The nematode Caenorhabditis elegans has two ADF (actin-depolymerizing factor)/cofilin isoforms, UNC-60A and UNC-60B, which are expressed by the unc60 gene by alternative splicing. UNC-60A has higher activity to cause net depolymerization, and to inhibit polymerization, than UNC-60B. UNC-60B, on the other hand, shows much stronger severing activity than UNC-60A. To understand the structural basis of their functional differences, we have determined the solution structures of UNC-60A and UNC-60B proteins and characterized their backbone dynamics. Both UNC-60A and UNC-60B show a conserved ADF/cofilin fold. The G-actin (globular actin)-binding regions of the two proteins are structurally and dynamically conserved. Accordingly, UNC-60A and UNC-60B individually bind to rabbit muscle ADP–G-actin with high affinities, with Kd values of 32.25 nM and 8.62 nM respectively. The primary differences between these strong and weak severing proteins were observed in the orientation and dynamics of the F-actin (filamentous actin)-binding loop (F-loop). In the strong severing activity isoform UNC-60B, the orientation of the F-loop was towards the recently identified F-loop-binding region on F-actin, and the F-loop was relatively more flexible with 14 residues showing motions on a nanosecond–picosecond timescale. In contrast, in the weak severing protein isoform UNC-60A, the orientation of the F-loop was away from the F-loop-binding region and inclined towards its own C-terminal and strand β6. It was also relatively less flexible with only five residues showing motions on a nanosecond–picosecond timescale. These differences in structure and dynamics seem to directly correlate with the differential F-actin site-binding and severing properties of UNC-60A and UNC-60B, and other related ADF/cofilin proteins.

INTRODUCTION

The proteins of the eukaryotic ADF (actin-depolymerizing factor)/cofilin family are essential and key regulators of actin filament dynamics [1]. The ADF/cofilin proteins regulate actin filament dynamics through G-actin (globular actin) binding, F-actin (filamentous actin) binding and depolymerization [2], F-actin severing [3], G-actin monomer sequestering activity [46], and controlling the rate of nucleotide exchange from actin monomers [3,79]. ADF/cofilin proteins increase the turnover rate of actin filaments by accelerating the dissociation of actin monomers from the pointed ends of filaments. In addition, F-actin filament severing generates new uncapped barded ends and pointed ends where polymerization and depolymerization can occur.

ADF-H (ADF-homology) domains are divided into five classes: ADF/cofilin, GMF (glia maturation factor), coactosin, twinfilin and Abp1/debrins [10]. The ADF/cofilins represent the highly conserved ADF-H fold, which is also observed in destrin, actophorin and gelsolin [11]. This canonical fold, which is well represented by the structure of the 143 residues of cofilin from Saccharomyces cerevisiae (ScCof), consists of a six-stranded mixed β-sheet in which the four central strands are anti-parallel, whereas the two edge strands run parallel to the neighbouring strands. The β-sheet is sandwiched between a pair of α-helices on each face. The longest helix (α3) is kinked at the position of a serine residue in a conserved segment [12].

ADF/cofilins have two distinct actin-binding sites. These have been named as the G/F-site and the F-site. The G/F-site is required for binding to both G-actin and F-actin. This site corresponds to the N-terminal flexible region, the long kinked helix (α3), the last β-strand (β5 or β6), and the loop before the C-terminal helix (α4) [2,10]. The long-kinked helix of ADF/cofilins binds at the groove between subdomain 1 and subdomain 3 of actin, which is also a site for the binding of many other actin-binding proteins [2,10]. The F-site is responsible for binding to F-actin and is comprised of the ‘F-loop’, which is a long loop between strand β4 and β5 (or β3 and β4) that typically protrudes out of the structure, the C-terminal α-helix and the C-terminal residues, all of which contain several basic or charged residues [2,10].

In the regular actin filament, residues of SD2 and SD4 of protomer n interact with SD1 and SD3 of protomer n+2 respectively, which are longitudinally connected with each other [13]. The ADF/cofilin molecule contacts with two longitudinally connected actin protomers (n and n+2) in an actin filament by binding in a cleft between them [13]. The F-site interacts with the protomer n, whereas the G/F-site interacts with the longitudinally succeeding protomer n+2. Rotation of the outer domain (SD1 and SD2) of the actin protomer in the actin filament, upon binding of ADF/cofilin, leads to disruption of all longitudinal contacts between longitudinally adjacent protomers. This change co-operatively continues to the bare region of the actin filament. As a result, severing of the actin filament takes place at the junction of the bare region and the ADF/cofilin-decorated region [13].

Actin dynamics has been shown to play an important role in oocyte development, fertilization and a wide range of important events during early embryogenesis, including proper chromosome segregation and cytokinesis, in the nematode Caenorhabditis elegans [14]. C. elegans has been used as the model organism to study the organization and function of myofibrils, because the body wall muscles of this organism are obliquely striated muscle and are structurally and biochemically related to vertebrate striated muscles [15]. Among the several actin dynamics and modulatory proteins that have been characterized for C. elegans, two important proteins are UNC-60A and UNC-60B, which belong to the ADF/cofilin family and are expressed from the unc60 gene by alternative splicing [14]. Mutation in the unc60 gene results in slow movement and paralysed nematodes [14]. UNC-60A has been shown to be essential for organized assembly of the contractile actin network in the myoepithelial sheath of C. elegans somatic gonads, whereas UNC-60B is expressed in body wall muscles, and is required for the proper assembly of actin in myofibrils [15,16].

UNC-60A is 165 amino acids in length and exhibits strong pointed end depolymerzation on C. elegans actin, strong inhibition of polymerization, strong monomer-sequestering activity, weak severing activity and low affinity for F-actin binding [9]. UNC-60B is 152 amino acids in length and exhibits strong pointed-end depolymerization on rabbit muscle actin, strong severing activity and high affinity for F-actin binding [9]. UNC-78, an AIP-1 isoform, disassembles UNC-60B-bound actin filaments, prevents formation of aggregates, and maintains the dynamic state of their interactions. Both UNC-60A and UNC-60B isoforms show only a slight effect of pH on their activity [9]. On the basis of activity, UNC-60A is classified in the ADF subgroup and UNC-60B is classified in the cofilin subgroup. UNC-60B has a greater effect on actin filament dynamics because of its strong severing activity, whereas UNC-60A maintains a pool of monomeric actin because of its high monomer-sequestering activity [9,16].

To understand the structure–activity relationship of these two ADF/cofilins, we have determined the solution structures of UNC-60A and UNC-60B using NMR spectroscopy. We have also characterized the dynamics of these proteins by measuring the 15N-relaxation rates and steady-state heteronuclear [1H]-15N NOE, and analysing these through the Lipari–Szabo formalism. The dynamics were also characterized by relaxation dispersion studies. Furthermore, we have performed NMR chemical-shift perturbation analysis of the effect of F-loop and C-terminal mutation on UNC-60A and UNC-60B structure. The UNC-60A and UNC-60B structures were docked on the crystal structure of the rabbit muscle G-actin monomer. The binding affinities, characterizing the interaction of UNC-60A and UNC-60B with rabbit muscle ADP–G-actin, were determined by ITC (isothermal titration calorimetry). Our results demonstrate that specific combinations of the F-loop orientation, flexibility and charge are the determining factors for both actin sequestering and F-actin binding.

EXPERIMENTAL

Sequence alignment of UNC-60B with UNC-60A and other ADF/cofilin proteins

Protein–protein BLAST was carried out at the NCBI (National Center for Biotechnology Information) server (http://www.ncbi.nlm.nih.gov/BLAST) by taking the UNC-60B sequence as a template. A multiple sequence alignment, including UNC-60A and other ADF/cofilin proteins described below, was generated using ClustalW. The following protein sequences (with GenBank® sequence numbers in parentheses) were retrieved from the NCBI and used in the alignment: AtADF1, Arabidopsis thaliana ADF1 (AAC72407); AcActophorin, Acanthamoeba castellani actophorin (AAA02909); ScCOF, S. cerevisiae cofilin (AAA13256); C. elegans UNC-60A (AAL02461); C. elegans UNC-60B (AAC14457), LdCof, Leishmania donovani ADF/cofilin (AAY99389), PfADF2, Plasmodium falciparum ADF2 (NP705497); PfADF1, P. falciparum ADF1 (NP703379), and TgADF, Toxoplasma gondii ADF (AAC47717). The alignment file was used as the input file for the program ESPript (http://espript.ibcp.fr/ESPript/ESPript/) which was used to prepare the alignment Figure.

Cloning and preparation of NMR samples

Clones of UNC-60A and UNC-60B were obtained from Dr Shoichiro Ono in expression vector pET-3d [15]. The C. elegans unc60a and unc60b genes were subcloned into pETNH6 vector using restriction sites NcoI and BamHI. The clones were overexpressed in the BL21 (λDE3) strain of Escherichia coli. The cloning procedure added extra residues at the N-terminal that included a His6 tag and a TEV (tobacco etch virus) protease site. Conditions for optimal overexpression and purification were standardized. The yield of purified UNC-60A protein was 30 mg/l and UNC-60B was 35 mg/l of culture medium. For isotopic labelling, overexpression of UNC-60A and UNC-60B were standardized in minimal medium containing [15N]ammonium sulphate and [13C]glucose (CIL) as the sole nitrogen and carbon sources respectively.

NMR samples of 13C/15N-labelled UNC-60A and UNC-60B were prepared at a concentration of approximately 0.65 mM and 1.0 mM respectively in NMR buffer (20 mM sodium phosphate, pH 6.5, 50 mM NaCl and 0.1% NaN3) containing 93% H2O/7% 2H2O. For the backbone dynamics study, the NMR sample of 15N-labelled UNC-60A and UNC-60B was prepared at a concentration of approximately 0.7 mM in NMR buffer containing 93% H2O/7% 2H2O.

For CSP (chemical shift perturbation) analysis, mutants K100T (F-loop) and K161N (C-terminal) were constructed for UNC-60A, and R80A, K91A and R80A–K91A (F-loop), and ΔRI, I152A, R151A (C-terminal) were constructed for UNC-60B and sequences were verified by DNA sequencing. Proteins were expressed and purified as described above for the wtUNC-60A (wild-type UNC-60A) and wtUNC-60B.

Structure calculations

Distance restraints were obtained from 3D 15N-edited NOESY-HSQC spectra (τmix 150 ms), 13C (aliphatic)-edited NOESY-HSQC (τmix 160 ms) and 13C (aromatic)-edited NOESY-HSQC spectra (τmix 160 ms) recorded on a Bruker 800 MHz NMR spectrometer. All of the spectra were processed using Topspin3.0 and all NOE were assigned manually using CARA-1.8.4 [17]. Integrated NOE peaks were calibrated and converted into distance restraints with the program CALIBA [18]. In the final structural determination, the program CYANA-3.0 [19] was used. The torsion angle restraints were obtained from the assigned backbone chemical shifts using the program TALOS+ [20]. In total, 2827 NOE and 2400 NOE distance restraints were identified, to which 74 and 60 hydrogen bond restrains were added for determination of UNC-60A and UNC-60B structures respectively. In total, for the structures of UNC-60A and UNC-60B, 200 randomized conformers were generated and ten conformers with lowest target function with no distance violation >0.5 Å (1 Å=0.1 nm) and no angle violations were selected for each. These ten structures with lowest target functions were further subjected to molecular dynamics simulation in explicit water with NMR-derived distance restraints and angle restraints using the CNS 1.21 program [21] and the standard water shell refinement protocol [22,23]. At this stage the distances were relaxed. This step improved the Ramachandran plot statistics and also the Z-score for the Procheck (phi-psi) and Procheck (all) for the ordered residues. The programs CING and PSVS v1.4 (http://www.psvs-1_4.nesg.org) were used to analyse the quality of the structures. The program PyMOL (http://www.pymol.org/) was used for generating protein structure Figures.

Effect of point mutation by chemical shift perturbation analysis

15N-labelled proteins of different mutants of UNC-60A and UNC-60B were prepared as described for wtUNC-60A and wtUNC-60B and 15N-HSQC spectra were recorded. CSPs were analysed to check the effect of point mutation on each residue. Combined CSP ∆Total of 1HN and 15N nuclei were weighted according to ΔTotal=[(Δ1H) 2+(0.2×Δ15N)2]1/2 to normalize the larger chemical shift range of 15N [24]. The resonance frequencies at pH 6.5 of wtUNC-60A and wtUNC-60B were taken as reference points for their respective mutants.

Docking of rabbit muscle actin monomer with UNC-60A and UNC-60B

The crystal structure of the C-terminal ADF-H domain of Twinfilin (Twf-C) in complex with a G-actin monomer (PDB code 3DAW) was taken as a template to build a docked model of UNC-60A and UNC-60B with the G-actin monomer [10]. The G/F-site required for binding of ADF/cofilins with G-actin is highly conserved in UNC-60A and UNC-60B. We used the ClusPro protein–protein docking web server which is publicly available (http://cluspro.bu.edu/login.php) [25] for docking studies. UNC-60B was taken as ligand and the G-actin monomer from the crystal structure of PDB code 3DAW, after removing the Twf-C from it, was taken as receptor and docked using the ClusPro protein–protein docking server. The details of the interface area were analysed by using the web-based server PDBePISA (http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html).

Isothermal titration calorimetry

ITC experiments were performed at 25°C on a VP-ITC MicroCal™ calorimeter. The calorimeter was calibrated according to the user manual of the instrument. All of the samples were briefly centrifuged and then degassed for 20 min before each of the ITC experiments. Titrations were performed at least in duplicate using the same set of stock solutions. The ITC experiments were performed by adding aliquots of ligands to rabbit muscle actin. The rabbit muscle actin for the experiments and samples for ITC (stock solution of UNC-60A, UNC-60B and rabbit muscle ADP–G-actin) were prepared as described previously [26,27]. The sample cell was filled with 1.459 ml of 0.01 mM rabbit muscle actin (titrand) and titrated against UNC-60A which was filled in the syringe of 290 μl at a concentration of 0.100 mM. For UNC-60B 0.005 mM of actin was titrated with 0.05 mM UNC-60B. The injectant volume was set at 10 μl per injection, and the duration of the injection was 20 s, with an interval of 180 s between injections. During the titration, the reaction mixture was continuously stirred at 351 rev./min. Control experiments were performed by injecting UNC-60A/UNC-60B into G-actin buffer under conditions exactly similar to the rabbit muscle actin/titrand titration, to take into account heats of dilution and viscous mixing. The heats of injection of the control experiment were subtracted from the raw data of rabbit muscle actin and titrand titration.

The ITC data were analysed using the ORIGIN version 7.0 software provided by MicroCal™. The heats of binding were normalized with respect to the titrand concentration, and a volume correction was performed to take into account dilution of titrand during each injection. The amount of heat produced per injection was calculated by integration of the area under each peak using a baseline selected by the ORIGIN program, assuming a one-site binding model. The dissociation constant (Kd) and molar enthalpy (∆H) for the binding of titrand to actin was determined by non-linear least square fitting to the data.

Relaxation measurements

To study the backbone dynamics of UNC-60A and UNC-60B, experiments for measurement of 15N longitudinal and transverse relaxation rate constants and [1H]-15N steady-state NOE were recorded as described previously [28]. For backbone amide 15N longitudinal relaxation rate constants, R1, 2D correlation spectra were measured for ten different relaxation delays: 0, 10, 70, 110, 190, 280, 450, 610, 890 and 1330 ms. Transverse relaxation rate constants, R2, were determined from correlation spectra measured with nine different relaxation delays: 10, 30, 50, 70, 90, 130, 170, 190 and 230 ms. [1H]-15N steady state NOE experiments were recorded in an interleaved manner with and without 3 s 1H saturation during the 5 s recycle delay. All of the relaxation parameters of UNC-60A and UNC-60B, along with the [1H]-15N NOE intensities for backbone 15N nuclei, were measured at 25°C, at a magnetic field strength of 14.1 T (corresponding to the resonance frequency of 599.721 MHz for 1H). All of the relaxation data were acquired with 96×1024 (t1×t2) complex points and spectral width of 2309.494 Hz and 8396.300 Hz respectively. Relaxation rate constants of UNC-60A and UNC-60B were determined from the single exponential decay of peak intensities in correlation spectra using the software Curvefit, while steady-state NOE values of UNC-60A and UNC-60B were determined from the ratio of peak heights for spectra recorded with and without 1H saturation.

The heteronuclear 15N relaxation parameters R1, R2 and the steady state heteronuclear [1H]-15N NOE values were analysed by Modelfree formalism as described previously [2831]. For Modelfree analysis of UNC-60A and UNC-60B, the N–H bond length was assumed to be 1.01 Å and a 15N chemical shift anisotropy value of −170 p.p.m. was considered. The uncertainties in R1 and R2 of UNC-60A were set to an upper limit of 3% and 5% respectively, although the uncertainties in steady-state heteronuclear [1H]-15N NOE of UNC-60A were fixed at 0.05. In UNC-60B, the uncertainties of R1 and R2 were set to 3.5% and 5% respectively, and the uncertainties in steady state heteronuclear [1H]-15N NOE of UNC-60B was fixed at 0.04. R1, R2 and steady-state heteronuclear [1H]-15N NOE were subjected to Modelfree analysis [3033] and Fast-Modelfree interface [33]. The dynamic parameters were extracted by using five simple models as proposed by Palmer et al. [34] using the Modelfree 4.1 program. The models considered were: (i) S2 only, τe and Rex are negligible; (ii) S2 and τe only, Rex is negligible; (iii) model 1 and Rex term; (iv) model 2 and Rex term; and (v) incorporation of an additional order parameter for anisotropic rotational diffusion. The 15N-1H vectors with relaxation data that can be fitted to model 1 usually have large S2 values and are more rigid. 15N-1H vectors with relaxation data that must be fitted to models 3 and 4 are those displaying conformational exchange processes on the millisecond to microsecond timescale. 15N-1H vectors with relaxation data fit to models 2, 4 or 5 display internal motions on the picosecond to nanosecond timescale.

RESULTS

Sequence comparison of UNC-60A and UNC-60B proteins with other members of the ADF/cofilin family

UNC-60A protein is 36% identical to UNC-60B (Figure 1, bit score 110 from BLAST) and this intra-species identity is low in comparison with the three mammalian ADF/cofilins, which are all approximately 70% identical to each other. Among nematodes, ADF-like UNC-60A shows high sequence identity (67–75%) with the ADF-like isoforms in Ascaris suum, Loa loa, and Brugia malayi, whereas the cofilin-like isoform UNC-60B shows high sequence identity with cofilin-like isoforms in A. suum, L. loa and B. malayi (77%). However, both UNC-60A and UNC-60B show lower sequence identity (<40%) with ADF/cofilins from other organisms. In comparison with the canonical 143-residue-long yeast cofilin sequence, UNC-60A and UNC-60B have extra residues after β3, in α2 and in the F-loop. Therefore, both proteins have longer F-loops and α2 in comparison with other ADF/cofilins [12]. In comparison with the yeast cofilin sequence, the vertebrate cofilins, which are all approximately 165 amino acids in length, have two inserts and an extended C-terminal. The first insert forms a helix between α1 and β2, which contains a NLS (nuclear localization signal). The second insert follows β3 and forms a bulge and β-strand, which pairs with the N-terminal of β2 [35]. In comparison with UNC-60B, UNC-60A protein is 13 amino acids longer. These extra residues in UNC-60A align in the form of two inserts. The first insert is of four residues from Lys36 to Val39, whereas the second insert is from Ile50 to Asp57, which, incidentally, includes four acidic residues.

Sequence alignment of ADF/cofilin proteins

Figure 1
Sequence alignment of ADF/cofilin proteins

A sequence alignment was constructed for UNC-60A, UNC-60B and other eukaryotic ADF/cofilins, for which 3D structures are known. Secondary structure elements are indicated for UNC-60B. Strictly conserved residues are shown in black boxes, and regions of residues with similar properties are indicated in open boxes. Sequences of ADF/cofilins are shown for C. elegans, T. gondii (TgADF), P. falciparum (PfADF1; PfADF2), Acanthamoeba Actophorin, S. cerevisiae (ScCof; yeast cofilin), A. thaliana (AtADF) and L. donovani (LdCof). The gaps in the alignment are represented as dots. The alignment Figures were made using program ESPRIPT.

Figure 1
Sequence alignment of ADF/cofilin proteins

A sequence alignment was constructed for UNC-60A, UNC-60B and other eukaryotic ADF/cofilins, for which 3D structures are known. Secondary structure elements are indicated for UNC-60B. Strictly conserved residues are shown in black boxes, and regions of residues with similar properties are indicated in open boxes. Sequences of ADF/cofilins are shown for C. elegans, T. gondii (TgADF), P. falciparum (PfADF1; PfADF2), Acanthamoeba Actophorin, S. cerevisiae (ScCof; yeast cofilin), A. thaliana (AtADF) and L. donovani (LdCof). The gaps in the alignment are represented as dots. The alignment Figures were made using program ESPRIPT.

Solution structures of UNC-60A and UNC-60B

The ribbon representations of the lowest energy structures and the wire representations of the final ensemble of ten structures of UNC-60A and UNC-60B are shown in Figure 2. The pairwise RMSDs for the secondary structure regions of the final ensemble of the ten lowest energy structures of UNC-60A and UNC-60B were 0.7 Å and 0.6 Å respectively. The atomic co-ordinates for all ten structures of both proteins have been deposited in the PDB (UNC-60A; PDB code 2MP4 and UNC-60B; PDB code 2LXX). Structural parameters for the solution structures of UNC-60A and UNC-60B are summarized in Table 1.

Solution structures of UNC-60A and UNC-60B

Figure 2
Solution structures of UNC-60A and UNC-60B

(A and C) Ribbon diagram of structure of UNC-60A and UNC-60B respectively. The individual β-strands and α-helices are labelled. In UNC-60A, the β-strands are β1 (Met6–Val7), β2 (Ile29–Ile32), β3 (Ile38–Ala42), β4 (Cys80–Cys91), β5 (Ser99–Ile109) and β6 (Leu139–Val143) and the α-helices are α1 (Asp10–Glu20), α2 (Lys60–Thr74), α3 (Ile116–Leu133) and α4 (His151–Lys161). In UNC-60B, the β-strands are β1 (Lys6–Val7), β2 (Ser25–Asp32), β3 (Thr36–Gly44), β4 (Arg68–Val78), β5 (Thr86–Val97) and β6 (Gln128–Ala131), and the four helices correspond to α1 (Pro10–Lys21), α2 (Tyr50–Glu62), α3 (Val104–Leu121) and α4 (Glu139–Gln150). (B and D) Superimposition of backbone traces from the final ensemble of ten structures of UNC-60A and UNC-60B respectively.

Figure 2
Solution structures of UNC-60A and UNC-60B

(A and C) Ribbon diagram of structure of UNC-60A and UNC-60B respectively. The individual β-strands and α-helices are labelled. In UNC-60A, the β-strands are β1 (Met6–Val7), β2 (Ile29–Ile32), β3 (Ile38–Ala42), β4 (Cys80–Cys91), β5 (Ser99–Ile109) and β6 (Leu139–Val143) and the α-helices are α1 (Asp10–Glu20), α2 (Lys60–Thr74), α3 (Ile116–Leu133) and α4 (His151–Lys161). In UNC-60B, the β-strands are β1 (Lys6–Val7), β2 (Ser25–Asp32), β3 (Thr36–Gly44), β4 (Arg68–Val78), β5 (Thr86–Val97) and β6 (Gln128–Ala131), and the four helices correspond to α1 (Pro10–Lys21), α2 (Tyr50–Glu62), α3 (Val104–Leu121) and α4 (Glu139–Gln150). (B and D) Superimposition of backbone traces from the final ensemble of ten structures of UNC-60A and UNC-60B respectively.

Table 1
Experimental restraints and structural statistics for final ensemble of ten structures of UNC-60A and UNC-60B

Ramachadran plot statistics, CING ROG analysis and WHAT IF summary were done for UNC-60A in the regions Met6–Val7, Asp10–Glu20, Ile27–Ile32, Val37–Ala42, Gln45–Leu48, Asp54–Asp56, Lys60–Ser72, Cys80–Arg93, Ala96–Ile109, Ile116–Ser132, Ile138–Val143 and His151–Lys161. For UNC-60B, these were done for the regions Lys6–Val7, Pro9–Leu18, Tyr26–Lys30, Ala37–Gly44, Lys50–Leu60, Tyr69–Gln79, Thr88–Val95, Val104–Ser125, Met134–Asp136 and Glu139–Ser148.

Parameter UNC-60A UNC-60B 
Distance restraint list 
 Sequential 548 456 
 Intra-residual 808 696 
 Medium-range 800 656 
 Long-range 671 582 
 Hydrogen bonds 74 60 
 Dihedral angle restraints (ϕ and φ) 266 241 
RMSD values (Å) 
 All backbone atoms 0.7 Å 0.6 
 All heavy atoms 1.1 Å 0.9 
Ramachandran plot statistics 
 Most favoured region (%) 94.1 94.7 
 Allowed region (%) 5.9 5.3 
 Additionally allowed region (%) 0.0 0.0 
 Disallowed region (%) 0.0 0.0 
CING ROG analysis 
 Red 13 (12%) 6 (6%) 
 Orange 24 (21%) 35 (38%) 
 Green 76 (67%) 72 (56%) 
WHAT IF Summary 
 Structure Z-scores   
  First generation packing quality 2.349±1.015 2.344±1.175 
  Second generation packing quality 4.417±1.932 5.478±1.961 
  Ramachandran plot appearance −2.555±0.343 −2.624±0.529 
  Chi-1/chi-2 rotamer normality −5.135±0.256 −4.122±0.273 
  Backbone conformation 0.124±0.603 0.694±0.601 
RMS Z-scores 
 Bond lengths 1.035±0.001 1.031±0.001 
 Bond angles 0.283±0.008 0.288±0.005 
 Omega angle restraints 0.745±0.053 0.663±0.067 
 Side chain planarity 0.393±0.039 0.381±0.041 
 Improper dihedral distribution 0.453±0.011 0.439±0.017 
 Inside/outside distribution 1.026±0.014 1.097±0.022 
Parameter UNC-60A UNC-60B 
Distance restraint list 
 Sequential 548 456 
 Intra-residual 808 696 
 Medium-range 800 656 
 Long-range 671 582 
 Hydrogen bonds 74 60 
 Dihedral angle restraints (ϕ and φ) 266 241 
RMSD values (Å) 
 All backbone atoms 0.7 Å 0.6 
 All heavy atoms 1.1 Å 0.9 
Ramachandran plot statistics 
 Most favoured region (%) 94.1 94.7 
 Allowed region (%) 5.9 5.3 
 Additionally allowed region (%) 0.0 0.0 
 Disallowed region (%) 0.0 0.0 
CING ROG analysis 
 Red 13 (12%) 6 (6%) 
 Orange 24 (21%) 35 (38%) 
 Green 76 (67%) 72 (56%) 
WHAT IF Summary 
 Structure Z-scores   
  First generation packing quality 2.349±1.015 2.344±1.175 
  Second generation packing quality 4.417±1.932 5.478±1.961 
  Ramachandran plot appearance −2.555±0.343 −2.624±0.529 
  Chi-1/chi-2 rotamer normality −5.135±0.256 −4.122±0.273 
  Backbone conformation 0.124±0.603 0.694±0.601 
RMS Z-scores 
 Bond lengths 1.035±0.001 1.031±0.001 
 Bond angles 0.283±0.008 0.288±0.005 
 Omega angle restraints 0.745±0.053 0.663±0.067 
 Side chain planarity 0.393±0.039 0.381±0.041 
 Improper dihedral distribution 0.453±0.011 0.439±0.017 
 Inside/outside distribution 1.026±0.014 1.097±0.022 

The solution structures of both UNC-60A and UNC-60B possess a typical ADF/cofilin fold, the core of which consists of a central six-stranded mixed β-sheet. The strand ordering is β1–β3–β2–β4–β5–β6, in which the four central strands are antiparallel, whereas the strands β1–β3 and β5–β6 run parallel to each other. The central β-sheet core is surrounded by helices α1 and α3 on one face, and by helices α2 and α4 on the opposite face. Solution structures of UNC-60A and UNC-60B show conserved features of both G/F-site and F-site, which include the characteristic long kinked helix α3 (G/F-site), the β4–β5 loop (F-loop and F-site), and the C-terminal helix (F-site). The G-actin-binding long helix α3 retains the structurally conserved kink in both of these structures.

As discussed above, UNC-60A protein possesses 13 extra residues subsequent to the strand β3 (post-β3 insert), in comparison with UNC-60B. This post-β3 insert forms two helical turns, Thr44–Leu48 and Asp54–Asp56. The first helical turn (Thr44–Leu48) is in close proximity to the N-terminal of α2, whereas the second helical turn (Asp54–Asp56) is in close proximity to β4 and β5 (Figure 3). This kind of structural feature has not been observed in the structure of any other ADF/cofilin. Residues Asp53 and Asp56 of this region form salt bridges with Lys88 and Lys100 of β4 and β5 respectively (Figure 3). In addition to this unique feature, UNC-60A has a longer α2 in comparison with UNC-60B and other ADF/cofilins.

Structure of UNC-60A protein showing unique structural features

Figure 3
Structure of UNC-60A protein showing unique structural features

(A and B) Ribbon diagram of the structure of UNC-60A at two different orientations showing the unique post-β3 insert, G/F-site and F-site. (C) Salt bridges that form between the residues of the post-β3 insert and the F-loop.

Figure 3
Structure of UNC-60A protein showing unique structural features

(A and B) Ribbon diagram of the structure of UNC-60A at two different orientations showing the unique post-β3 insert, G/F-site and F-site. (C) Salt bridges that form between the residues of the post-β3 insert and the F-loop.

Upon alignment of structures of these two proteins, finer structural differences can be distinguished in the F-sites of UNC-60A and UNC-60B (Figure 4A). In UNC-60A, the inclination of the F-loop towards the C-terminal is ~57°, whereas in UNC-60B the inclination of the F-loop towards the C-terminal is ~43°, as shown in Figures 4(B) and 4(C). Therefore, the F-loop is closer to the C-terminal in the structure of UNC-60A than UNC-60B. Residues Phe89 (β4) and Leu154 (α4) of UNC-60A are positionally correlated to the residues Val76 (β4) and Val142 (α4) respectively of UNC-60B. The distance between Cα of Phe89 and Leu154 is 8.9 Å in UNC-60A (Figure 4B). However, as a consequence of the difference in the inclination of the F-loop towards the C-terminal in UNC-60B, the distance between Cα of Val76 and Val142 is increased to 11.12 Å (Figure 4C). In addition to the above, a slight change is also observed in the orientations of the C-terminals of these proteins. In UNC-60B, the C-terminal is almost parallel to β6, whereas in UNC-60A it forms an angle of ~200 with respect to β6. Furthermore, the composite F-site (F-loop and C-terminal helix) is more inclined towards and closer to β6 in the case of UNC-60A, in comparison with UNC-60B (Figure 4A). Similar differences in the F-sites are also observed upon comparison of structures of yeast cofilin and LdCof (Figure 4D). The F-loop is inclined by 52° towards the C-terminal in LdCof (PDB code 2KVK), whereas this inclination is 42° in yeast cofilin (PDB code 1COF) (Figure 4D). Similarly, the C-terminus is almost parallel to β6 in yeast cofilin, whereas it forms an angle of ~200 with β6 in LdCof [12,26].

Comparison of the structure of UNC-60A with UNC-60B, yeast cofilin and LdCof

Figure 4
Comparison of the structure of UNC-60A with UNC-60B, yeast cofilin and LdCof

(A) Overlap of UNC-60A with UNC-60B showing the different orientation of the F-site (F-loop and C-terminal). (B and C) Structure of UNC-60A and UNC-60B showing detailed description of difference in the F-site in terms of angle and distance. (D) Overlap of yeast cofilin with Leishmania cofilin showing different orientation of F-site. (E) Overlap of UNC-60B with yeast cofilin showing same orientation of F-site. (F) Overlap of UNC-60A with Leishmania cofilin showing same orientation of F-site. Structures of UNC-60A, UNC-60B, yeast cofilin and Leishmania cofilin are shown in cyan, purple, green and magenta colour respectively.

Figure 4
Comparison of the structure of UNC-60A with UNC-60B, yeast cofilin and LdCof

(A) Overlap of UNC-60A with UNC-60B showing the different orientation of the F-site (F-loop and C-terminal). (B and C) Structure of UNC-60A and UNC-60B showing detailed description of difference in the F-site in terms of angle and distance. (D) Overlap of yeast cofilin with Leishmania cofilin showing different orientation of F-site. (E) Overlap of UNC-60B with yeast cofilin showing same orientation of F-site. (F) Overlap of UNC-60A with Leishmania cofilin showing same orientation of F-site. Structures of UNC-60A, UNC-60B, yeast cofilin and Leishmania cofilin are shown in cyan, purple, green and magenta colour respectively.

Overall, on the basis of the comparison of the F-site, various ADF/cofilin structures can be divided into two categories. The inclination of the F-loop towards the C-terminal is ~40° in the case of UNC-60B, yeast cofilin (PDB code 1COF), Acanthamoeba actophorin (PDB code 1CNU), human cofilin (PDB code 4BEX) and chick cofilin (PDB code 1TVJ). On the other hand, the inclination of the F-loop towards the C-terminal is ~55° in the case of UNC-60A and LdCof (PDB code 2KVK) [12,26,3537]. In UNC-60B, yeast cofilin, Acanthamoeba actophorin, human cofilin and chick cofilin, the C-terminal is almost parallel to β6, whereas in UNC-60A and LdCof, it forms an angle of approximately ~20° with respect to β6 [12,26,3537]. Superposition of UNC-60B and yeast cofilin structure, and superposition of UNC-60A and LdCof structures showing similar orientation of F-site are shown in Figures 4(E) and 4(F) respectively. In UNC-60A, F-loop residues are involved in seven salt bridges, whereas in UNC-60B F-loop residues are involved in only two salt bridges (Table 2). In LdCof, the residues of the F-loop are involved in nine salt bridges. On the other hand, F-loop residues are involved in one to three salt bridges in yeast cofilin (ScCof), Acanthamoeba actophorin, human cofilin and chick cofilin [12,26,3537] (Table 2).

Table 2
Salt bridges formed from the residues of the F-loop with other regions in different ADF/cofilins
(a) Salt bridge from residues of F-loop in UNC-60A 
Lys23 (NZ) 2.67 Å Asp102 (OD1) 
Lys88 (NZ) 2.68 Å Asp53 (OD2) 
Lys100 (NZ) 2.62 Å Asp56 (OD1) 
Lys100 (NZ) 3.63 Å Asp56 (OD2) 
Lys103 (NZ) 3.36 Å Asp86 (OD1) 
Lys103 (NZ) 2.62 Å Asp86 (OD2) 
Lys103 (NZ) 3.85 Å Asp102 (OD1) 
(b) Salt bridge from residues of F-loop in UNC-60B 
Asp73 (OD1) 2.55 Å His19 (ND1) 
Glu75 (OE1) 3.57 Å Lys46 (NZ) 
(c) Salt bridge from residues of F-loop in LdCof 
Asp68 (OD1) 2.66 Å Lys22 (NZ) 
Asp68 (OD2) 2.80 Å Lys22 (NZ) 
Glu79 (OE1) 2.63 Å Lys22 (NZ) 
Asp68 (OD2) 2.66 Å Arg25 (NH1) 
Glu70 (OE1) 2.73 Å Lys77 (NZ) 
Asp73 (OD1) 2.60 Å Arg134 (NH1) 
Asp73 (OD1) 3.67 Å Arg134 (NH2) 
Asp73 (OD1) 3.40 Å Arg137 (NH2) 
Asp73 (OD2) 2.76 Å Arg137 (NH2) 
(d) Salt bridge from residues of F-loop in yeast cofilin 
Asp68 (OD1) 3.85 Å Lys23 (NZ) 
Asp68 (OD2) 3.01 Å Lys23 (NZ) 
(a) Salt bridge from residues of F-loop in UNC-60A 
Lys23 (NZ) 2.67 Å Asp102 (OD1) 
Lys88 (NZ) 2.68 Å Asp53 (OD2) 
Lys100 (NZ) 2.62 Å Asp56 (OD1) 
Lys100 (NZ) 3.63 Å Asp56 (OD2) 
Lys103 (NZ) 3.36 Å Asp86 (OD1) 
Lys103 (NZ) 2.62 Å Asp86 (OD2) 
Lys103 (NZ) 3.85 Å Asp102 (OD1) 
(b) Salt bridge from residues of F-loop in UNC-60B 
Asp73 (OD1) 2.55 Å His19 (ND1) 
Glu75 (OE1) 3.57 Å Lys46 (NZ) 
(c) Salt bridge from residues of F-loop in LdCof 
Asp68 (OD1) 2.66 Å Lys22 (NZ) 
Asp68 (OD2) 2.80 Å Lys22 (NZ) 
Glu79 (OE1) 2.63 Å Lys22 (NZ) 
Asp68 (OD2) 2.66 Å Arg25 (NH1) 
Glu70 (OE1) 2.73 Å Lys77 (NZ) 
Asp73 (OD1) 2.60 Å Arg134 (NH1) 
Asp73 (OD1) 3.67 Å Arg134 (NH2) 
Asp73 (OD1) 3.40 Å Arg137 (NH2) 
Asp73 (OD2) 2.76 Å Arg137 (NH2) 
(d) Salt bridge from residues of F-loop in yeast cofilin 
Asp68 (OD1) 3.85 Å Lys23 (NZ) 
Asp68 (OD2) 3.01 Å Lys23 (NZ) 

UNC-60A and UNC-60B display high G-actin binding affinities

In order to understand the structural basis of interaction of UNC-60A and UNC-60B with actin, the structures of these proteins were docked on the rabbit muscle G-actin crystal structure [10]. In both UNC-60A–G-actin and UNC-60B–G-actin docked models, several hydrogen bonds and salt bridges were observed between the residues of the proteins forming the pairs. The details of the interacting residues of UNC-60A–G-actin and UNC-60B–G-actin pairs in the docked models are given in Supplementary Table S1. In the UNC-60B–G-actin docked model, the orientation of α3 of UNC-60B, in the cleft between subdomain1 and subdomain3 of actin, is similar to the orientation of the long helix of Twf-C in the crystal structure of the G-actin and Twf-C complex (Figure 5C). However, in the UNC-60A–G-actin model, the orientation of α3 is slightly different from that of Twf-C (Figure 5A). The interacting regions of the actin monomer are almost similar for the UNC-60A–G-actin and UNC-60B–G-actin models.

Molecular docking of UNC-60A/UNC-60B with G-actin and comparison of interfacial residues with G/F-site residues displaying motion on NMR timescale

Figure 5
Molecular docking of UNC-60A/UNC-60B with G-actin and comparison of interfacial residues with G/F-site residues displaying motion on NMR timescale

(A and C) UNC-60A–G-actin and UNC-60B–G-actin docked models showing residues of UNC-60A and UNC-60B involved in interaction with G-actin respectively. (B and D) UNC-60A–G-actin and UNC-60B–G-actin docked models showing dynamic residues of UNC-60A and UNC-60B on the NMR timescale respectively. This Figure shows that the interacting orientation of the UNC-60A and UNC-60B at the cleft between the subdomain 1 and subdomain 3 is slightly different. This Figure also displays the backbone dynamics of the G/F-site that correlate well with the docking study as residues involved in interaction with G-actin also show dynamic flexibility on the NMR timescale.

Figure 5
Molecular docking of UNC-60A/UNC-60B with G-actin and comparison of interfacial residues with G/F-site residues displaying motion on NMR timescale

(A and C) UNC-60A–G-actin and UNC-60B–G-actin docked models showing residues of UNC-60A and UNC-60B involved in interaction with G-actin respectively. (B and D) UNC-60A–G-actin and UNC-60B–G-actin docked models showing dynamic residues of UNC-60A and UNC-60B on the NMR timescale respectively. This Figure shows that the interacting orientation of the UNC-60A and UNC-60B at the cleft between the subdomain 1 and subdomain 3 is slightly different. This Figure also displays the backbone dynamics of the G/F-site that correlate well with the docking study as residues involved in interaction with G-actin also show dynamic flexibility on the NMR timescale.

In the UNC-60B–G-actin model, residues Arg104, Arg105 and Arg107 of α3 show interaction with Asp25 (subdomain1), Ser125, Arg147 (helix connecting subdomain1 and subdomain3) and Glu167 (subdomain3) of the actin monomer. In addition to this, Asn34 and Thr36 (loop connecting β2–β3) of UNC-60B form hydrogen bonds with Ser350 and Thr351 (subdomain3) of actin. Residue Asp100 (β5–α3 loop) of UNC-60B forms many hydrogen bonds and salt bridges with Arg147 (helix connecting subdomain1 and subdomain3) and Lys328 (subdomain3) of actin. Details of hydrogen bonds and salt bridges in the UNC-60A–G-actin docked model and in the UNC-60B–G-actin docked model are given in Supplementary Table S1.

The structural features of UNC-60A and UNC-60B are well supported by a direct demonstration of their binding with rabbit muscle G-actin in the presence of ADP. ITC titration of UNC-60A with ADP–G-actin reveals 1:1 stoichiometry and a dissociation constant Kd of 32.25 nM, with ∆G of −7.899×103 cal/mol (1 cal≈4.184 J), ΔH of −2.305×103±71.14 cal/mol and ΔS of 26.5 cal/mol per K, whereas titration of UNC-60B with ADP–G-actin reveals a Kd of 8.62 nM with ∆G of −1.1484×104 cal/mol, ΔH of −6.716×103±116.9 cal/mol and ΔS of 16.0 cal/mol per K. The ITC curves are shown in Figures 6(A) and 6(B).

ITC characterization of binding of UNC-60A/UNC-60B with G-actin

Figure 6
ITC characterization of binding of UNC-60A/UNC-60B with G-actin

Left-hand panels, probing UNC-60A–ADP–G-actin interactions by ITC. Right-hand panels, Probing UNC-60B–ADP-G-Actin interactions by ITC. The negative peaks indicate an exothermic reaction. The area under each peak represents the heat released after an injection. The lower panels show the binding isotherms obtained by plotting peak areas against the molar ratio of titrands. The lines represent the best-fit curves obtained from least-squares regression analyses assuming a one-site binding model.

Figure 6
ITC characterization of binding of UNC-60A/UNC-60B with G-actin

Left-hand panels, probing UNC-60A–ADP–G-actin interactions by ITC. Right-hand panels, Probing UNC-60B–ADP-G-Actin interactions by ITC. The negative peaks indicate an exothermic reaction. The area under each peak represents the heat released after an injection. The lower panels show the binding isotherms obtained by plotting peak areas against the molar ratio of titrands. The lines represent the best-fit curves obtained from least-squares regression analyses assuming a one-site binding model.

15N relaxation and Modelfree analysis for UNC-60A and UNC-60B

The backbone dynamics of UNC-60A and UNC-60B were characterized by measurements of the backbone 15N longitudinal relaxation rates (R1), transverse relaxation rates (R2), and steady-state heteronuclear [1H]-15N NOEs. The longitudinal relaxation rates R1 are sensitive to a nanosecond–picosecond timescale. The transverse relaxation rates R2 are more sensitive to nanosecond motions, but they also reflect contributions from slower millisecond–microsecond exchange processes [3840]. The heteronuclear [1H]-15N NOEs are typically most sensitive to picosecond motions with lower values, indicating increased local flexibility of the polypeptide [3840]. R2/R1 ratios characterize the different timescale local motions. Residues with a low value of R2/R1 reflect fast fluctuations on the nanosecond–picosecond timescale, whereas residues with a high value of R2/R1 reflect contributions from slower millisecond–microsecond exchange processes [3840].

The backbone relaxation rates were determined for 137 out of 163 non-proline residues of UNC-60A, and 116 out of 149 non-proline residues of UNC-60B, by using the program Curvefit. The average values of relaxation parameters for residues of UNC-60A are R1=1.35 s−1, R2=14.29 s−1, [1H]-15N NOE=0.70. For residues of UNC-60B, R1=1.32 s−1, R2=13.68 s−1, [1H]-15N NOE=0.59. The residue-specific R1, R2 and steady-state heteronuclear [1H]-15N NOE values obtained for UNC-60A and UNC-60B at 600 MHz are shown in Figures 7(A) and 7(B).

Sequence-dependent variations of backbone 15N relaxation parameters and Modelfree internal mobility parameters of UNC-60A and UNC-60B

Figure 7
Sequence-dependent variations of backbone 15N relaxation parameters and Modelfree internal mobility parameters of UNC-60A and UNC-60B

(A and B) R1 and R2, steady state [1H]-15N NOE and R2/R1 ratio values against the amino acid sequence of UNC-60A and UNC-60B respectively. All experiments with UNC-60A and UNC-60B were performed on 0.7 mM 15N-labelled sample at 25°C on a Varian Inova spectrometer at 600 MHz. (C and D) Plots of Modelfree parameters including generalized order parameter (S2), the effective correlation time (τe) and the chemical exchange parameter (Rex) as a function of residue number for UNC-60A and UNC-60B respectively.

Figure 7
Sequence-dependent variations of backbone 15N relaxation parameters and Modelfree internal mobility parameters of UNC-60A and UNC-60B

(A and B) R1 and R2, steady state [1H]-15N NOE and R2/R1 ratio values against the amino acid sequence of UNC-60A and UNC-60B respectively. All experiments with UNC-60A and UNC-60B were performed on 0.7 mM 15N-labelled sample at 25°C on a Varian Inova spectrometer at 600 MHz. (C and D) Plots of Modelfree parameters including generalized order parameter (S2), the effective correlation time (τe) and the chemical exchange parameter (Rex) as a function of residue number for UNC-60A and UNC-60B respectively.

The relaxation parameters were analysed using the Modelfree formalism with isotropic models for both proteins [2831], which yielded optimized values of molecular rotational correlation times, τm, of 10.098 ns and 10.088 ns for UNC-60A and UNC-60B respectively, both of which correspond to the correlation time expected for a monomeric protein of ~20 kDa. All 137 residues of UNC-60A, for which the relaxation rates were determined, could be fitted to one of the five models of Modelfree. However, for UNC-60B, out of 116 residues, only 108 residues could be fitted, whereas eight residues, Met1, Ala2, Ser3, Glu55, Asp63, Ala71, Asn101 and Ala102, could not be fitted to any of the five models.

Three dynamic parameters were extracted from Modelfree analysis: generalized order parameter (S2), effective correlation time (τe), and chemical exchange rate (Rex) [3033]. S2 and τe are measures of the amplitude and correlation time of wobbling of N-H vectors in space, with respect to overall molecular tumbling, on picosecond-to-nanosecond time scales. Motions on this timescale reflect local flexibility and entropic contributions to protein–ligand interactions. Rex provides a measure of the rate of local exchange processes on a millisecond-to-microsecond timescale for the residues involved in such processes [3033,38,41], and is particularly valuable in understanding how dynamics drives enzyme catalysis. The S2, τe and Rex for each backbone amide NH vector that could be determined using the Modelfree formalism for UNC-60A and UNC-60B are shown in Figures 7(C) and 7(D). The ribbon representations of UNC-60A and UNC-60B structures shaded according to residue-specific S2, τe and conformational exchange term (Rex) are shown in Figure 8 and details of residues showing chemical exchange (Rex) and effective correlation time (τe) are given in Table 3.

Ribbon diagram representing the dynamics properties of UNC-60A and UNC-60B

Figure 8
Ribbon diagram representing the dynamics properties of UNC-60A and UNC-60B

(A and C) The ribbon representations of UNC-60A and UNC-60B solution structures shaded according to the S2 values derived from Modelfree analysis respectively. The colour coding is from blue for S2=1 to red for S2=0.2. Proline residues and residues that are not included in Modelfree analysis are coloured grey. (B and D) The ribbon representations of UNC-60A and UNC-60B shaded according to chemical exchange (Rex) terms, effective correlation (τe) times from Modelfree analysis respectively. The residues displaying motion on picosecond-to-nanosecond timescale (τe) are represented in blue, residues displaying conformational exchange (Rex) from Modelfree analysis are represented in green.

Figure 8
Ribbon diagram representing the dynamics properties of UNC-60A and UNC-60B

(A and C) The ribbon representations of UNC-60A and UNC-60B solution structures shaded according to the S2 values derived from Modelfree analysis respectively. The colour coding is from blue for S2=1 to red for S2=0.2. Proline residues and residues that are not included in Modelfree analysis are coloured grey. (B and D) The ribbon representations of UNC-60A and UNC-60B shaded according to chemical exchange (Rex) terms, effective correlation (τe) times from Modelfree analysis respectively. The residues displaying motion on picosecond-to-nanosecond timescale (τe) are represented in blue, residues displaying conformational exchange (Rex) from Modelfree analysis are represented in green.

Table 3
Residues of UNC-60A and UNC-60B showing effective correlation time (τe) and chemical exchange (Rex) from Modelfree analysis and relaxation dispersion analysis
Analysis Residues of UNC-60A Residues of UNC-60B 
Modelfree 
 Residues in effective correlation time (τeGly4–Val7 (N-terminal and β1), Gln12 (α1), Gly21, Lys23–Glu24 (loop after α1), Gly52–Asp53 (insert after β3), Arg93–Gly97 (F-loop), Lys117 (α3), Thr135 (loop after α3), Ser150 (loop before C-terminal helix) and Tyr162–His165 (C-terminal region) Gly4–Lys6 (N-terminal and β1), Asn34 (loop between β2 and β3), Val61 (α2), Glu66 (loop after α2), Thr77–Gln79 (β4), Gln81–Asn90, Val92 (F-loop), Cys98, Asp100 (loop after β5), Ser148 and Gln150–Ile152 (C-terminal) 
 Residues in chemical exchange (RexAsp10–Phe15, Lys17–Ser19 (α1), Ser59–Ala61(second helical turn of extra region), Phe63–Lys65, Val67–Thr74 (α2 and loop after α2), Cys80 (β4), Cys110 (loop after β5), Lys130 (loop after α3), Val143 (loop after β6) and H165 (C-terminal) Lys12–Asn13, Tyr15, Leu17–Leu18 (α1), Lys21 (loop after α1), Phe29 (β2), Val43–Gly44 (β3), Tyr50–Ala51, Phe53–Val54, Met57–Lys58, Val61 (α2), Val74 (β4), Cys98, Asp100 (loop after β5), Arg105 (α3), Gly122–Leu123, Leu126 (loop after α3), Asn130 (β6), Asp136 (helical turn) and Ser144 (α4) 
Relaxation dispersion 
 Residues in chemical exchange (RexGln12–Ser14, Lys17, Ser19, Glu20 (α1), Glu40, Thr44, Thr51, Gly52, Asp54, Asp57 (insert after β3), Ala61, Lys65, Val67–Val70 (α2), Asp75–Leu77 (loop after α2), Gly95–Thr98, Lys100–Asp102 (F-loop), Cys110, Asp112, Gly113 (β5–α3 loop), Lys119, Tyr122, Ser125, Thr131 (α3), Leu133, Thr135 (α3–β6 loop), Asp145, Glu146, Glu148 (helical turn), Lys152–Leu154, Asn156, Leu158 and Lys161 (C-terminal) Ser10, Lys12–Ala14, His19 (α1), Asn34–Asp35 (loop between β2 and β3), Glu52–Glu55, Met57, Lys59 (α2), Glu62, Gly64, Glu66 (loop after α2), Arg105, Leu109, Tyr110, Leu117, Lys118, Leu121 (α3), Leu123, Leu126 (α3–β6 loop), Asp138 (loop after β6), Val142–Ser144 and Leu146 (C-terminal) 
Analysis Residues of UNC-60A Residues of UNC-60B 
Modelfree 
 Residues in effective correlation time (τeGly4–Val7 (N-terminal and β1), Gln12 (α1), Gly21, Lys23–Glu24 (loop after α1), Gly52–Asp53 (insert after β3), Arg93–Gly97 (F-loop), Lys117 (α3), Thr135 (loop after α3), Ser150 (loop before C-terminal helix) and Tyr162–His165 (C-terminal region) Gly4–Lys6 (N-terminal and β1), Asn34 (loop between β2 and β3), Val61 (α2), Glu66 (loop after α2), Thr77–Gln79 (β4), Gln81–Asn90, Val92 (F-loop), Cys98, Asp100 (loop after β5), Ser148 and Gln150–Ile152 (C-terminal) 
 Residues in chemical exchange (RexAsp10–Phe15, Lys17–Ser19 (α1), Ser59–Ala61(second helical turn of extra region), Phe63–Lys65, Val67–Thr74 (α2 and loop after α2), Cys80 (β4), Cys110 (loop after β5), Lys130 (loop after α3), Val143 (loop after β6) and H165 (C-terminal) Lys12–Asn13, Tyr15, Leu17–Leu18 (α1), Lys21 (loop after α1), Phe29 (β2), Val43–Gly44 (β3), Tyr50–Ala51, Phe53–Val54, Met57–Lys58, Val61 (α2), Val74 (β4), Cys98, Asp100 (loop after β5), Arg105 (α3), Gly122–Leu123, Leu126 (loop after α3), Asn130 (β6), Asp136 (helical turn) and Ser144 (α4) 
Relaxation dispersion 
 Residues in chemical exchange (RexGln12–Ser14, Lys17, Ser19, Glu20 (α1), Glu40, Thr44, Thr51, Gly52, Asp54, Asp57 (insert after β3), Ala61, Lys65, Val67–Val70 (α2), Asp75–Leu77 (loop after α2), Gly95–Thr98, Lys100–Asp102 (F-loop), Cys110, Asp112, Gly113 (β5–α3 loop), Lys119, Tyr122, Ser125, Thr131 (α3), Leu133, Thr135 (α3–β6 loop), Asp145, Glu146, Glu148 (helical turn), Lys152–Leu154, Asn156, Leu158 and Lys161 (C-terminal) Ser10, Lys12–Ala14, His19 (α1), Asn34–Asp35 (loop between β2 and β3), Glu52–Glu55, Met57, Lys59 (α2), Glu62, Gly64, Glu66 (loop after α2), Arg105, Leu109, Tyr110, Leu117, Lys118, Leu121 (α3), Leu123, Leu126 (α3–β6 loop), Asp138 (loop after β6), Val142–Ser144 and Leu146 (C-terminal) 

Overall, for both UNC-60A and UNC-60B, residues in secondary structure elements display high order parameter (S2) (Figures 8A and 8C), whereas the N-terminal, the C-terminal and the F-loop residues are flexible. Interestingly, the helix α1 and α2 are comparatively more flexible than the remaining secondary structure elements, as shown by their lower values of S2 in comparison with the average value for the regions with secondary structure.

In UNC-60A, residues showing motion on a nanosecond-to-picosecond scale (τe) are present in the five main clusters, i.e. N-terminal, loop after α1, post-β3 insert, F-loop and C-terminal. In UNC-60B, residues showing motion on a nanosecond-to-picosecond scale (τe) are present in the four main clusters i.e. N-terminal, β2–β3 loop, F-loop and C-terminal. A clear difference in the dynamics of the F-loop is seen between UNC-60A and UNC-60B. In UNC-60A, five residues (Arg93–Gly97) of the F-loop show fluctuations on a fast picosecond-to-nanosecond time scale, whereas in UNC-60B, 14 residues (Thr77–Gln79, Gln81–Asn90 and Val92) show fluctuations on a fast picosecond-to-nanosecond time scale. Therefore, the F-loop of UNC-60B is more dynamic in comparison with the F-loop of UNC-60A.

Residues involved in a millisecond-to-microsecond time scale exchange processes characterized by Rex were also identified from the Modelfree analysis. In UNC-60A, residues displaying Rex were present in four main clusters i.e. α1, second helical turn of the post-β3 insert, α2 and loop after α2. In UNC-60B, residues displaying Rex were present in five main clusters i.e. α1, β3, α2, β5-α3 loop and α3–β6 loop. Millisecond-to-microsecond timescale motions of UNC-60A and UNC-60B were subsequently examined quantitatively by using the 15N-CPMG relaxation dispersion NMR method, which is able to probe the chemical exchange phenomena in proteins [4244]. Details of 15N-CPMG relaxation dispersion NMR method are given in the Supplementary online data and Supplementary Figures S1–S4. In UNC-60A, residues displaying chemical exchange rate analysed from relaxation dispersion [Rex (RD)] were mainly present in one cluster of G/F-site (α3), two clusters of F-site (F-loop, C-terminal), and five other clusters (α1, post-β3 insert, α2, β5–α3 loop, α3–β6 loop) (Figure 9A). In UNC-60B, residues displaying Rex (RD) were mainly present in one cluster of G/F-site (α3), one cluster of F-site (C-terminal) and four other clusters (α1, α2, β5–α3 loop and α3–β6 loop) (Figure 9B). In UNC-60A, Leu133 and Thr135 of the α3–β6 loop show conformational exchange on the NMR timescale. Similarly, the residues Gly122, Leu123 and Leu126 of this loop in UNC-60B are also in conformational exchange. The backbone dynamics of the G/F-site correlate well with the docking study as residues involved in interaction with G-actin also show dynamic flexibility on the NMR timescale. To illustrate this point, interfacial residues of UNC-60A and UNC-60B (shown in red) are labelled in Figure 5(A) and 5(C) respectively, whereas the residues displaying dynamics on the NMR timescale of the G/F-site of UNC-60A and UNC-60B are labelled in Figures 5(B) and 5(D) respectively (red for τe and yellow for Rex).

Slow time scale motion characterization for UNC-60A and UNC-60B by the relaxation dispersion method

Figure 9
Slow time scale motion characterization for UNC-60A and UNC-60B by the relaxation dispersion method

(A and B) Plot of residue-wise Rex (s−1) values for UNC-60A and UNC-60B respectively, determined by fitting the relaxation dispersion data to eqn. (2). The residues showing significantly higher than the average Rex values are labelled.

Figure 9
Slow time scale motion characterization for UNC-60A and UNC-60B by the relaxation dispersion method

(A and B) Plot of residue-wise Rex (s−1) values for UNC-60A and UNC-60B respectively, determined by fitting the relaxation dispersion data to eqn. (2). The residues showing significantly higher than the average Rex values are labelled.

A comparison of slow time scale motions for UNC-60A and UNC-60B, which were derived from CPMG-based relaxation dispersion experiments, shows that almost similar clusters of residues, which are spatially comparable, are involved in exchange process. Except for the post-β3 insert, which is unique for UNC-60A, the prominent difference between these two proteins is in the F-loop. Residues of the F-loop (Gly95–Thr98, Lys100–Asp102) in UNC-60A were found to be in chemical exchange, whereas no residues of the F-loop of UNC-60B were in chemical exchange (Figure 9). This further shows that the F-loop is more dynamic in UNC-60B where fast nanosecond-to-picosecond timescale motions dominate, whereas slow millisecond to microsecond timescale motions are observed for the F-loop of UNC-60A.

DISCUSSION

Structure and dynamics rationale for F-actin binding and different activities of ADF/cofilins

In the present study, we have examined the solution structures and dynamics of the two ADF/cofilin isoforms of C. elegans, UNC-60A and UNC-60B, which differ significantly in their F-actin-severing activities, monomer-sequestering activities and co-sedimentation properties.

Structurally, both UNC-60A and UNC-60B proteins have similar F-site features, characterized by the presence of a long F-loop, which protrudes beyond the core globular structure, C-terminal helix, and presence of conserved basic residue on the F-loop (Lys103 in UNC-60A and Lys91 in UNC-60B) [12]. Among all the known ADF/cofilin structures, structures of PfADF1 and TgADF display the shortest F-loop that does not protrude out of the structure [27,45]. In both of these proteins, there is a single basic residue (Lys72 in PfADF1; Lys68 in TgADF), which is essential for their weak severing activity. The K72A mutation leads to complete loss of severing activity for PfADF1 [45]. The orientation of this basic residue is conserved in the structures of various ADF/cofilins, and it represents the minimal structural feature required for weak severing activity. In the case of human cofilin, a point mutation of the corresponding Lys96 residue in the F-loop leads to a loss of severing activity and increased depolymerizing activity [46]. As mentioned above, although this feature is conserved in the structures of both UNC-60A and UNC-60B, it is not sufficient to explain the stronger severing activity of UNC-60B as well as the weaker severing activity of UNC-60A. In UNC-60A and LdCof, both of which show weak severing activities [26,47], the F-loop has three basic residues and the C-terminal has five basic residues. On the other hand, in UNC-60B, which shows strong severing activity, the F-loop has only two basic residues and the C-terminal has only three basic residues. This suggests that severing activity does not linearly correlate with the number of positively charged residues on the F-loop and the C-terminal.

Since the finer structure and dynamics differences between these two isoforms have been mapped in the present study, it is pertinent to overlay these structures with the structures of ADF/cofilins that have strong and weak severing activities, namely yeast cofilin and LdCof respectively [2,12,26,47]. Structural overlays of various combinations of pairs of these four proteins are shown in Figure 4. It is seen that the structural features of the F-site (F-loop and C-terminal) of UNC-60A, i.e. inclination of the F-loop towards the C-terminus, and inclination of the F-site towards β6, match closely to that of LdCof (Figure 4F) [26], and are different from the F-site of UNC-60B and yeast cofilin (Figure 4A) [12]. Concurrently, the orientations of F-sites of UNC-60B and yeast cofilin match closely to each other (Figure 4E), and to other strong severing proteins such as Acanthamoeba actophorin, human cofilin and chick cofilin [12,3537]. Furthermore, the F-sites of LdCof and yeast cofilin differ structurally in the same manner as they differ for UNC-60A and UNC-60B (Figures 4A and 4D).

In order to extend this understanding, we have individually superimposed the structures of UNC-60A, UNC-60B and LdCof on the structure of HsCof (Homo sapiens cofilin) in the HsCof–F-actin structure determined from cryo-electron microscopy (PDB code 3J0S) [13]. These overlays are shown in Figure 10. From the overlays, it is seen that the F-loops of UNC-60B and yeast cofilin have similar orientations as the F-loop of human cofilin in its bound form. This orientation enables the interaction of the F-loop with residues 21–28 and 90–96 of actin, which have been identified as the putative F-loop-binding site on the actin surface (Figure 10). On the other hand, because of more inclination, F-loops of UNC-60A and LdCof are oriented away from this F-loop binding site, and are oriented towards the interface of actin protomers n and n+2 (Figure 10). This suggests that correct positioning of the F-loop, as in the case of UNC-60B and HsCof, is necessary for stable association of ADF/cofilin with the filamentous form of actin, which further leads to efficient F-actin severing.

Superimposition of UNC-60A, UNC-60B and LdCof on HsCof in the structure of HsCof–F-actin

Figure 10
Superimposition of UNC-60A, UNC-60B and LdCof on HsCof in the structure of HsCof–F-actin

To model the binding of different ADF/cofilins with F-actin, the structures of UNC-60A, UNC-60B and LdCof were superimposed on HsCOF in the cryo-electron microscopy structure of human cofilin decorated F-actin to observe the binding of these proteins on F-actin. (A) Overlays of structure of unc-60a with cryoelectron microscopy structure of HsCof with F-actin. (B) Overlay of structure of UNC-60B and LdCOF on HsCof/F-actin. In overlays, actin protomers are shown in different colours. Structure of HsCof, UNC-60A, UNC-60B and LdCof are shown in magenta, green, red and cyan respectively. ADF/cofilins are shown in cyan, residues of F-loop showing motion on nanosecond-to-picosecond scale are shown in blue. The structure of HsCof was omitted from overlays of UNC-60B and LdCOF for clarity.

Figure 10
Superimposition of UNC-60A, UNC-60B and LdCof on HsCof in the structure of HsCof–F-actin

To model the binding of different ADF/cofilins with F-actin, the structures of UNC-60A, UNC-60B and LdCof were superimposed on HsCOF in the cryo-electron microscopy structure of human cofilin decorated F-actin to observe the binding of these proteins on F-actin. (A) Overlays of structure of unc-60a with cryoelectron microscopy structure of HsCof with F-actin. (B) Overlay of structure of UNC-60B and LdCOF on HsCof/F-actin. In overlays, actin protomers are shown in different colours. Structure of HsCof, UNC-60A, UNC-60B and LdCof are shown in magenta, green, red and cyan respectively. ADF/cofilins are shown in cyan, residues of F-loop showing motion on nanosecond-to-picosecond scale are shown in blue. The structure of HsCof was omitted from overlays of UNC-60B and LdCOF for clarity.

Comparison of dynamics of UNC-60A and UNC-60B with other ADF/cofilins reveals that the dynamic behaviour of UNC-60A is similar to LdCof in terms of the number of flexible residues present in the F-loop [26]. In UNC-60A five residues (Arg93–Gly97) and in LdCof four residues (Asp73–Ser76) of the F-loop show motion on a nanosecond–picosecond timescale [26]. In UNC-60B, 14 residues of the F-loop show motion on a nanosecond-to-picosecond timescale. In weak severing proteins such as UNC-60A and LdCof, seven and nine salt bridges are formed by the residues of the F-loop with the residues of other regions respectively [27]. In strong severing proteins such as UNC-60B, yeast cofilin, Acanthamoeba actophorin, human cofilin and chick cofilin, only one to three salt bridges are formed by the residues of the F-loop with the residues of other regions [2,12,3537] (Table 2). It is possible that the presence of a higher number of salt bridges in the weak severing proteins UNC-60A and LdCof makes the F-loop relatively rigid in comparison with the strong severing proteins UNC-60B, Acanthamoeba actophorin, human cofilin and chick cofilin. It seems that the weaker severing activity conformation of the F-loop, which is oriented towards the interface of two actin protomers in F-actin, is relatively more rigid. On the other hand, a stronger severing conformation of the F-loop, which is oriented towards the binding region of the F-loop in F-actin, is relatively more flexible. This orientation and flexibility appears to facilitate stable association with F-actin and stronger severing activity towards actin filaments. Overall, these comparisons suggest that both F-loop orientation and flexibility are important determinants of the severing activities and co-sedimentation properties of UNC-60A and UNC-60B.

The G-actin sequestration property of ADF/cofilins may be dependent on their two activities, namely strong pointed end depolymerization and strong inhibition of F-actin polymerization. Inhibition of polymerization/nucleation does not directly correlate with the binding affinity of ADF/cofilins with G-actin, as this affinity is lower for UNC-60A in comparison with UNC-60B. It is possible that the F-loop plays an important role in inhibition of polymerization/nucleation activity as observed in the case of Schizosaccharomyces pombe cofilin (SpCof) where point mutation of Arg78 in the F-loop resulted in a loss of nucleating activity [3]. As described above, juxtaposition of the F-site of UNC-60A at the interface of two longitudinally attached protomers may obstruct the interaction of the helix (Ile287–Leu293) of subdomain 3 of one protomer with the helix (Thr203–Lys215) of subdomain 4 of the other protomer of actin, as shown schematically in Figure 11. This implies that the F-loop and C-terminal of UNC-60A bound to G-actin may present a steric restriction for the approach of a second G-actin monomer longitudinally, which is necessary for polymerization and/or nucleation. Additionally, this region is also more rigid in UNC-60A in comparison with UNC-60B, and this higher rigidity may also contribute towards the inhibition of polymerization and/or nucleation. Therefore, it seems that the properties of weak severing activity and inhibition of polymerization and/or nucleation may share a common basis in terms of F-loop structure and dynamics.

Schematic representation of the role of the F-loop of UNC-60A in inhibiting polymerization/nucleation

Figure 11
Schematic representation of the role of the F-loop of UNC-60A in inhibiting polymerization/nucleation

(A) The actin monomer. (B) The UNC-60A–G-actin docked model. (C and D) An actin monomer is placed on the surface of the UNC-60A–G-actin model, the F-loop of UNC-60A gets in close proximity with the helix (Thr203–Lys215) of subdomain 4 of actin (yellow), which interacts with the helix (Ile287–Leu293) of subdomain 3 of the adjacent protomer (blue) in the co-filament structure of ADF and F-actin.

Figure 11
Schematic representation of the role of the F-loop of UNC-60A in inhibiting polymerization/nucleation

(A) The actin monomer. (B) The UNC-60A–G-actin docked model. (C and D) An actin monomer is placed on the surface of the UNC-60A–G-actin model, the F-loop of UNC-60A gets in close proximity with the helix (Thr203–Lys215) of subdomain 4 of actin (yellow), which interacts with the helix (Ile287–Leu293) of subdomain 3 of the adjacent protomer (blue) in the co-filament structure of ADF and F-actin.

Functional importance of the α3–β6 loop

Residues of the α3–β6 loop (Leu133 and Thr135) of UNC-60A and (Gly122, Leu123 and Leu126) of UNC-60B show conformational flexibility on the millisecond-to-microsecond time scale in both proteins, and similar dynamic behaviour for this region was also observed for TgADF [27]. In the F-loop mutant K91A of UNC-60B, in addition to the residues of the F-site, some residues of the α3–β6 loop also show CSPs. In the K96Q mutant of human cofilin, in addition to the residues of the F-loop and C-terminus, CSPs were also observed for the residues of the α3–β6 loop (Leu132 and His133) [35]. Residues of the α3–β6 loop of human cofilin are close to the residues Lys50, Glu57 and Asn92 of actin protomer n, in the co-filament structure as observed in the cryo-electron microscopy image reconstruction (Supplementary Figure S5). This suggests that, in addition to the previously recognized F-actin-binding site, the α3–β6 loop of ADF/cofilin may be involved in interaction with F-actin.

Structure and dynamics rationale for ADP–G-actin-binding affinity of ADF/cofilins

Structures of many ADF/cofilins and other related ADF-H domains have been characterized and they show conserved common features for G-actin binding, but with different affinities. It indicates that, besides conserved biochemical and structural features, protein dynamics also contributes to binding affinity. G-actin-binding sites in UNC-60A and UNC-60B are well formed and 15N-relaxation dynamics of residues of the G-actin-binding site show desired interfacial flexibility as having a flexible N-terminal (Gly4–Val7 in UNC-60A and Gly4–Lys6 in UNC-60B) and N-terminal of α3 (Lys117, Lys119 and Tyr122 in UNC-60A and Arg105, Leu109 and Tyr110 in UNC-60B) to facilitate the interaction with G-actin. Although the N-terminal is flexible in almost all of the ADF/cofilins, the flexibility at the N-terminal of α3 appears to enhance the binding affinity [27]. As described above, backbone dynamics of G/F-site correlate well with the docking study (Figure 5). In UNC-60A/G-actin and UNC-60B/G-actin docking models, one acidic residue (Asp112 in UNC-60A and Asp100 in UNC-60B) of β5–α3 loop is involved in interaction with the G-actin by forming an H-bond and salt-bridge, and this residue is dynamic in nature at NMR timescale. The presence of an acidic residue at corresponding position is highly conserved in all ADF/cofilins, which strongly suggests that this residue may be involved in binding with G-actin.

In conclusion, we have presented the first solution structures of C. elegans ADF/cofilins, UNC-60A and UNC-60B, which are also the first representatives from the nematode phylum. The main finding of the present study is that, with reference to their putative binding region on F-actin, the relatively flexible vertical orientation of F-site, as observed for UNC-60B, is associated with stronger severing activity and co-sedimentation property, whereas the relatively rigid inclined orientation of F-site, as observed for UNC-60A, is associated with weak severing activity. This conclusion is further corroborated by structural comparisons with other strong and weak severing ADF/cofilin proteins such as yeast cofilin, Acanthamoeba actophorin, human cofilin, chick cofilins and LdCof. UNC-60A and UNC-60B display strong affinity with ADP–G-actin, which results from a structurally and dynamically conserved G/F-site, especially the flexibility of the N-terminal of the long-kinked helix α3.

AUTHOR CONTRIBUTION

Solution structures of UNC-60A and UNC-60B, molecular docking study, mutational analysis, and relaxation dispersion analysis were done by Vaibhav Kumar Shukla. Ashish Kabra and Diva Maheshwari assisted in sample preparation and data analysis. Subcloning of unc60a and unc60b genes was done by Rahul Yadav. Modelfree analysis and ITC experiments were done by Anupam Jain and Sarita Tripathi respectively. Shoichiro Ono provided the clones of unc60a and unc60b in pET3d vector, and gave useful suggestions. Acquisition of relaxation dispersion data and NOESY-HSQC data was done by Dinesh Kumar. Ashish Arora conceived, designed and directed the study, acquired NMR data, analysed the results and wrote the paper.

We are thankful to Dr C.L. Khetrapal for use of 800 MHz NMR spectrometer at the Centre for Biomedical Research, Lucknow. This is the communication number 8812 from CSIR-CDRI.

FUNDING

This work was supported by the Council of Scientific and Industrial Research (CSIR) Network Project UNSEEN and a National Biosciences Award Grant from Department of Biotechnology (DBT) to A.A. V.K.S. and D.M. are recipients of research fellowships from CSIR, New Delhi, India. R.Y. and A.K. are recipients of research fellowships from DBT and Indian Council for Medical Research, New Delhi, India respectively. Work relating to unc-60a and unc-60b clones was supported by a grant from the National Institutes of Health [grant number AR048615 (to S.O.)].

Abbreviations

     
  • ADF

    actin-depolymerizing factor

  •  
  • ADF-H

    ADF-homology

  •  
  • CSP

    chemical shift perturbation

  •  
  • F-actin

    filamentous actin

  •  
  • G-actin

    globular actin

  •  
  • HsCof

    Homo sapiens cofilin

  •  
  • ITC

    isothermal titration calorimetry

  •  
  • LdCof

    Leishmania donovani ADF/cofilin

  •  
  • NCBI

    National Center for Biotechnology Information

  •  
  • PfADF

    Plasmodium falciparum ADF

  •  
  • ScCof

    Saccharomyces cerevisiae cofilin

  •  
  • TgADF

    Toxoplasma gondii ADF

  •  
  • wtUNC

    wild-type UNC

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Author notes

The structural co-ordinates reported for UNC-60A and UNC-60B will appear in the PDB under code 2MP4 and 2LXX respectively.

Supplementary data