Crystal structure of crosslinked WxActin
In two previous studies, we reported low-resolution SAXS structures of actin nucleation complexes formed by the Arp2/3 complex and tandem W domains 22; 23
. Barbed end polymerization in these studies was blocked by crosslinking of the W domain to Cys-374 of the actin subunit located at the barbed end of the complexes. This approach was based on analysis of the structures of various W-actin complexes 11; 12; 13
, which placed the N-terminus of the W domain within disulfide bond distance to actin Cys-374. In each case, a Cys residue was introduced into the W domain at the most favorable position for crosslinking to actin Cys-374. Here, this approach was used again to obtain the low-resolution crystal structure of 3W-Actin. However, it remained unclear whether the crosslink altered the structure of actin and/or the W domain in a significant way, which prompted us to pursue the determination of a crosslinked WxActin structure. It later became apparent that this structure also provided the best molecular replacement model for determination of the structure of 3W-Actin.
After testing crystallization with various W domains, good diffracting crystals were obtained of the crosslinked complex of actin with a synthetic peptide corresponding to the first W domain (amino acids 130–160) of Vibrio parahemolyticus VopL. During synthesis, residue Val-131 of this W domain was replaced by Cys and crosslinked to actin Cys-374 (Materials and Methods). The crystal structure of WxActin was determined by molecular replacement to 2.9 Å resolution ( and ).
FIG. 1 Structure of WxActin. (A) Two perpendicular views of the structure of WxActin. The inset shows the 2Fo-Fc electron density map (contoured at 1σ) in the region around the crosslink. Although the crosslink was visualized, this is one of less well-defined (more ...)
Crystallographic Data and Refinement Statistics
The structure of WxActin is very similar to those of non-crosslinked W-Actin complexes determined with bound DNase I 11; 13
and that of Drosophila
ciboulot bound to actin-latrunculin A 12
. shows a comparison of the structure of WxActin with that of the non-crosslinked complex of actin with the W domain WASP (PDB code 2A3Z). The two structures superimpose with r.m.s deviation of 0.66 Å for 358 equivalent Cα atoms. The most important differences occur in regions that were visualized in one of the structures but not the other, including the DNase I-binding loop (D-loop), the C-terminus of actin, and the N-terminus of the W domain. The D-loop is disordered in most structures of actin, as well as in the structure of WxActin described here, but forms an extended β-sheet with β-strands of DNase I in the non-crosslinked structure. The C-terminus of actin is also disordered in most crystal structures, except complexes with profilin, which interacts with the C-terminus of actin 24; 25; 26
. In the non-crosslinked W-Actin complex, the last 10 amino acids of actin (Gly-366 to Phe-375) are disordered and the W domain is only visualized starting from WASP residue Arg-431 (corresponding to VopL Asn-132). In contrast, in the crosslinked structure only the last amino acid of actin (Phe-375) is unresolved in the electron density map, whereas the W domain of VopL is visualized from residue 130 to 151, i.e. the last nine amino acids of the synthetic peptide were not resolved. The disulfide bond between actin Cys-374 and VopL Cys-131 is visualized in the electron density map (inset in ), although it is poorly defined compared to the rest of the structure.
The similarity of the structures suggests that the crosslinking approach used here and in previous studies 22; 23
as a tool to cap the barbed end of actin polymerization nuclei for structural investigation does not introduce significant structural distortions. Furthermore, as we show next, the availability of the structure of WxActin aided the determination of the structure of 3W-Actin.
Crystal structure of 3W-Actin
The solution SAXS study of 3W-Actin revealed an elongated molecule, consistent with the presence of three actin subunits, somewhat similar to the long-pitch helix of the actin filament 22
. However, the nature of actin-actin contacts in the complex could not be determined. We had suggested that subdomain 2 of actin could move slightly, which combined with a helical conformation in the D-loop, would make the binding of tandem W domains fully compatible with intersubunit contacts in the actin filament 3; 4
. Other investigators had suggested that the W domain would probably interfere with intersubunit contacts in the filament 27; 28
. Knowing which proposal is correct is important, because it may shed light on the mechanism of nucleation, and possibly explain why tandem W domain-based nucleators do not influence elongation the way formins do. It may also answer important questions about differences in the activities of tandem W domain-based nucleators, and the mechanism of action of NPFs of the Arp2/3 complex, which also contain tandem W domains 7
. Therefore, we set out to crystallize the complexes of 2W-Actin and 3W-Actin. While both complexes were crystallized readily, the crystals did not diffract the X-rays. Additional search for conditions led to the identification of additives, such as RbCl and polyvinylpyrrolidone K15, which improved diffraction somewhat. After several attempts, the best result consisted of a rather complete and highly redundant X-ray dataset collected from crystals of 3W-Actin to 7 Å resolution. While we initially considered not reporting this structure, we later recognized that significant information could be obtained by positioning high-resolution W-Actin structures into the unit cell of the 3W-Actin crystals by molecular replacement. Because the individual structures are known at high-resolution, this approach overcomes some of the typical limitations of low-resolution structures in which the content of the unit cell is totally unknown. The limitations, however, are that individual atomic positions cannot be refined and the inter-W linkers cannot be visualized.
Consistent with the design and mass measurements in solution of the complex of 3W-Actin 22
, three copies of the W-Actin basic unit were expected in the asymmetric unit of the crystal. The volume of the asymmetric unit was also compatible with it containing three copies of the W-Actin unit (corresponding to a solvent content of 43%). However, weak diffraction is typically consistent with a higher solvent content. Not surprisingly, the molecular replacement solution, performed independently with the programs Phenix 29; 30
and AMoRe 31
, located only two W-Actin complexes in the asymmetric unit, for a solvent content of 62% (see Material and Methods and detailed description in Supplementary Material
). We do not understand why one of the actin molecules dissociates during crystallization, although it could simply be that this molecule is bound loosely and is therefore displaced by favorable crystal contacts. Analysis of the crystal packing demonstrates why a third actin molecule was never found. Consecutive actin dimers are stacked head-to-tail, forming a helix along the crystallographic c
axis (Movie S1
). Two such helices assemble tightly in anti-parallel fashion (see Movie S2
). Each anti-parallel pair comprises 24 actin subunits along the length of the c
axis, which constitutes the basic building block of the crystal lattice. Adjacent pairs of helices crossover twice in a repeat (or helical turn), corresponding to the length of the c
axis (see Movies S3
), thus assuring the connectivity of the crystal lattice and leaving no extra-space for the missing third actin subunit (or rather 12 actin subunits, when the P65
22 symmetry of the crystal is taken into consideration).
Because of the limited resolution, we could not identify which of the actin subunits is lost during crystallization, or whether the crystals consist solely of the actin subunit crosslinked to the long 3W polypeptide. Note that any non-crosslinked actin dissociated from the complex would be expected to polymerize during crystallization, and would therefore not be present in the crystals. To address this question, a large number of crystals were collected, washed multiple times in the crystallization solution by transferring them with a cryo-loop, and then dissolved in water for analysis by non-reducing gel electrophoresis and mass spectrometry (Materials and Methods). The results clearly illustrate that the crystals consist of a 50/50 mixture of actin crosslinked to construct 3W and non-crosslinked actin (). Therefore, we conclude that one of the non-crosslinked actins was lost during crystallization which, based on the arrangement of actin subunits in the asymmetric unit, is most likely that bound to the last W domain.
FIG. 2 Structure of 3W-Actin. (A) Non-reducing gel electrophoresis and mass spectrometry analysis indicate that the crystals of 3W-Actin consist of a 50/50 mixture of actin crosslinked to construct 3W (expected mass 53,021 Da) and non-crosslinked actin. Actin (more ...)
The disposition of the actin subunits in the structure of 3W-Actin () is somewhat similar to the longitudinal arrangement of actin subunits in the long-pitch helix of the actin filament model 3; 4
(). However, important differences are observed. To better understand these differences, it is important to discuss what is currently known about longitudinal contacts in the actin filament. Multiple crystal structures of actin show similar longitudinal contacts between actin subunits (including both non-crystallographic dimers and symmetry-related dimers), which are thought to mimic inter-subunit contacts in the actin filament (). However, because of constraints imposed by crystal symmetry, these dimers are unwound, i.e. they lack the natural twist of the actin filament. A detailed analysis of these structures and their implications for our understanding of the actin filament has been carried out by other investigators 32
, and will not be repeated here. However, it is important to compare the structure of 3W-Actin to both the actin filament model 3; 4
and the longitudinal dimers observed in crystal structures, with the understanding that the structure of 3W-Actin does not address the conformation of the actin filament per se,
but rather the mechanism of recruitment of actin subunits by tandem W domain proteins.
Structures of Longitudinal Actin Dimers.
The dimers observed in crystal structures are generally similar and often crystallographically isomorphous. Based on a superimposition of their structures, we have identified three subgroups that diverge more significantly (represented by PDB entries 2FXU, 1Y64 and 2HMP) (). Compared to a long-pitch dimer of the actin filament model in which consecutive subunits are rotated by ~27° 3; 4
, these three subgroups present flat structures, i.e. rotated counterclockwise with respect to the filament dimer by approximately −27° () (although the orientation of the axis of rotation is markedly different for entry 2HMP). Remarkably, the longitudinal contacts between subdomains 4 and 3 of neighboring actin subunits are well conserved in the three subgroups (). It thus appears that longitudinal contacts between actin subunits in the filament have a strong tendency to reemerge as crystal contacts in actin structures 32
. It is important to note that these structures offer the most accurate view of longitudinal contacts currently available 32
, because the resolution of the actin filament model 3; 4
is still insufficient to address specific atomic interactions. Additional longitudinal contacts are thought to involve the D-loop in subdomain 2 33
, which is proposed to bind in the hydrophobic cleft between subdomains 1 and 3 of the actin subunit immediately above it 4
. However, the D-loop is disordered in all the crystal structures containing longitudinal actin dimers, and its conformation(s) and actual contacts in the filament are unknown.
FIG. 3 Inter-subunit contacts in the structure of 3W-Actin compared to those of crystallographic actin dimers. Insets show that longitudinal contacts between subdomains 4 and 3 of adjacent actin subunits observed in various crystal structures (right) are mostly (more ...)
On the other hand, the rotation between the two actin subunits in the structure of 3W-Actin is approximately −33°, i.e. −60° compared with a longitudinal dimer of the actin filament 3; 4
(). As a result, the longitudinal contacts observed in other crystal dimers are generally broken in the structure of 3W-Actin (), whereas the contacts involving subdomain 2 are unresolved. Therefore, it appears that the presence of the W domain at the interface between actin subunits breaks the natural tendency that actin has to preserve filament-like longitudinal contacts in crystal structures, and induces a rotation between actin subunits that is of similar magnitude but opposite direction to that of the filament (−33° vs 27°). These results are generally consistent with our previous SAXS studies 22
, which revealed an extended (pseudo long-pitch) arrangement of the actin subunits stabilized by tandem W domains. However, the SAXS envelope lacked the resolution to distinguish between the dimer observed here in the crystal structure of 3W-actin and a longitudinal dimer of the actin filament model.
It is interesting to note that there is also a crystal contact in the structure of 3W-Actin (between two adjacent dimers) that resembles the dimer of the asymmetric unit. This so-called ‘crystal’ dimer differs even more significantly from both the actin filament and the other actin dimers described above, due to an overall translation of ~8 Å between actin subunits compared to the dimer of the asymmetric unit (). In the crystal dimer, the crosslink with construct 3W is at the interface between actin subunits, which may explain the added translation. However, it is significant that the actin subunits of both the non-crystallographic and crystal dimers are rotated counterclockwise by about the same amount compared to all the other actin dimers observed in crystal structures, suggesting that this is a general constraint imposed by the W domain at the interface between actin subunits.
FIG. 4 Comparison of the non-crystallographic and crystallographic dimers in the structure of 3W-Actin. (A) Representation of two consecutive dimers related by crystal symmetry. (B) Superimposition of the non-crystallographic (yellow-blue and red W domains) (more ...)
We conclude that while Spire-like tandem W domains can bring actin subunits into close proximity for nucleation, the conformation of the polymerization nucleus that they form differs significantly from that of the actin filament. This may explain their weak nucleation activity as analyzed next.
Long-pitch nucleation by tandem W domains is suboptimal
The structural results prompted us to test the polymerization activity of construct 3W as compared to those of the prototypical tandem W domain nucleator Spire, which stabilizes a long-pitch nucleus, and the Arp2/3 complex, which forms a short-pitch nucleus. The nucleation activity of Drosophila
has been mapped to the fragment Spire366–482
comprising the four W domains (), which was used in the current study. We used the pyrene-actin polymerization assay to study the effect of Spire366–482
on the polymerization of 2 µM actin (6% pyrene labeled) by monitoring the fluorescence increase resulting from the incorporation of labeled actin monomers into the filament (). At the concentration of 25 nM, Spire366–482
had very little effect on actin polymerization (polymerization rate 1.0±0.2 nM/sec as compared to 0.8±0.1 nM/sec for actin alone), whereas the Arp2/3 complex activated by the WCA fragment of mouse N-WASP showed a major increase in polymerization (polymerization rate 31.5±1 nM/sec). However, the nucleation activity of Spire366–482
increased with concentration, becoming a stronger nucleator at 250 nM (polymerization rate 4.8±0.2 nM/sec). The opposite effect was observed with construct 3W, which had no effect on actin polymerization at the concentration of 25 nM, but inhibited polymerization when used at 250 nM. This could be an indication that construct 3W, like Tβ4, sequesters actin monomers ().
FIG. 5 Different effects of Spire, 3W, and Tβ4 on actin polymerization. (A) Schematic diagram of Tβ4, the four W domain region of Drosophila Spire, and construct 3W. Note that construct 3W consists of three W domains (occurring naturally in mouse (more ...)
Tβ4 is a short 43-aa polypeptide related to the W domain 8; 9
, but it contains an additional helix at the C-terminus that binds atop actin subdomains 2 and 4 34
(). As a result, and despite the apparent simplicity of its helix-loop-helix design, Tβ4 has the ability to block actin monomer addition to both the pointed and barbed ends of the actin filament, making it an extremely effective monomer sequestering protein 35; 36
. Some proteins contain tandem repeats of the Tβ4 fold. Examples include, Acanthamoeba castellanii
, Drosophila melanogaster
and Caenorhabditis elegans
, which respectively contain two-and-a-half, three and four copies of the Tβ4 fold. Contrary to tandem repeats of the W domain, that frequently mediate filament nucleation 7
, tandem Tβ4 proteins are characterized by their ability to sequester actin monomers 37; 38
. Therefore, we asked whether 3W, consisting of a tandem repeat of three W domains followed by the C-terminal helix of Tβ4 (), would sequester actin monomers. A concentration-dependence analysis of steady-state actin polymerization revealed that construct 3W sequesters actin monomers even more effectively than Tβ4 (). We had previously shown, using analytical ultracentrifugation, light scattering and native gel electrophoresis, that 3W binds three actin monomers in solution 22
, which may explain its stronger sequestering activity compared to Tβ4. Therefore, the effect of 3W on actin polymerization is more closely related to that of tetrathymosin, which binds and sequesters multiple actin monomers 38
. Although actobindin and ciboulot also sequester actin monomers, perhaps surprisingly they form 1:1 complexes with actin, indicating that only one of their actin-binding sites is fully functional 12; 37
. It thus appears that the simple addition of the pointed end capping helix of Tβ4 to tandem W domains changes their activity from nucleation, as in Spire 14
, to monomer sequestration as in Tβ4 35; 36