Conformational Changes In Subdomain Research Articles

Mechanism of actin polymerization revealed by cryo-EM structures of actin filaments with three different bound nucleotides

We used cryo-electron microscopy (cryo-EM) to reconstruct actin filaments with bound AMPPNP (β,γ-imidoadenosine 5'-triphosphate, an ATP analog, resolution 3.1 Å), ADP-Pi (ADP with inorganic phosphate, resolution 3.1 Å), or ADP (resolution 3.6 Å). Subunits in the three filaments have similar backbone conformations, so assembly rather than ATP hydrolysis or phosphate dissociation is responsible for their flattened conformation in filaments. Polymerization increases the rate of ATP hydrolysis by changing the positions of the side chains of Q137 and H161 in the active site. Flattening during assembly also promotes interactions along both the long-pitch and short-pitch helices. In particular, conformational changes in subdomain 3 open up multiple favorable interactions with the DNase-I binding loop in subdomain 2 of the adjacent subunit. Subunits at the barbed end of the filament are likely to be in this favorable conformation, while monomers are not. This difference explains why filaments grow faster at the barbed end than the pointed end. When phosphate dissociates from ADP-Pi-actin through a backdoor channel, the conformation of the C terminus changes so it distorts the DNase binding loop, which allows cofilin binding, and a network of interactions among S14, H73, G74, N111, R177, and G158 rearranges to open the phosphate release site.

Functional Characterization of Disease-Related Human β-Actin Mutants

Mutations in sarcomeric actins have been linked to various skeletal and cardiac myopathies. In contrast, only a small number of mutations in the cytoplasmic isoforms β-actin and γ-actin have been identified and linked to disease processes. Here, we describe the characterization of disease-related human β-actin mutants R183W and E364K. Mutation R183W is associated with developmental malformations, deafness and dystonia. The residue is located in subdomain 4 in the nucleotide binding cleft and participates in forming a gate-like structure for the nucleotide. Mutation E364K was found in a patient with neutrophil dysfunction. E364 is a surface residue in subdomain 1. We produced both mutants in Sf9 cells and characterized their properties in vitro. The midpoints of the thermal transition Tm, which correspond to the temperatures at which half of all protein molecules are in the native state and the remaining half is in the denatured state, are unaffected by the mutations suggesting proper folding and normal protein stability. Both mutants display increased affinity for DNase-I indicating conformational changes in subdomain 2 that appear to alter the binding interface between monomers. Activated neutrophils are highly motile phagocytic cells and they are thus highly sensitive to changes that affect actin polymerization and F-actin stability. We observed reduced rates of nucleotide exchange and elongation velocities during polymerization for R183W and E364K β-actin. Mutation R183W accelerates ATP hydrolysis by F-actin. R183W β-actin shows impaired ability to activate non-muscle myosin 2A ATPase.

Regulation of Myosin Motility by D-Loop of Actin

Subdomain 2 of actin, which contains the DNase I binding loop (D-loop, residues 38-52), slightly changes its conformation during actin polymerization and interacts with the C-terminus of the adjacent subunit in actin filament. This region is suggested to be important for actin-myosin interaction: it was found that binding of myosin II induces conformational changes in subdomain 2 and the proteolytic digestion of the D-loop disturbs the motor function of myosin II. However, although as many as 24 classes of myosin have already been found and their in vivo roles are completely different, the contribution of the D-loop to actin-myosin interaction has so far been studied only for myosin II.In this study, to determine whether the D-loop contributes to the interaction with myosin V and if so, in what way it affects its motor function, we prepared actins modified in the D-loop and analyzed the effects of modifications on the motile properties of myosins II and V. We found that the D-loop modifications, namely, the proteolytic digestion with subtilisin and the M47A point mutation, significantly decreased the gliding velocity on myosin II-HMM in an in vitro motility assay, due to a weaker generated force. On the other hand, single molecules of myosin V walked with the same velocity on both the wild-type and modified actins; however, the run lengths decreased sharply, correlating with a lower affinity of myosin for actin due to the D-loop modifications.These results show that the D-loop strongly modulates the force generation by myosin II and the processivity of myosin V, presumably affecting actin-myosin interaction in the A.M.ADP.Pi state of both myosins. Our findings are important to understand the principles how an actin molecule may regulate diverse in vivo functions of various myosin isoforms.

Mutations in Subdomain B of the Minichromosome Maintenance (MCM) Helicase Affect DNA Binding and Modulate Conformational Transitions

Minichromosome maintenance (MCM) proteins are believed to provide the replicative helicase activity in eukaryotes and archaea. The single MCM orthologue from Methanothermobacter thermautotrophicus (MthMCM) has been extensively characterized as a model of the eukaryotic heterohexameric MCM complex. MthMCM forms high molecular weight complexes in solution consistent with a dodecamer. Visualization of this complex by electron microscopy suggests that single and double heptameric or hexameric rings can form. We have mutated two arginine residues (Arg-137, Arg-160) in the N-terminal subdomain B of MthMCM based on their apparent potential to form inter-ring hydrogen bonds. Both the single R137A and the double R137A,R160A mutants were characterized by a combination of biophysical, biochemical, and electron microscopy techniques. Biophysical analysis coupled with electron microscopy studies shows that the R137A mutant forms a double heptameric ring, whereas the R137A,R160A protein assembles as a single heptamer. They both show a defect in DNA binding and a concomitant conformational change in subdomain A, with the double mutant displaying significant defects in helicase activity as well. We propose a model in which MCM loading and the subsequent activation of the helicase activity involve a conformational transition that is connected to a DNA binding event.

Structural Basis for NHERF Recognition by ERM Proteins

The Na+/H+ exchanger regulatory factor (NHERF) is a key adaptor protein involved in the anchoring of ion channels and receptors to the actin cytoskeleton through binding to ERM (ezrin/radixin/moesin) proteins. NHERF binds the FERM domain of ERM proteins, although NHERF has no signature Motif-1 sequence for FERM binding found in adhesion molecules. The crystal structures of the radixin FERM domain complexed with the NHERF-1 and NHERF-2 C-terminal peptides revealed a peptide binding site of the FERM domain specific for the 13 residue motif MDWxxxxx(L/I)Fxx(L/F) (Motif-2), which is distinct from Motif-1. This Motif-2 forms an amphipathic alpha helix for hydrophobic docking to subdomain C of the FERM domain. This docking causes induced-fit conformational changes in subdomain C and affects binding to adhesion molecule peptides, while the two binding sites are not overlapped. Our studies provide structural paradigms for versatile ERM linkages between membrane proteins and the cytoskeleton.

A Point Mutation in the Cargo-Binding Domain of Myosin V Affects Its Interaction with Multiple Cargoes

Class V myosins move diverse intracellular cargoes, which attach via interaction of cargo-specific proteins to the myosin V globular tail. The globular tail of the yeast myosin V, Myo2p, contains two structural and functional subdomains. Subdomain I binds to the vacuole-specific protein, Vac17p, while subdomain II likely binds to an as yet unidentified secretory vesicle-specific protein. All functions of Myo2p require the tight association of subdomains I and II, which suggests that binding of a cargo to one subdomain may inhibit cargo-binding to a second subdomain. Thus, two types of mutations are predicted to specifically affect a subset of Myo2p cargoes: first are mutations within a cargo-specific binding region; second are mutations that mimic the inhibited conformation of one of the subdomains. Here we analyze a point mutation in subdomain I, myo2-2(G1248D), which is likely to be this latter type of mutation. myo2-2 has no effect on secretory vesicle movement. The secretory vesicle binding site is in subdomain II. However, myo2-2 is impaired in several Myo2p-related functions. While subdomains I and II of myo2-2p tightly associate, there are measurable differences in the conformation of its globular tail. Based solely on the ability to restore vacuole inheritance, a set of intragenic suppressors of myo2-2 were identified. All suppressor mutations reside in subdomain I. Moreover, subdomain I and II interactions occurred in all suppressors, demonstrating the importance of subdomain I and II association for Myo2p function. Furthermore, 3 of the 10 suppressors globally restored all tested defects in myo2-2. This large proportion of global suppressors strongly suggests that myo2-2(G1248) causes a conformational change in subdomain I that simultaneously affects multiple cargoes.

Crystal Structure of Monomeric Actin in the ATP State: STRUCTURAL BASIS OF NUCLEOTIDE-DEPENDENT ACTIN DYNAMICS

A nucleotide-dependent conformational change regulates actin filament dynamics. Yet, the structural basis of this mechanism remains controversial. The x-ray crystal structure of tetramethylrhodamine-5-maleimide-actin with bound AMPPNP, a non-hydrolyzable ATP analog, was determined to 1.85-A resolution. A comparison of this structure to that of tetramethylrhodamine-5-maleimide-actin with bound ADP, determined previously under similar conditions, reveals how the release of the nucleotide gamma-phosphate sets in motion a sequence of events leading to a conformational change in subdomain 2. The side chain of Ser-14 in the catalytic site rotates upon Pi release, triggering the rearrangement of the loop containing the methylated His-73, referred to as the sensor loop. This in turn causes a transition in the DNase I-binding loop in subdomain 2 from a disordered loop in ATP-actin to an ordered alpha-helix in ADP-actin. Despite this conformational change, the nucleotide cleft remains closed in ADP-actin, similar to ATP-actin. An analysis of the existing structures of members of the actin superfamily suggests that the cleft is open in the nucleotide-free state.

Cofilin and DNase I Affect the Conformation of the Small Domain of Actin

Cofilin binding induces an allosteric conformational change in subdomain 2 of actin, reducing the distance between probes attached to Gln-41 (subdomain 2) and Cys-374 (subdomain 1) from 34.4 to 31.4 Å (pH 6.8) as demonstrated by fluorescence energy transfer spectroscopy. This effect was slightly less pronounced at pH 8.0. In contrast, binding of DNase I increased this distance (35.5 Å), a change that was not pH-sensitive. Although DNase I-induced changes in the distance along the small domain of actin were modest, a significantly larger change (38.2 Å) was observed when the ternary complex of cofilin-actin-DNase I was formed. Saturation binding of cofilin prevents pyrene fluorescence enhancement normally associated with actin polymerization. Changes in the emission and excitation spectra of pyrene-F actin in the presence of cofilin indicate that subdomain 1 (near Cys-374) assumes a G-like conformation. Thus, the enhancement of pyrene fluorescence does not correspond to the extent of actin polymerization in the presence of cofilin. The structural changes in G and F actin induced by these actin-binding proteins may be important for understanding the mechanism regulating the G-actin pool in cells.

The crystal structure of uncomplexed actin in the ADP state.

The dynamics and polarity of actin filaments are controlled by a conformational change coupled to the hydrolysis of adenosine 5'-triphosphate (ATP) by a mechanism that remains to be elucidated. Actin modified to block polymerization was crystallized in the adenosine 5'-diphosphate (ADP) state, and the structure was solved to 1.54 angstrom resolution. Compared with previous ATP-actin structures from complexes with deoxyribonuclease I, profilin, and gelsolin, monomeric ADP-actin is characterized by a marked conformational change in subdomain 2. The successful crystallization of monomeric actin opens the way to future structure determinations of actin complexes with actin-binding proteins such as myosin.

Divalent Cation-, Nucleotide-, and Polymerization-Dependent Changes in the Conformation of Subdomain 2 of Actin

Conformational changes in subdomain 2 of actin were investigated using fluorescence probes dansyl cadaverine (DC) or dansyl ethylenediamine (DED) covalently attached to Gln41. Examination of changes in the fluorescence emission spectra as a function of time during Ca2+/Mg2+ and ATP/ADP exchange at the high-affinity site for divalent cation-nucleotide complex in G-actin confirmed a profound influence of the type of nucleotide but failed to detect a significant cation-dependent difference in the environment of Gln41. No significant difference between Ca- and Mg-actin was also seen in the magnitude of the fluorescence changes resulting from the polymerization of these two actin forms. Evidence is presented that earlier reported cation-dependent differences in the conformation of the loop 38–52 may be related to time-dependent changes in the conformation of subdomain 2 in DED- or DC-labeled G-actin, accelerated by substitution of Mg2+ for Ca2+ in CaATP-G-actin and, in particular, by conversion of MgATP- into MgADP-G-actin. These spontaneous changes are associated with a denaturation-driven release of the bound nucleotide that is promoted by two effects of DED or DC labeling: lowered affinity of actin for nucleotide and acceleration of ATP hydrolysis on MgATP-G-actin that converts it into a less stable MgADP form. Evidence is presented that the changes in the environment of Gln41 accompanying actin polymerization result in part from the release of Pi after the hydrolysis of ATP on the polymer. A similarity of this change to that accompanying replacement of the bound ATP with ADP in G-actin is discussed.

Spectroscopic study of conformational changes in subdomain 1 of G-actin: influence of divalent cations

Temperature dependence of the fluorescence intensity and anisotropy decay of N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine attached to Cys374 of actin monomer was investigated to characterize conformational differences between Ca- and Mg-G-actin. The fluorescence lifetime is longer in Mg-G-actin than that in Ca-G-actin in the temperature range of 5-34 degrees C. The width of the lifetime distribution is smaller by 30% in Mg-saturated actin monomer at 5 degrees C, and the difference becomes negligible above 30 degrees C. The semiangle of the cone within which the fluorophore can rotate is larger in Ca-G-actin at all temperatures. Electron paramagnetic resonance measurements on maleimide spin-labeled (on Cys374) monomer actin gave evidence that exchange of Ca2+ for Mg2+ induced a rapid decrease in the mobility of the label immediately after the addition of Mg2+. These results suggest that the C-terminal region of the monomer becomes more rigid as a result of the replacement of Ca2+ by Mg2+. The change can be related to the difference between the polymerization abilities of the two forms of G-actin.

Conformational changes in actin induced by its interaction with gelsolin

Actin cleaved by the protease from Escherichia coli A2 strain between Gly42 and Val43 (ECP-actin) is no longer polymerizable when it contains Ca2+ as a tightly bound cation, but polymerizes when Mg2+ is bound. We have investigated the interactions of gelsolin with this actin with regard to conformational changes in the actin molecule induced by the binding of gelsolin. ECP-(Ca)actin interacts with gelsolin in a manner similar to that in which it reacts with intact actin, and forms a stoichiometric 2:1 complex. Despite the nonpolymerizability of ECP-(Ca)actin, this complex can act as a nucleus for the polymerization of intact actin, thus indicating that upon interaction with gelsolin, ECP-(Ca)actin undergoes a conformational change that enables its interaction with another actin monomer. By gel filtration and fluorometry it was shown that the binding of at least one of the ECP-cleaved actins to gelsolin is considerably weaker than of intact actin, suggesting that conformational changes in subdomain 2 of actin monomer may directly or allosterically affect actin-gelsolin interactions. On the other hand, interaction with gelsolin changes the conformation of actin within the DNase I-binding loop, as indicated by inhibition of limited proteolysis of actin by ECP and subtilisin. Cross-linking experiments with gelsolin-nucleated actin filaments using N,N-phenylene-bismaleimide (which cross-links adjacent actin monomers between Cys374 and Lys191) reveal that gelsolin causes a significant increase in the yield of the 115-kDa cross-linking product, confirming the evidence that gelsolin stabilizes or changes the conformation of the C-terminal region of the actin molecule, and these changes are propagated from the capped end along the filament. These results allow us to conclude that nucleation of actin polymerization by gelsolin is promoted by conformational changes within subdomain 2 and at the C-terminus of the actin monomer.

Conformational changes in subdomain I of actin induced by proteolytic cleavage within the DNase I-binding loop: energy transfer from tryptophan to AEDANS

Alteration of the actin polypeptide chain within the DNase I-binding loop by cleavage with E. coli A2 protease or subtilisin was shown to increase the efficiency of energy transfer from tryptophan residues to AEDANS attached to Cys-374. Analysis of structural and fluorescence data suggested that only two of four actin tryptophan residues, namely, Trp-340 and/or Trp-356, can be energy transfer donors. It was also found that labelling with AEDANS induces perturbations in the environment of the tryptophan residues, these perturbations being smaller in the cleaved actin. These changes are consistent with a shift of the C-terminal segment of actin monomer upon cleavage and confirm the existence of high conformational coupling between subdomains 1 and 2 of actin monomer. We also suggest that tryptophan residues 340 and/or 356 are located in the focus of this coupling.

Conformational changes in subdomain 2 of G-actin: fluorescence probing by dansyl ethylenediamine attached to Gln-41

Gln-41 on G-actin was specifically labeled with a fluorescent probe, dansyl ethylenediamine (DED), via transglutaminase reaction to explore the conformational changes in subdomain 2 of actin. Replacement of Ca2+ with Mg2+ and ATP with ADP on G-actin produced large changes in the emission properties of DED. These substitutions resulted in blue shifts in the wavelength of maximum emission and increases in DED fluorescence. Excitation of labeled actin at 295 nm revealed energy transfer from tryptophans to DED. Structure considerations and Cu2+ quenching experiments suggested that Trp-79 and/or Trp-86 serves as energy donors to DED. Energy transfer from these residues to DED on Gln-41 increased with the replacement of Ca2+ with Mg2+ and ATP with ADP. Polymerization of Mg-G-actin with MgCl2 resulted in much smaller changes in DED fluorescence than divalent cation substitution. This suggests that the conformation of loop 38-52 on actin is primed for the polymerization reaction by the substitution of Ca2+ with Mg2+ on G-actin.