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. 2015 Feb 27;427(4):824-839.
doi: 10.1016/j.jmb.2014.09.002. Epub 2014 Sep 16.

Filament assembly by Spire: key residues and concerted actin binding

Amy S Rasson  1 , Justin S Bois  1 , Duy Stephen L Pham  1 , Haneul Yoo  1 , Margot E Quinlan  2
Affiliations

Filament assembly by Spire: key residues and concerted actin binding

Amy S Rasson et al. J Mol Biol. .

Abstract

The most recently identified class of actin nucleators, WASp homology domain 2 (WH2) nucleators, use tandem repeats of monomeric actin-binding WH2 domains to facilitate actin nucleation. WH2 domains are involved in a wide variety of actin regulatory activities. Structurally, they are expected to clash with interprotomer contacts within the actin filament. Thus, the discovery of their role in nucleation was surprising. Here we use Drosophila Spire (Spir) as a model system to investigate both how tandem WH2 domains can nucleate actin and what differentiates nucleating WH2-containing proteins from their non-nucleating counterparts. We found that the third WH2 domain in Spir (Spir-C or SC) plays a unique role. In the context of a short nucleation construct (containing only two WH2 domains), placement of SC in the N-terminal position was required for the most potent nucleation. We found that the native organization of the WH2 domains with respect to each other is necessary for binding to actin with positive cooperativity. We identified two residues within SC that are critical for its activity. Using this information, we were able to convert a weak synthetic nucleator into one with activity equal to a native Spir construct. Lastly, we found evidence that SC binds actin filaments, in addition to monomers.

Keywords: Spir; WH2; actin; cytoskeleton; nucleation.

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Figures

Figure 1
Figure 1
Spir and N-WASp domains and constructs used. (A) Schematic of Drosophila Spir domain structure: KIND, kinase noncatalytic C-lobe domain (dark blue), , Spir box (light green), mFYVE, modified Fab1/YOTB/Vac1/EEA1 zinc-binding domain (light blue). Expanded: WH2, WASp homology-2 cluster (red, orange, yellow, and dark green) and linker domain regions (grey). Schematic of Rattus N-WASp domain structure: EVH1, Ena/VASP homology-1 domains (orange), B, basic region (light orange), G, GTPase binding domain (yellow), PolyPro, proline rich region (light green), C, central region (green), A, acidic domain (dark green). Expanded: WH2 domains (dark and light purple). (B) Schematic of the constructs used. Colored boxes represent the WH2 domains reported in (A), the black line represents Linker 3 from Spir, and mutated residues are indicated by an asterisk and shown is (E). The color of the construct name is used in future figures. (C) We compared the amino acid sequence of WH2 domains from proteins containing tandem WH2 domains with ClustalW [44]. The bottom line (consensus>60) reflects the consensus as follows: uppercase is identity; lowercase is consensus level great than 60%. (D) Alignment of Sc from nine species of Spir. The entire domain is highly conserved, including the residues flanking the isoleucine and arginine commonly found in the alpha helix of WH2 domains. (E) Sequences of the mutated WH2 domains and their wild type counterparts. Mutated residues in red are represented by asterisks in (B).
Figure 2
Figure 2
Domain order and key residues affect nucleation. (A) Representative traces of actin polymerization assays monitored by pyrene fluorescence. Minimal nucleation construct variants (0.25 µM) were added to 4 µM actin. (B) Gain of function point mutations convert NaS3d into a construct as potent as Sc3d (Na[fs]S3d). The converse mutations in Sc3d (Sc[qe]3d) result in loss of activity. (A,B) The time until half-maximum polymerization (t1/2) reported is the average of three independent trials +/− standard deviations.
Figure 3
Figure 3
Cooperative binding by tandem WH2 domains depends on domain order. (A) The reported Kd values of WH2 constructs bound to latB-actin are the mean of three independent trials of competition fluorescence anisotropy with Sd-AlexaFluor488 (Figure S3). Na[fs] binds too weakly to determine its affinity for actin monomers. Error bars represent one standard deviation. (B) Representative competition fluorescence anisotropy with Sd-AlexaFluor488 and latB-actin as a function of added Sc3d (red circles), Sd3c (purple circles) or Sc[gs]d (aqua circles). Data are fit with a two-site equilibrium binding model. Regressions are in the same color as the data set. The dashed lines represent modeling assuming the Kd for a second actin monomer is infinite for Sd3c or Sc[gs]d. The grey trace is a theoretical plot based on measured affinities for Sc and Sd, with independent binding. (C) Anisotropy curves of Sc3d, Sc3 and Sc based on measured affinities were normalized for comparison. The grey trace is a theoretical plot based on measured affinities for Sc and Sd, with independent binding.
Figure 4
Figure 4
Inhibition of actin polymerization by WH2 domains does not correlate with actin binding affinity. (A) Representative traces of actin polymerization in the presence of the four wild-type Spir-WH2 domains group together despite very different affinities for actin. (B) Adding Linker 3 has little effect on Sc but markedly decreases inhibition by Sd. (C) Mutations in Na decrease inhibition, making it behave more like Sc and mutations in Sc increase inhibition, making it behave more like Na. (A–C) 3 µM WH2 added to 4 µM actin is shown in all cases.
Figure 5
Figure 5
Effects of individual WH2 domains on actin nucleation. (A) Representative pyreneactin polymerization assays of different WH2 domain constructs (1.5 µM) added to actin (2 µM) during nucleation. Each trace is colored according to the actin binding affinity of the added WH2 domain according to the inset heat map (red is high affinity). If the effect on nucleation correlated to affinity, the traces would have a rainbow-like trend, but it does not.
Figure 6
Figure 6
Elongation rates of actin determined by TIRF microscopy. (A) Example of seeded actin (red) and elongation (green) in the presence of Sc observed by TIRF. (B) Actin elongation rates in the presence of 1 µM of the indicated WH2 domains. The values in parentheses are the predicted concentrations of free actin, given the Kds determined in this study. Asterisk indicates statistical significance (p < 0.01) when compared with actin alone (n is the number of filaments analyzed for each case; error bars are one standard deviation).
Figure 7
Figure 7
Effect of individual WH2 domains on actin at steady state. Raw data (black symbols) and analysis (blue and red traces) of steady state polymerization titration (SSPT) assays for all nine constructs are shown. Each concentration was tested three times, represented by different symbols. Predicted values based on the model described in the appendix, with Kd fixed by our anisotropy measurements, are shown in blue. Extending this model to include filament binding (Kdf) is shown in red. Due to extremely weak binding, we could not analyze the Na[fs] curves. Sb was not analyzed because it did not reached steady state, as evidenced by the concavity of the titration curves. No red trace is shown for Na because the regression did not converge.
Figure 8
Figure 8
Effects of Kdb and Kdf on steady state actin polymerization. For these illustrative curves, Kda is that determined for 10% pyrene labeled actin and Kd = 0.2 µM. The black curves have Kdb or Kdf → ∞. (A) SSPT curves for various values of Kdb with Kdf → ∞. (B) SSPT curves for various values of Kdf.
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