases in beta-sheet content. EM and AFM data suggest that dimerization and subsequent monomer addition are processes in which significant and asymmetric monomer conformational changes occur. Oligomer secondary structure and order correlated directly with fibril nucleation activity. Neurotoxic activity increased disproportionately (order dependence >1) with oligomer order. The structure-activity correlations reported here significantly extend our understanding of the conformational dynamics, structure, and relative toxicity of pure Abeta oligomers of specific order.

G Protein Activation by the Leukotriene B4 Receptor Dimer: EVIDENCE FOR AN ABSENCE OF TRANS-ACTIVATION

There is compelling evidence that G protein-coupled receptors exist as homo- and heterodimers, but the way these assemblies function at the molecular level remains unclear. We used here the purified leukotriene B(4) receptor BLT1 stabilized in its dimeric state to analyze how a receptor dimer activates G proteins. For this, we produced heterodimers between the wild-type BLT1 and a BLT1/ALXR chimera. The latter is no longer activated by leukotriene B(4) but is still activated by ALXR agonists. In this heterodimer, agonist binding to either one of the two protomers induced asymmetric conformational changes within the receptor dimer. Of importance, no G protein activation was observed when using a dimer where the ligand-loaded protomer was not able to trigger GDP/GTP exchange due to specific mutations in its third intracellular loop, establishing that the conformation of the agonist-free protomer is not competent for G protein activation. Taken together, these data indicate that although ligand binding to one protomer in the heterodimer is associated with cross-conformational changes, a trans-activation mechanism where the ligand-free subunit would trigger GDP/GTP exchange cannot be considered in this case for G protein activation. This observation sheds light into the way GPCR dimers, in particular heterodimers, could activate their cognate G proteins.

Direct Visualization of Asymmetric Adenine Nucleotide-Induced Conformational Changes in MutLα

MutL alpha, the heterodimeric eukaryotic MutL homolog, is required for DNA mismatch repair (MMR) in vivo. It has been suggested that conformational changes, modulated by adenine nucleotides, mediate the interactions of MutL alpha with other proteins in the MMR pathway, coordinating the recognition of DNA mismatches by MutS alpha and the activation of MutL alpha with the downstream events that lead to repair. Thus far, the only evidence for these conformational changes has come from X-ray crystallography of isolated domains, indirect biochemical analyses, and comparison to other members of the GHL ATPase family to which MutL alpha belongs. Using atomic force microscopy (AFM), coupled with biochemical techniques, we demonstrate that adenine nucleotides induce large asymmetric conformational changes in full-length yeast and human MutL alpha and that these changes are associated with significant increases in secondary structure. These data reveal an ATPase cycle in which sequential nucleotide binding, hydrolysis, and release modulate the conformational states of MutL alpha.

Asymmetric conformational changes in a GPCR dimer controlled by G-proteins

G-protein-coupled receptors (GPCRs) are key players in cell communication. Although long considered as monomeric, it now appears that these heptahelical proteins can form homo- or heterodimers. Here, we analyzed the conformational changes in each subunit of a receptor dimer resulting from agonist binding to either one or both subunits by measuring the fluorescent properties of a leukotriene B(4) receptor dimer with a single 5-hydroxytryptophan-labeled protomer. We show that a receptor dimer with only a single agonist-occupied subunit can trigger G-protein activation. We also show that the two subunits of the receptor dimer in the G-protein-coupled state differ in their conformation, even when both are liganded by the agonist. No such asymmetric conformational changes are observed in the absence of G-protein, indicating that the interaction of the G-protein with the receptor dimer brings specific constraints that prevent a symmetric functioning of this dimer. These data open new options for the differential signaling properties of GPCR dimers.

Asymmetric recognition of nonconsensus AP-1 sites by Fos-Jun and Jun-Jun influences transcriptional cooperativity with NFAT1.

Many regulatory elements in eukaryotic promoters do not correspond to optimal recognition sequences for the transcription factors that regulate promoter function by binding to the elements. The sequence of the binding site may influence the structural and functional properties of regulatory protein complexes. Fos-Jun heterodimers were found to bind nonconsensus AP-1 sites in a preferred orientation. Oriented Fos-Jun heterodimer binding was attributed to nonidentical recognition of the two half-sites by Fos and Jun. Jun bound preferentially to the consensus half-site, whereas Fos was able to bind nonconsensus half-sites. The orientation of heterodimer binding affected the transcriptional cooperativity of Fos-Jun-NFAT1 complexes at composite regulatory elements in mammalian cells. Jun dimerization with Fos versus ATF2 caused it to bind opposite half-sites at nonconsensus AP-1 elements. Similarly, ATF2 bound to opposite half-sites in Fos-ATF2-NFAT1 and ATF2-Jun-NFAT1 complexes. The orientations of nonconsensus AP-1 sites within composite regulatory elements affected the cooperativity of Fos-Jun as well as Jun-Jun binding with NFAT1. Since Jun homodimers cannot bind to AP-1 sites in a preferred orientation, the effects of the orientations of nonconsensus AP-1 sites on the stabilities of Jun-Jun-NFAT1 complexes are likely to be due to asymmetric conformational changes in the two subunits of the homodimer. Nonconsensus AP-1 site orientation also affected the synergy of transcription activation between Jun homodimers and NFAT1 at composite regulatory elements. The asymmetric recognition of nonconsensus AP-1 sites can therefore influence the transcriptional activities of Fos and Jun both through effects on the orientation of heterodimer binding and through differential conformational changes in the two subunits of the dimer.

Mitochondrial Lon of Saccharomyces cerevisiae is a ring-shaped protease with seven flexible subunits.

Lon (or La) is a soluble, homooligomeric ATP-dependent protease. Mass determination and cryoelectron microscopy of pure mitochondrial Lon from Saccharomyces cerevisiae identify Lon as a flexible ring-shaped heptamer. In the presence of ATP or 5'-adenylylimidodiphosphate, most of the rings are symmetric and resemble other ATP-driven machines that mediate folding and degradation of proteins. In the absence of nucleotides, most of the rings are distorted, with two adjacent subunits forming leg-like protrusions. These results suggest that asymmetric conformational changes serve to power processive unfolding and translocation of substrates to the active site of the Lon protease.

Structure and function in GroEL-mediated protein folding.

Recent structural and biochemical investigations have come together to allow a better understanding of the mechanism of chaperonin (GroEL, Hsp60)-mediated protein folding, the final step in the accurate expression of genetic information. Major, asymmetric conformational changes in the GroEL double toroid accompany binding of ATP and the cochaperonin GroES. When a nonnative polypeptide, bound to one of the GroEL rings, is encapsulated by GroES to form a cis ternary complex, these changes drive the polypeptide into the sequestered cavity and initiate its folding. ATP hydrolysis in the cis ring primes release of the products, and ATP binding in the trans ring then disrupts the cis complex. This process allows the polypeptide to achieve its final native state, if folding was completed, or to recycle to another chaperonin molecule, if the folding process did not result in a form committed to the native state.