The goal of this study was to get a better insight into the structural basis of the interaction of vertebrate profilins with PI(4,5)-P₂. To investigate the possible role of the above mentioned residues (see Background) in PI(4,5)-P₂-binding and to obtain profilins that have reduced PI(4,5)-P₂-binding capacity, we created a set of single and double mutants in the residues Ser56, Arg74, Arg88, Arg135 and Arg136 of human profilin I (Figure 1 and Table 1) and a mutant W3A defective in poly(L-proline) binding.

Wild type human profilin I as well as the mutants listed in Table 1 were expressed in E. coli and all could be purified by poly(L-proline) affinity chromatography, except for the W3A mutant which does not bind poly(L-proline) (see below). We initially included the R88E mutant, but due to its instability, we were unable to purify this protein in sufficient amounts for biochemical analysis.

We first probed whether the introduced mutations did not affect the conformation and stability by analyzing the conformational integrity of the mutants using circular dichroism (CD) spectra. We measured and compared spectra for wild type and mutant profilins between 184 and 260 nm (Figure 2). All mutants adopt a very similar fold as wild type profilin I. The wavelengths at which maximal and minimal peak values are observed do not or only slightly change. The small shoulders at lower wavelength, observed for the double mutants with R136D, suggests that mutation of this residue to aspartic acid affects in some way the stability or the position of the carboxy terminal α-helix. The differences are however too small to be interpreted quantitatively.

To further test the stability of the mutants, especially the ones that show greatly altered binding to PI(4,5)-P₂ (see below), we measured urea denaturation curves (Figure 3). For R136D and R88A/R136D we observed a very small shift of the transition to lower urea concentration when compared to wild type profilin I. On the contrary, R88E/R136D, which has the most pronounced phenotype (see below) displays a denaturation curve very similar to that of wild type profilin I. Together, these data show that the mutants are stable and correctly folded under the conditions used in the assays described below.

To sensor more subtle effects on the poly(L-proline) binding of the mutants, we used surface plasmon resonance technology to monitor the binding of the mutants to the (GP₅)₃ peptide derived from VASP. The measured reasonance units (RU) for each mutant at three different concentrations are given in Table 1. Although it is not possible to calculate a K_d for profilin I by this method 36, from the obtained RU-values we can deduce relative affinities for the mutants as compared to wild type profilin I (Table 1). The most severe effects are observed for R135D, R136D and double mutants containing one of these mutations. This is logical because Arg135 and Arg136 are located in the carboxy terminal α-helix, which is involved in poly(L-proline) binding. These residues do, however, not directly contact the proline-rich peptide nor do they stabilize any of the crucial poly(L-proline) binding residues 1718. Instead they are oriented outward, away from the poly(L-proline) moiety in the co-crystal. Therefore, the mutations may induce a conformational change in the carboxy terminal helix, which distorts correct orientation of the poly(L-proline) binding residues. But as judged from the CD-spectra and modeling experiments, this structural change is probably very subtle (Figure 2). In addition, mutations may inhibit or facilitate the previously observed conformational changes that occur in profilin upon binding of poly(L-proline) 27. Even though mutations at positions 56, 74 or 88 and combinations thereof are distant from the poly(L-proline) binding site, they also result in lowered poly(L-proline) binding. Remarkably, mutations in this region in yeast profilin caused a similar phenotype 14. Apparently these mutations cause allosteric conformational changes, resulting in less efficient binding of the proline-rich peptide.

We determined the dissociation constants of our mutants for α-skeletal muscle actin using capped filaments (Table 2). Under these conditions, profilin displays only G-actin sequestering activity. In addition, we studied the effect of each mutant on non-steady state actin polymerization (Figure 4). To analyze the obtained curves we determined the amount of F-actin formed at a time point (indicated in Figure 4 as T_1/2) where the amount of F-actin in the absence of profilin is 50% of the amount formed after 1500 sec. In the presence of WT profilin I, only 12% of F-actin is formed at this time point. The values for the mutant profilins are given in Table 2.

We could not calculate a K_d value for R74A, R74E, S56E/R74E, S56E/R74A and R74E/R88E because the concentration of the actin-profilin complex was nearly zero, leading to very high K_d estimates. This is consistent with the observation that these mutants have no activity in the time course polymerization assay. As determined from the crystal structure of the actin-profilin complex 1 and mutagenesis studies 37, Arg74 is a crucial residue for actin binding, since it forms a salt bridge with the carboxyl group of Phe375. Consequently, changing the arginine to an alanine or glutamic acid abolishes this interaction completely. Arg88 is also part of the actin-profilin interface, but changing it to alanine decreases the affinity only three-fold, indicating that the binding is less stringent than for Arg74. Mutating Arg88 to leucine 32 or to glutamic acid in combination with S56E (which on its own has no effect), however, abolished actin binding completely. Arg135 and Arg136 locate in the carboxy terminal helix on the opposite side of the molecule (Figure 1) and do not participate in actin binding. As a consequence, mutations in these residues do not affect the affinity for actin to a significant extent (Figure 4 and Table 2).

We used microfiltration and gel filtration to assay the ability of the mutants to bind PI(4,5)-P₂ (Figure 5, Table 3). The results of both assays were comparable. Based on analogy with invertebrate profilins (see background) and combined with sequence comparison of profilin I and IIa, we expected S56E to contribute negatively to PI(4,5)-P₂-binding. This is, however, not the case and thus this amino acid difference between profilin I and IIa cannot explain the different affinities of the two profilin isoforms for PI(4,5)-P₂. A further difference with invertebrate profilins is the observation that mutating Arg74 to leucine, glutamic acid or alanine (this study and 32) does not significantly affect PI(4,5)-P₂-binding. Since substitution to an acidic residue at this position results in only a slight effect, we consider the contribution of Arg74 in PI(4,5)-P₂-binding to be of minor importance. Consequently, also the double mutants S56E/R74A and S56E/R74E show nearly wild type PI(4,5)-P₂-binding. Previously, it was shown that Arg88 is involved in PI(4,5)-P₂-binding of human profilin I 32, in agreement with several crystal structures showing a phosphate or sulfate anion associated with Arg88 and surrounding residues 3839. In our assays, R88A has a small effect on PI(4,5)-P₂-binding (Figure 5C). Unfortunately we were unable to purify mutant R88E for which we expected a more pronounced phenotype. The effect of the latter mutation can, however, be inferred from the double mutants R74E/R88E and S56E/R88E. Both mutants show reduced PI(4,5)-P₂-binding, compared to S56E, R74E and S56E/R74E which display nearly wild type binding capacity (Table 3).

Interestingly, mutant R136D has a more pronounced effect than R88A (Figure 5C and Table 3). In contrast, mutating the neighboring residue Arg135 has only a small effect on PI(4,5)-P₂-binding. Combining mutations in Arg88 and Arg136 has an additive effect : R88A/R136D and R88E/R136D show a much larger reduction in PI(4,5)-P₂-binding than the single mutants (Figure 5C). This suggests that the reduced PI(4,5)-P₂-binding seen for R136D is due to a direct loss of an interaction. Although we cannot exclude contribution from allosteric effects, modeling experiments substituting R136 with an aspartic acid (data not shown) show no significant change in position of the side-chain or of the carboxy terminal α-helix. We conducted gel filtration experiments at high profilin to PI(4,5)-P₂ ratio's for wild type profilin I and the R136D mutant to assess if the mutation affects overall saturable binding ability. This seems, however, not to be the case (data not shown), since we found for both wild type and mutant a ratio of ten profilin molecules per PI(4,5)-P₂ micelle, suggesting a stoichiometry of 1:8 profilin : PI(4,5)-P₂ molecules, consistent with a previous report 21. Depending on the assay conditions used, variable values for the stoichiometry of the profilin : PI(4,5)-P₂ complex were found, varying between 1:4 and 1:10 321222526. Given this 1:8 stoichiometry, it is difficult to observe the loss of one interaction using PI(4,5)-P₂ micelles. We note, however, that in case of the mutant higher concentrations of profilin and PI(4,5)-P₂ than for wild type profilin were required to obtain saturation, in agreement with the lower affinity of this R136D mutant

Lassing and Lindberg 3 showed that the inhibition on actin polymerization of wild type human profilin I decreases in the presence of PI(4,5)-P₂. If Arg136 is involved in PI(4,5)-P₂-binding, then this mutant should be less affected in its inhibitory activity in the presence of PI(4,5)-P₂. This is indeed what we observe (Figure 6). R136D behaves similar to wild type profilin I in the absence of PI(4,5)-P₂ (Figure 2 and 6). In the presence of a 9-fold molar excess of PI(4,5)-P₂ we observe, however, a significant difference. For R136D we measure only a small reduction in sequestering activity compared to an almost complete inhibition of the sequestering activity of wild type profilin I. In the presence of a 25-fold molar excess of PI(4,5)-P₂, however, R136D loses its sequestering activity completely (data not shown), indicating that the mutation did not entirely abolish PI(4,5)-P₂-binding. This is consistent with the results from the gel filtration experiment (Figure 3C) and implicates a role for other residues such as Arg88.

Recently we demonstrated that profilin IIa has a lower affinity for PI(4,5)-P₂ than profilin I 28. This can be explained with the data presented in this paper. In profilin I, Arg136 is important for PI(4,5)-P₂-binding. In profilin IIa, there is an aspartic acid at this position (Asp136) and the profilin I R136D mutant thus mimics the profilin IIa isoform with respect to PI(4,5)-P₂-binding.

Based on experiments with photoactivatable PI(4,5)-P₂ analogues, Chaudhary and coworkers (1998) 34 suggested that hydrophobic residues in the amino terminal helix are involved in the interaction with PI(4,5)-P₂. Trp3, the fluorescence of which is quenched in the presence of PI(4,5)-P₂40, is spatially close to Arg136 (see Figure 1). Therefore we mutated the former residue to alanine, thereby reducing the hydrophobic moiety. Trp3 is a crucial residue for the interaction of profilin with poly(L-proline) 21415161741 and as expected the W3A mutants of profilin I and IIa lack poly(L-proline) binding and were thus purified using alternative methods (see Materials and Methods). The dissociation constant for the actin-profilin I W3A-complex was similar to that of wild type profilin I (Table 2). The profilin I W3A mutant did not show a significant decrease in PI(4,5)-P₂-binding, suggesting this residue does not directly contribute to the interaction. Interestingly, the profilin IIa W3A mutant shows increased affinity for PI(4,5)-P₂ and the affinity is comparable with that of wild type profilin I (Figure 7). Given the profilin I W3A data presented here and in view of the conformational changes observed upon ligand binding 2740, we propose that mutating Trp3 in profilin IIa promotes/induces a conformation which is more competent for PI(4,5)-P₂-binding (see below).

The data presented here show that in addition to Arg88, Arg136 is involved in PI(4,5)-P₂-binding of mammalian profilin I. Based on our quantitative gel filtration assay, the contribution of Arg136 is in fact more important than that of Arg88 and the double mutant hardly binds PI(4,5)-P₂ micelles. We conclude that the PI(4,5)-P₂ binding sites of profilin are located in two distinct regions of the molecule that are approximately 31 Å apart (see Figure 1). It is remarkable that there are no corresponding positively charged residue(s) in the carboxy terminus of yeast and Acanthamoeba profilins that could account for a similar interaction as found here for human profilin I. This may indicate that the structural basis for the interaction of PI(4,5)-P₂ with profilins from lower and higher eukaryotes is partially different. We also note that Acanthamoeba profilin II has a ten fold lower affinity for PI(4,5)-P₂ than human profilin I 26.

Both PI(4,5)-P₂-binding regions in vertebrate profilins are implicated in the interaction with another profilin ligand. Arg136 is close to several poly(L-proline) binding residues. Not surprisingly, mutations in Arg136 have also strongly decreased poly(L-proline) affinity, although Arg136 itself is not directly contacting proline-rich ligands. On the other hand, Arg88, involved in PI(4,5)-P₂-binding is also part of the actin binding site 1. A partial overlap of actin- and PI(4,5)-P₂-binding sites was also observed for actophorin 42 and gelsolin 334344, suggesting this is the basis for a general regulatory mechanism for several actin binding proteins, whose function is inhibited by PI(4,5)-P₂. Our data thus offer an explanation for the previously observed competition between PI(4,5)-P₂ and the two other profilin ligands : actin 3 and poly(L-proline) 27. This offers a nice model for the regulation of profilin with its different ligands. Since PI(4,5)-P₂ inhibits both actin and poly(L-proline) binding 327, it is conceivable that PI(4,5)-P₂ may have a master regulatory function in the cell. When PI(4,5)-P₂ is hydrolyzed after cell stimulation, profilin may be set free to interact both with proteins containing proline-rich regions and with actin to regulate actin dynamics. The concerted action in vivo of profilin-actin complexes with several proline-rich proteins such as Ena/VASP proteins, N-WASP and formins for the promotion of actin polymerization was suggested previously 91145.

Several of our mutants suggest allosteric communication within vertebrate profilins. Arg88 mutants have reduced poly(L-proline) binding, although this residue is not part of the poly(L-proline) binding pocket. Conversely, W3A (in profilin IIa) influences PI(4,5)-P₂-binding, but appears not to be directly involved in PI(4,5)-P₂-binding as suggested by the data on profilin I W3A, although a W3N mutation in profilin I results in a higher affinity for PI(4,5)-P₂46. These results suggest that the interaction of profilin with PI(4,5)-P₂ and poly(L-proline) involve conformational changes, which have been experimentally observed before 2740. The interaction of profilin with PI(4,5)-P₂ induces an increase in α-helical content 2240. We propose that the local structure of the neighboring binding sites may change upon binding of PI(4,5)-P₂ and poly(L-proline). The fact that W3A of profilin I binds PI(4,5)-P₂ similar to wild type, suggests that profilin I already has the correct conformation for optimal binding of PI(4,5)-P₂ and that mutating Trp3 to alanine does not ameliorate this conformation further (see also below), while an asparagine at position 3 does have a positive effect 46. In contrast, the W3A mutation in profilin IIa increases the affinity for PI(4,5)-P₂, suggesting that this mutation induces a conformational change which optimizes the interaction with PI(4,5)-P₂ despite the presence of an aspartic acid at the nearby position 136. The profilin IIa structure is, however, optimal for strong poly(L-proline) binding. In modeled and energy minimized profilin IIa structures we observed that the terminal α-helices are further apart from each other suggesting better access to the poly(L-proline) binding cleft 27. From this point of view, it is logical to assume that changing the position of these terminal α-helices has dramatic effects on ligand binding. This idea is consistent with our observation that the addition of a myc-tag to the carboxy terminal end of profilin IIa results in the dramatic loss of poly(L-proline) binding despite the fact that all known proline interacting residues are present (Figure 8). The suggested conformational change in profilin IIa-myc does, however, not significantly influence the affinity for actin (Table 2). Similarly, mouse profilin IIb, which has six additional amino acids at its carboxy terminus, does not bind poly(L-proline) 47. In addition, it has been reported that both amino- and carboxy terminal GFP fusion proteins of mammalian profilins display a dramatic loss in poly(Lproline) binding 4849. Some fusion proteins even lack complete poly(L-proline) binding. Therefore we believe that the correct positioning of the terminal α-helices of profilin is a primary requirement for ligand interaction. It is clear that any distortion of the α-helices will reduce the interaction with poly(L-proline).

The profilin I cDNA amplified by polymerase chain reaction from a human cDNA library was subcloned into pET11d 28. Site directed mutagenesis was performed by polymerase chain reaction with mutated oligonucleotide primers and pfu polymerase. Mutations were verified by sequencing. MC1061 E. coli harboring the pT7POL26 plasmid 50 were used for expression of wild type and mutant profilin I, Proteins were subsequently purified by poly(L-proline) affinity chromatography 28. W3A mutants do not bind poly(L-proline), thus the flow-through of the poly(L-proline) column was loaded onto a DEAE column equilibrated in buffer A (20 mM Tris-HCl, pH 8.1, 1 mM EDTA, 1 mM DTT). The column was eluted with a 0 to 500 mM NaCl gradient in buffer A. Profilin eluted with 60 to 130 mM NaCl. The profilin containing fractions were pooled and loaded on a MonoQ column. The flowthrough of this column contained profilin and only very few other proteins. These contaminating proteins were then removed by gel filtration in buffer A.

We purified actin from rabbit skeletal muscle and isolated it as calcium G-actin by Sephadex G200 chromatography in G-buffer (5 mM Tris-HCl, pH 7.7, 0.1 mM CaCl₂, 0.2 mM ATP, 0.2 mM dithiothreitol, 0.01% sodium azide) 5152. Actin was pyrene labeled on cysteine 375 53. Gelsolin was purified from human plasma 54.

We performed CD measurements in the far UV region (184–260 nm) for WT and mutant profilins at a concentration of 15 μM in 7 mM TRIS/HCl, pH 8 in a JASCO J-170 spectropolarimeter using a 1 cm pathway cell. The step resolution was 0.5 nm and the scan speed 20 nm/min. For each sample the average of 9 scans was obtained and spectra were normalized for concentrations.

Profilin was diluted to 2 μM in increasing concentrations of urea (0 to 8 M) in 20 mM Tris-HCl pH 8.1, 1 mM EDTA, 1 mM DTT. The samples were incubated for 15 min. at room temperature and the intrinsic fluorescence change during a wavelength scan between 300 and 400 nm was measured in a Hitachi F4500 spectrophotometer with the excitation wavelength set at 295 nm. We recorded a shift of the emission peak from 332 nm to 352 nm upon denaturation with urea. For each sample we plotted the ratio F(352 nm)/F(332 nm) versus the concentration of urea in that sample (see Figure 3) 55.

A (GP₅)₃ peptide, derived from VASP, was used to compare the affinities of the profilin I mutants on a BiacoreX (Pharmacia). The amino terminally biotinylated peptide was coupled to a streptavidin coated Biacore biosensor chip (Pharmacia). The experiments were carried out and analyzed as described in 36.

The affinity of the profilin mutants for α-actin was determined using gelsolin capped filaments as described in 6. To determine the effect on non-steady state actin polymerization we pre-incubated 10 μM actin (5 % pyrene labeled) with or without 5 μM profilin for 15 minutes at room temperature prior to the addition of a final concentration of 2 mM MgCl₂ and 100 mM KCl. The fluorescence change was recorded using a Hitachi F4500 spectrophotometer.

Microfiltration was performed as described 27 using 4 μM profilin and different concentrations of PI(4,5)-P₂ (Sigma) as indicated in Figure 3A. For gel filtration experiments, 10 μM profilin was pre-incubated with PI(4,5)-P₂ micelles for 30 min on ice prior to loading on a Superdex75 gel filtration column (SMART, Pharmacia). The peak surface of free profilin was determined and used to calculate the percentage of bound and free profilin in each sample.

The competition experiment between actin and PI(4,5)-P₂ was performed with 8 μM Mg²⁺-ATP-G-α-actin (5% pyrene labeled), 4 μM profilin and 36 μM PI(4,5)-P₂ in 5 mM Tris-HCl, 0.2 mM ATP, 0.2 mM dithiothreitol, pH 7 in the absence of Ca²⁺ and Mg²⁺ to avoid precipitation of PI(4,5)-P₂. Profilin and PI(4,5)-P₂-micelles were incubated for 10 minutes on ice prior to addition of actin and subsequent incubation for 10 minutes at room temperature. Polymerization was started by adding KCl to a final concentration of 50 mM.