Nucleotide Binding Cleft Research Articles

Preferential binding of ADP-bound mitochondrial HSP70 to the nucleotide exchange factor GRPEL1 over GRPEL2.

Human nucleotide exchange factors GRPEL1 and GRPEL2 play pivotal roles in the ADP-ATP exchange within the protein folding cycle of mitochondrial HSP70 (mtHSP70), a crucial chaperone facilitating protein import into the mitochondrial matrix. Studies in human cells and mice have indicated that while GRPEL1 serves as an essential co-chaperone for mtHSP70, GRPEL2 has a role regulated by stress. However, the precise structural and biochemical mechanisms underlying the distinct functions of the GRPEL proteins have remained elusive. In our study, we present evidence revealing that ADP-bound mtHSP70 exhibits remarkably higher affinity for GRPEL1 compared to GRPEL2, with the latter experiencing a notable decrease in affinity upon ADP binding. Additionally, Pi assay showed that GRPEL1, but not GRPEL2, enhanced the ATPase activity of mtHSP70. Utilizing Alphafold modeling, we propose that the interaction between GRPEL1 and mtHSP70 can induce the opening of the nucleotide binding cleft of the chaperone, thereby facilitating the release of ADP, whereas GRPEL2 lacks this capability. Additionally, our findings suggest that the redox-regulated Cys87 residue in GRPEL2 does not play a role in dimerization but rather reduces its affinity for mtHSP70. Our findings on the structural and functional disparities between GRPEL1 and GRPEL2 may have implications for mitochondrial protein folding and import processes under varying cellular conditions.

New insights into the structure and function of the complex between the Escherichia coli Hsp70, DnaK, and its nucleotide-exchange factor, GrpE

The 70 kDa heat shock proteins (Hsp70s) play a pivotal role in many cellular functions using allosteric communication between their nucleotide-binding domain (NBD) and substrate-binding domain, mediated by an interdomain linker, to modulate their affinity for protein clients. Critical to modulation of the Hsp70 allosteric cycle, nucleotide-exchange factors (NEFs) act by a conserved mechanism involving binding to the ADP-bound NBD and opening of the nucleotide-binding cleft to accelerate the release of ADP and binding of ATP. The crystal structure of the complex between the NBD of the Escherichia coli Hsp70, DnaK, and its NEF, GrpE, was reported previously, but the GrpE in the complex carried a point mutation (G122D). Both the functional impact of this mutation and its location on the NEF led us to revisit the DnaK NBD/GrpE complex structurally using AlphaFold modeling and validation by solution methods that report on protein conformation and mutagenesis. This work resulted in a new model for the DnaK NBD in complex with GrpE in which subdomain IIB of the NBD rotates more than in the crystal structure, resulting in an open conformation of the nucleotide-binding cleft, which now resembles more closely what is seen in other Hsp/NEF complexes. Moreover, the new model is consistent with the increased ADP off-rate accompanying GrpE binding. Excitingly, our findings point to an interdomain allosteric signal in DnaK triggered by GrpE binding.

Dose-dependent effect of cyclophosphamide treatment on actin

The actin is the essential unit protein of cytoskeleton and muscle sarcomeres. The continuous management of filaments is the key machinery of eukaryotic cytoskeletal plasticity which based on the different complexes with divalent cations (Ca2+ or Mg2+) and nucleotides (ATP, ADP). Any structural modification of nucleotide-binding sites in G actin can bind ATP or ADP under different cation conditions and can initialize the remodelling of the cleft and change the stiffness of two main domains. The evolutionary important nucleotide-binding cleft as a bridge between the two domains needs more investigation to can express its importance in the development of actin functions. The cyclophosphamide (CP) is a cytostatic drug applied in chemotherapy it can alkylate the long residues in the ATP binding sites thus change the structure of the binding cleft. Our previous study explained that the actin filaments show less sensitivity to the CP treatment than monomers. Here we investigate the CP dose-response effect on the thermodynamic stability of actin monomers and polymers in the presence of Ca2+ or Mg2+ to know the minimum effective concentration for the interpretation of any relevant dosage at level of tissues. The previously expressed “titled state” EM model of filamentous actin based on the same structural change of monomers as we found here where the domains react to any modification with taking apart sd4 from sd2 results in a more exposed nucleotide-binding cleft.

Alterations of inter-domain flexibility in actin monomers during cyclophosphamide treatment

The actin is one of the main component of the eukaryotic cytoskeleton. The continuous rearrangement of actin filaments is provided by the different complexes with divalent cations (Ca2+ or Mg2+) and nucleotides (ATP, ADP). In the medical routine, cyclophosphamide (CP) is applied as cytostatic and it was shown that in vivo muscle filament system was changed by the CP treatment and it has direct interaction with actin monomers as well. The evolutionary importance of physical links between domains is one of the most interesting question to understand the multi-domain development of protein functions. Here, we analyse the thermal stability modifier act of inter-domain links in proteins, monitored by DSC, with the concept of that how did the nucleotide binding cleft between the two main domains of actin monomers affect the activation energy of domains if it was blocked or released by CP binding or dissociation, respectively. We investigated the importance of inter-domain linkers on the thermodynamic properties of actin. Ca2+ and Mg2+ bound G-actin can be stabilized by CP binding or polymerization. CP treatment of Ca2+-F actin lacks the structural integrity of the more flexible polymer and shows same stability as CP bound monomers. However, Mg2+-F actin did not show any kinetic response to the CP treatment. We can assume that the inter-domain linker of actin reduces the stability of the domains which leads to a more reactive and variable structure as a thermodynamic advantage for the development of a multi-domain protein can be blocked by CP treatment.

Cyclophosphamide treatment evoked side effect on skeletal muscle actin, monitored by DSC

Several kind of drugs—used in cancer treatments—such as cyclophosphamide (CP) can also trigger a disease classified as toxic polyneuropathy. Polyneuropathy is a simultaneous malfunction of several peripheral nerves, typical side effect of a cancer therapy. In our previous study, we used CP treated in vitro animal model (Guinea pig) with a comparable dosage and time handling of human protocol to show evidences of this drug-induced effects. We could show a dose-dependent difference between in Tm and ΔHcal of untreated and treated samples assigned to their intact muscle and nerve, blood plasma and red blood cells. In our current study we analyze this side effect on skeletal muscle actin (prepared from m. psoas of rabbit) by DSC (differential scanning calorimetry), to follow the possible consequence of drug treatment on the “activator” of muscle contraction. We have demonstrated that run of DSC curves, Tms together with the ΔHcal exhibit clear CP effect. In case of Ca2+ G actin it is manifested in a well separated second high denaturing temperature as a consequence of CP binding into the cleft. This way the nucleotide binding cleft with subdomains 1 and 3 becomes less flexible, indicating clear sensitivity to CP treatment. In F-actin samples, the main peak represents the thermal denaturation of subdomains 1 and 3, and the increased calorimetric enthalpy administrating Ca2+ as well as CP refers to a more rigid structure. These alterations can be the molecular background in the malfunction of muscle in case of polyneuropathy after CP treatment.

Identification of Tractable Drug-like eIF4Al Inhibitors with Potent Anti-Tumor Activity

Clinical efficacy of targeted signaling inhibitors for hematologic malignancies is limited bynoutgrowth of subpopulations with alternative pathways independent of the drug target. The eIF4F complex responsible for translation initiation is a convergence point for cancer-promoting signaling pathways and its inhibition leads to decreased expression of key oncoproteins and apoptosis. Lymphomas and leukemias show particular dependence on constitutive eIF4F activation. Indeed, natural compounds targeting the eIF4F enzymatic component, eIF4A1, demonstrate activities in vitro and in vivo against lymphoma and leukemia model systems, among other tumor types. The natural compound silvestrol is a potent inhibitor of eIF4A1, results in cancer cell cytotoxicity, and has an established therapeutic window in vivo. Silvestrol shows potent antitumor activity against 924 pan-cancer tumor cell lines with 830/924 (90%) sensitive at IC50 <100nM with lymphoma and leukemia cell lines being particularly sensitive. Silvestrol and other natural compounds, however, lack core drug-like properties and synthetic tractability. To discover new, specific and tractable inhibitors of eIF4A1 that are more drug-like, we have constructed several molecular models that we used to virtually screen more than 20 million compounds. eIF4A1 is the founding member of the DEAD-box RNA helicases, which include its paralogs eIF4A2 (91% amino-acid identity with eIF4A1) and eIF4A3 (60% identity). All DEAD-box helicases contain two RecA-like domains separated by a flexible linker. The cleft between these domains is lined with helicase motifs that mediate nucleotide binding and hydrolysis. In an absence of RNA or nucleotide, eIF4A proteins adopt diffuse open conformations; binding of RNA and ATP triggers transition to a more stable closed state. Modeling small-molecule interactions in the nucleotide cleft of eIF4A1 therefore assesses ability of molecules to lock eIF4A1 in a conformation unable to cycle through ATPase and helicase activities. A new crystal structure of eIF4A1 has become available (2019) with a resolution of 2 angstroms. The protein is co-crystallized with ANP in the nucleotide binding site at the interface of the N and C-terminal domains and with known inhibitor, Rocaglamide, bound to the interface of the eIF4A1and a polypurine RNA. We used this high-resolution crystal structure to build models predicting interactions of small molecules in the interdomain nucleotide-binding cleft. We then performed all-atom explicit-water molecular dynamics (MD) simulations for 500-700 ns to study conformational dynamics and atomic interactions of ATP-bound and ATP-unbound states. Extended molecular dynamic simulations confirm the hypothesis that rocaglamide stabilizes the interaction between the helicase and a polypurine sequence on RNA, thus preventing further ATPase activity and RNA unwinding. Pooling these results, we constructed two homology models of human eIF4A1 with both open and closed conformations as structural templates. Over 50 compounds identified as hits in silicowere ordered and tested thus far in our biochemical and cell-based validation platform. Using our machine learning and virtual screening approach targeted to the ATP binding site of eIF4A1, we identified a promising piperazine-amide fragment scaffold (UM107; ~300 MW) with similar electronics to nucleotide triphosphates. UM107 caused cellular toxicities with an LD50 of 50 uM and was weakly active in the biochemical screen against eIF4A1 with an IC50 of 250 uM. We will increase molecular weight by adding more groups to maximize hydrogen bond interactions in the active site. These analogs will be synthesized and screened virtually building on the core using established medicinal chemistry optimization tools followed by biochemical and cellular validation. We therefore have developed an accurate and novel in silico models of eIF4A1 highly useful in assessing interactions of small-molecule ATPase inhibitors, with focus on the ATP-binding cleft. Disclosures No relevant conflicts of interest to declare.

Conformational coupling by trans-phosphorylation in calcium calmodulin dependent kinase II.

The calcium calmodulin-dependent protein kinase II (CaMKII) is a dodecameric holoenzyme important for encoding memory. Its activation, triggered by binding of calcium-calmodulin, persists autonomously after calmodulin dissociation. One (receiver) kinase captures and subsequently phosphorylates the regulatory domain peptide of a donor kinase forming a chained dimer as the first stage of autonomous activation. Protein dynamics simulations examined the conformational changes triggered by dimer formation and phosphorylation, aimed to provide a molecular rationale for human mutations that result in learning disabilities. Ensembles generated from X-ray crystal structures were characterized by network centrality and community analysis. Mutual information related collective motions to local fragment dynamics encoded with a structural alphabet. Implicit solvent tCONCOORD conformational ensembles revealed the dynamic architecture of inactive kinase domains was co-opted in the activated dimer but the network hub shifted from the nucleotide binding cleft to the captured peptide. Explicit solvent molecular dynamics (MD) showed nucleotide and substrate binding determinants formed coupled nodes in long-range signal relays between regulatory peptides in the dimer. Strain in the extended captured peptide was balanced by reduced flexibility of the receiver kinase C-lobe core. The relays were organized around a hydrophobic patch between the captured peptide and a key binding helix. The human mutations aligned along the relays. Thus, these mutations could disrupt the allosteric network alternatively, or in addition, to altered binding affinities. Non-binding protein sectors distant from the binding sites mediated the allosteric signalling; providing possible targets for inhibitor design. Phosphorylation of the peptide modulated the dielectric of its binding pocket to strengthen the patch, non-binding sectors, domain interface and temporal correlations between parallel relays. These results provide the molecular details underlying the reported positive kinase cooperativity to enrich the discussion on how autonomous activation by phosphorylation leads to long-term behavioural effects.

Computational Identification of Tractable Drug-like Inhibitors of eIF4A1

Targeted signaling inhibitors for hematologic malignancies often lead to limited clinical efficacy due to the outgrowth of subpopulations with alternative pathways independent of the drug target. The eIF4F complex responsible for translation initiation is a convergence point for many cancer-promoting signaling pathways and its inhibition leads to decreased expression of key oncoproteins and apoptosis. Lymphomas and leukemias show particular dependence on constitutive eIF4F activation. Indeed, natural compounds targeting the eIF4F enzymatic component, eIF4A1, demonstrate activities both in vitro and in vivo against lymphoma and leukemia model systems, among other tumor types. eIF4A1 is a noteworthy target for hematologic malignancies based on the finding that BCR stimulation leads to increased mRNA translation primary CLL patient samples. Additionally, eIF4F components eIF4A1 and eIF4G1 had increased expression upon IgM-induced BCR activation. The natural compound silvestrol is a potent inhibitor of eIF4A1, results in cancer cell cytotoxicity, and has an established therapeutic window in vivo. Silvestrol shows potent antitumor activity against 924 pan-cancer tumor cell lines with 830/924 (90%) sensitive at IC50 <100nM with lymphoma and leukemia cell lines being particularly sensitive. Silvestrol and other natural compounds, however, lack core drug-like properties and synthetic tractability.To discover new, specific and tractable inhibitors of eIF4A1 that are more drug-like, we have constructed several molecular models that we used to virtually screen more than 20 million compounds. eIF4A1 is the founding member of the DEAD-box RNA helicases, which include its paralogs eIF4A2 (91% amino-acid identity with eIF4A1) and eIF4A3 (60% identity). All DEAD-box helicases contain two RecA-like domains separated by a flexible linker. The cleft between these domains is lined with helicase motifs that mediate nucleotide binding and hydrolysis. In an absence of RNA or nucleotide, eIF4A proteins adopt diffuse open conformations; binding of RNA and ATP triggers transition to a more stable closed state. Modeling small-molecule interactions in the nucleotide cleft of eIF4A1 therefore assesses ability of molecules to lock eIF4A1 in a conformation unable to cycle through ATPase and helicase activities. Although no experimentally derived structure of human eIF4A1 co-crystalized with ATP exists, crystal structures of other DEAD-box family members with similar motifs permit detailed studies of nucleotide and ligand-binding and the development of homology models. We have used four available high-resolution crystal structures to build models predicting interactions of small molecules in the interdomain nucleotide-binding cleft. We identified nucleotide binding-site residues and accurately reproduced ATP interactions for all four models (derived from PDB: 2J0S, 1FUU, 2VSO, 2DB3). We then performed all-atom explicit-water molecular dynamics (MD) simulations for 500-700 ns to study conformational dynamics and atomic interactions of ATP-bound and ATP-unbound states. Yeast eif4A crystal structure (PDB:1FUU) in the open state, for example, illustrated closure of the two RecA domains upon ATP binding. As expected, ATP makes strong interactions with the N-terminal, while phosphate groups extend to the C-terminal interacting with arginines, bringing the two RecA domains together. In contrast, we did not observe domain closure in the same simulation with 1FUU without ATP bound. We also assessed 2J0S, a crystal structure of human closed eIF4A3 bound to ANP. ATP docked to this active site followed by 500 ns MD held the protein in the closed state with several interdomain interactions. Upon nucleotide removal, marked RecA separation occurred. We observed similar domain closure and opening for PDB: 2VSO and 2DB3. Pooling these results, we constructed two homology models of human eIF4A1 with both open (2VSO, 1FUU) and closed conformations (2J0S) as structural templates. We therefore have developed accurate and novel in silico models of eIF4A1 highly useful in assessing interactions of small-molecule ATPase inhibitors, with focus on the ATP-binding cleft. DisclosuresNo relevant conflicts of interest to declare.

A Local Allosteric Network in Heat Shock Protein 70 (Hsp70) Links Inhibitor Binding to Enzyme Activity and Distal Protein-Protein Interactions.

Allosteric inhibitors can be more difficult to optimize without an understanding of how their binding influences the conformational motions of the target. Here, we used an integrated computational and experimental approach to probe the molecular mechanism of an allosteric inhibitor of heat shock protein 70 (Hsp70). The anticancer compound, MKT-077, is known to bind a conserved site in members of the Hsp70 family, which favors the ADP-bound state and interferes with a protein-protein interaction (PPI) at long range. However, the binding site does not overlap with either the nucleotide-binding cleft or the PPI contact surface, so its mechanism is unclear. To this end, we modeled Hsp70's internal dynamics and studied how MKT-077 alters local sampling of its allosteric states. The results pointed to a set of concerted motions between five loops in Hsp70's nucleotide-binding domain (NBD), surrounding the MKT-077 binding site. To test this prediction, we mutated key residues and monitored chaperone activities in vitro. Together, the results indicate that MKT-077 interacts with loop222 to favor a pseudo-ADP bound conformer of Hsp70's NBD, even when ATP is present. We used this knowledge to synthesize an analog of MKT-077 that would better prevent motions of loop222 and confirmed that it had improved antiproliferative activity in breast cancer cells. These results provide an example of how to unlock and leverage the complex mechanisms of allosteric inhibitors.

Conformational changes in Arp2/3 complex induced by ATP, WASp-VCA, and actin filaments

We used fluorescence spectroscopy and EM to determine how binding of ATP, nucleation-promoting factors, actin monomers, and actin filaments changes the conformation of Arp2/3 complex during the process that nucleates an actin filament branch. We mutated subunits of Schizosaccharomyces pombe Arp2/3 complex for labeling with fluorescent dyes at either the C termini of Arp2 and Arp3 or ArpC1 and ArpC3. We measured Förster resonance energy transfer (FRET) efficiency (ETeff) between the dyes in the presence of the various ligands. We also computed class averages from electron micrographs of negatively stained specimens. ATP binding made small conformational changes of the nucleotide-binding cleft of the Arp2 subunit. WASp-VCA, WASp-CA, and WASp-actin-VCA changed the ETeff between the dyes on the Arp2 and Arp3 subunits much more than between dyes on ArpC1 and ArpC3. Ensemble FRET detected an additional structural change that brought ArpC1 and ArpC3 closer together when Arp2/3 complex bound actin filaments. VCA binding to Arp2/3 complex causes a conformational change that favors binding to the side of an actin filament, which allows further changes required to nucleate a daughter filament.

Abstract 918: A secondary RET mutation allosterically conferring resistance to vandetanib

Abstract Targeted therapy against oncogenic ALK and ROS1 fusion using type I tyrosine kinase inhibitors (TKIs), which bind to the ATP-binding cleft of kinases, is highly effective in lung adenocarcinoma (LADC); however, such cancers inevitably acquire resistance, which severely limits the efficacy of cancer treatments. The identification of these resistance mutations led to the development of novel TKIs to overcome acquired resistance. Here we report resistance to vandetanib, a type I RET kinase inhibitor, developed in a patient with metastatic LADC harboring a CCDC6-RET fusion that initially exhibited a response to treatment. The resistant tumor acquired a secondary mutation resulting in substitution of an amino acid located outside the nucleotide binding cleft. The mutation conferred resistance to vandetanib by increasing the ATP affinity and auto-phosphorylation activity of RET kinase. A crystal structure of the mutant revealed a small hydrophobic core around the mutated amino acid likely to enhance basal kinase activity by stabilizing an active conformer. In addition, a thermal shift assay suggested that the mutation was less able to bind vandetanib. Molecular dynamics simulation supported the mutant underwent conformational changes decreasing the binding affinity of vandetanib. Our findings indicate that the missense mutation is able to increase kinase activity and confers drug resistance through allosteric effects. Citation Format: Takashi Nakaoku, Takashi Kohno, Mitsugu Araki, Seiji Nino, Rakhee Chauhan, Phillip P. Knowles, Katsuya Tsuchihara, Shingo Matsumoto, Yoko Shimada, Sachiyo Mimaki, Genichiro Ishii, Hitoshi Ichikawa, Yasushi Okuno, Kiyotaka Yoh, Neil Q. McDonald, Koichi Goto. A secondary RET mutation allosterically conferring resistance to vandetanib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 918.

Arachidonic Acid and Oxidation in the Myosin II Motor Domain

Myosin is the motor protein responsible for movement and muscle contraction in all eukaryotic cells. The catalytic motor domain of myosin II contains three functionally important subdomains: the actin‐binding cleft, the force‐generating domain, and the nucleotide binding pocket. The chemomechanical coupling in the myosin catalytic domain allows for hydrolysis of adenosine triphosphate (ATP) into adenosine diphosphate (ADP) and inorganic phosphate (Pi), producing the energy needed for the contraction of muscle. This chemical reaction of ATP hydrolysis in the nucleotide binding cleft must be accompanied by a crucial interaction with actin to allow for the proper production of mechanical force. Various physiological and pharmacological effector molecules interact with myosin, specifically in its actinbinding cleft, and affect the actomyosin functional interaction. For example, oxidation of a methionine amino acid residue in the actin‐binding cleft, M394, decreases myosin's actin‐activated ATPase activity and changes the structural dynamics of the actin‐binding cleft of myosin (1). Conversely, arachidonic acid, a bioactive lipid produced naturally in eukaryotic cells, acts as a positive allosteric effector of myosin and is postulated to bind near the actin‐binding cleft of myosin, increasing the rate at which myosin hydrolyzes ATP (2). We have investigated the combined functional and structural effects of oxidation and arachidonic acid on the myosin motor domain in a Dictyostelium myosin II model system. Changes in actomyosin functional interaction in the presence of arachidonic acid after in vitro oxidation was assessed using a myosin ATPase activity assay and an actomyosin cosedimentation assay while changes in structural dynamics of the actin‐binding cleft was investigated using electron paramagnetic resonance (EPR). The results illustrate the importance of myosin's actin‐binding cleft structural dynamics and how allosteric effectors effect actomyosin functional interaction.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The crystal structure of pseudokinase PEAK1 (Sugen kinase 269) reveals an unusual catalytic cleft and a novel mode of kinase fold dimerization

The pseudokinase group encompasses some 10% of protein kinases, but pseudokinases diverge from canonical kinases in key motifs. The two members of the small new kinase family 3 (NKF3) group are considered pseudokinases. These proteins, pseudopodium-enriched atypical kinase 1 (PEAK1, Sugen kinase 269, or SgK269) and pragmin (Sugen kinase 223 or SgK223), act as scaffolds in growth factor signaling pathways, and both contain a kinase fold with degraded kinase motifs at their C termini. These kinases may harbor regions that mediate oligomerization or control other aspects of signal transduction, but a lack of structural information has precluded detailed investigations into their functional roles. In this study, we determined the X-ray crystal structure of the PEAK1 pseudokinase domain to 2.3 Å resolution. The structure revealed that the PEAK1 kinase-like domain contains a closed nucleotide-binding cleft that in this conformation may deleteriously affect nucleotide binding. Moreover, we found that N- and C-terminal extensions create a highly unusual all α-helical split-dimerization region, termed here the split helical dimerization (SHED) region. Sequence conservation analysis suggested that this region facilitates a dimerization mode that is conserved between PEAK1 and pragmin. Finally, we observed structural similarities between the PEAK1 SHED region and the C-terminal extension of the Parkinson's disease-associated kinase PINK1. In summary, PEAK1's kinase cleft is occluded, and its newly identified SHED region may promote an unexpected dimerization mode. Similarities of PEAK1 with the active kinase PINK1 may reclassify the latter as a member of the new kinase family 3 group.

High-resolution cryo-EM structures of actin-bound myosin states reveal the mechanism of myosin force sensing

Myosins adjust their power outputs in response to mechanical loads in an isoform-dependent manner, resulting in their ability to dynamically adapt to a range of motile challenges. Here, we reveal the structural basis for force-sensing based on near-atomic resolution structures of one rigor and two ADP-bound states of myosin-IB (myo1b) bound to actin, determined by cryo-electron microscopy. The two ADP-bound states are separated by a 25° rotation of the lever. The lever of the first ADP state is rotated toward the pointed end of the actin filament and forms a previously unidentified interface with the N-terminal subdomain, which constitutes the upper half of the nucleotide-binding cleft. This pointed-end orientation of the lever blocks ADP release by preventing the N-terminal subdomain from the pivoting required to open the nucleotide binding site, thus revealing how myo1b is inhibited by mechanical loads that restrain lever rotation. The lever of the second ADP state adopts a rigor-like orientation, stabilized by class-specific elements of myo1b. We identify a role for this conformation as an intermediate in the ADP release pathway. Moreover, comparison of our structures with other myosins reveals structural diversity in the actomyosin binding site, and we reveal the high-resolution structure of actin-bound phalloidin, a potent stabilizer of filamentous actin. These results provide a framework to understand the spectrum of force-sensing capacities among the myosin superfamily.

Sodium fluoride as a nucleating factor for Mg-actin polymerization

Dynamic instability of actin filaments can be inhibited by Pi analogs beryllium fluoride and aluminium fluoride that mimic the intermediate ADP-Pi state and stabilize actin filaments. On the other hand, the phosphoryl transfer enzymes can be activated in the absence of aluminium by magnesium fluoride if magnesium ions and sodium fluoride (NaF) were present in the solution. Whether magnesium fluoride promotes functional activities of actin is not known. Here we show, for the first time, that sodium fluoride strongly accelerates polymerization of highly dynamic Mg-F-actin assembled from the monomers proteolytically cleaved between Gly42 and Val43 within the D-loop with actin-specific protease protealysin (Pln-actin), apparently due to stabilization of nuclei formed at the initial step of actin polymerization. Thereby, NaF did not inhibit the ATPase activity (subunit exchange) on Pln-F-actin, did not increase the amount of Pln-F-actin sedimented by ultracentrifugation, and did not stabilize the inter-strand contacts of Pln-F-actin. On the other hand, NaF diminished accessibility of the nucleotide binding cleft of Mg-G-actin to trypsin, pointing to an additional cleft closure, and additionally protected the D-loop from the protealysin cleavage in Mg-F-actin, thus indicating that the longitudinal contacts are stabilized. We also demonstrate that in cultured cells NaF can directly promote assembly of F-actin structures under conditions when the corresponding activity of the RhoA pathway is inhibited. These data suggest that the NaF-induced assembly of actin filaments is promoted by magnesium fluoride that can be formed by the NaF-originating fluoride and the actin tightly bound magnesium.

The effect of mouse twinfilin-1 on the structure and dynamics of monomeric actin

The effect of twinfilin-1 on the structure and dynamics of monomeric actin was investigated with fluorescence spectroscopy and differential scanning calorimetry experiments. Fluorescence anisotropy measurements proved that G-actin and twinfilin-1 could form a complex. Due to the formation of the complexes the dissociation of the nucleotide slowed down from the nucleotide-binding pocket of actin. Fluorescence quenching experiments showed that the accessibility of the actin bound ε-ATP decreased in the presence of twinfilin-1. Temperature dependent fluorescence resonance energy transfer and differential scanning calorimetry experiments revealed that the protein matrix of actin becomes more rigid and more heat resistant in the presence of twinfilin-1. The results suggest that the nucleotide binding cleft shifted into a more closed and stable conformational state of actin in the presence of twinfilin-1.

Structure of the Legionella Effector, lpg1496, Suggests a Role in Nucleotide Metabolism

Pathogenic Gram-negative bacteria use specialized secretion systems that translocate bacterial proteins, termed effectors, directly into host cells where they interact with host proteins and biochemical processes for the benefit of the pathogen. lpg1496 is a previously uncharacterized effector of Legionella pneumophila, the causative agent of Legionnaires disease. Here, we crystallized three nucleotide binding domains from lpg1496. The C-terminal domain, which is conserved among the SidE family of effectors, is formed of two largely α-helical lobes with a nucleotide binding cleft. A structural homology search has shown similarity to phosphodiesterases involved in cleavage of cyclic nucleotides. We have also crystallized a novel domain that occurs twice in the N-terminal half of the protein that we term the KLAMP domain due to the presence of homologous domains in bacterial histidine kinase-like ATP binding region-containing proteins and S-adenosylmethionine-dependent methyltransferase proteins. Both KLAMP structures are very similar but selectively bind 3',5'-cAMP and ADP. A co-crystal of the KLAMP1 domain with 3',5'-cAMP reveals the contribution of Tyr-61 and Tyr-69 that produces π-stacking interactions with the adenine ring of the nucleotide. Our study provides the first structural insights into two novel nucleotide binding domains associated with bacterial virulence.

The effect of toxofilin on the structure and dynamics of monomeric actin

The effects of toxofilin (an actin binding protein of Toxoplasma gondii) on G-actin was studied with spectroscopy techniques. Fluorescence anisotropy measurements proved that G-actin and toxofilin interact with 2:1 stoichiometry. The affinity of toxofilin to actin was also determined with a fluorescence anisotropy assay. Fluorescence quenching experiments showed that the accessibility of the actin bound ε-ATP decreased in the presence of toxofilin. The results can be explained by the shift of the nucleotide binding cleft into a closed conformational state. Differential scanning calorimetry measurements revealed that actin monomers become thermodynamically more stable due to the binding of toxofilin.

Coronin Enhances Actin Filament Severing by Recruiting Cofilin to Filament Sides and Altering F-Actin Conformation

High rates of actin filament turnover are essential for many biological processes and require the activities of multiple actin-binding proteins working in concert. The mechanistic role of the actin filament severing protein cofilin is now firmly established; however, the contributions of other conserved disassembly-promoting factors including coronin have remained more obscure. Here, we have investigated the mechanism by which yeast coronin (Crn1) enhances F-actin turnover. Using multi-color total internal reflection fluorescence microscopy, we show that Crn1 enhances Cof1-mediated severing by accelerating Cof1 binding to actin filament sides. Further, using biochemical assays to interrogate F-actin conformation, we show that Crn1 alters longitudinal and lateral actin–actin contacts and restricts opening of the nucleotide-binding cleft in actin subunits. Moreover, Crn1 and Cof1 show opposite structural effects on F-actin yet synergize in promoting release of phalloidin from filaments, suggesting that Crn1/Cof1 co-decoration may increase local discontinuities in filament topology to enhance severing.

Structural basis of thymosin-β4/profilin exchange leading to actin filament polymerization.

Thymosin-β4 (Tβ4) and profilin are the two major sequestering proteins that maintain the pool of monomeric actin (G-actin) within cells of higher eukaryotes. Tβ4 prevents G-actin from joining a filament, whereas profilin:actin only supports barbed-end elongation. Here, we report two Tβ4:actin structures. The first structure shows that Tβ4 has two helices that bind at the barbed and pointed faces of G-actin, preventing the incorporation of the bound G-actin into a filament. The second structure displays a more open nucleotide binding cleft on G-actin, which is typical of profilin:actin structures, with a concomitant disruption of the Tβ4 C-terminal helix interaction. These structures, combined with biochemical assays and molecular dynamics simulations, show that the exchange of bound actin between Tβ4 and profilin involves both steric and allosteric components. The sensitivity of profilin to the conformational state of actin indicates a similar allosteric mechanism for the dissociation of profilin during filament elongation.