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• W2005473412 abstract "A major factor in profilin regulation of actin cytoskeletal dynamics is its facilitation of G-actin nucleotide exchange. However, the mechanism of this facilitation is unknown. We studied the interaction of yeast (YPF) and human profilin 1 (HPF1) with yeast and mammalian skeletal muscle actins. Homologous pairs (YPF and yeast actin, HPF1 and muscle actin) bound more tightly to one another than heterologous pairs. However, with saturating profilin, HPF1 caused a faster etheno-ATP exchange with both yeast and muscle actins than did YPF. Based on the -fold change in ATP exchange rate/Kd, however, the homologous pairs are more efficient than the heterologous pairs. Thus, strength of binding of profilin to actin and nucleotide exchange rate are not tightly coupled. Actin/HPF interactions were entropically driven, whereas YPF interactions were enthalpically driven. Hybrid yeast actins containing subdomain 1 (sub1) or subdomain 1 and 2 (sub12) muscle actin residues bound more weakly to YPF than did yeast actin (Kd = 2 μm versus 0.6 μm). These hybrids bound even more weakly to HPF than did yeast actin (Kd = 5 μm versus 3.2 μm). sub1/YPF interactions were entropically driven, whereas the sub12/YPF binding was enthalpically driven. Compared with WT yeast actin, YPF binding to sub1 occurred with a 5 times faster koff and a 2 times faster kon. sub12 bound with a 3 times faster koff and a 1.5 times slower kon. Profilin controls the energetics of its interaction with nonhybrid actin, but interactions between actin subdomains 1 and 2 affect the topography of the profilin binding site. A major factor in profilin regulation of actin cytoskeletal dynamics is its facilitation of G-actin nucleotide exchange. However, the mechanism of this facilitation is unknown. We studied the interaction of yeast (YPF) and human profilin 1 (HPF1) with yeast and mammalian skeletal muscle actins. Homologous pairs (YPF and yeast actin, HPF1 and muscle actin) bound more tightly to one another than heterologous pairs. However, with saturating profilin, HPF1 caused a faster etheno-ATP exchange with both yeast and muscle actins than did YPF. Based on the -fold change in ATP exchange rate/Kd, however, the homologous pairs are more efficient than the heterologous pairs. Thus, strength of binding of profilin to actin and nucleotide exchange rate are not tightly coupled. Actin/HPF interactions were entropically driven, whereas YPF interactions were enthalpically driven. Hybrid yeast actins containing subdomain 1 (sub1) or subdomain 1 and 2 (sub12) muscle actin residues bound more weakly to YPF than did yeast actin (Kd = 2 μm versus 0.6 μm). These hybrids bound even more weakly to HPF than did yeast actin (Kd = 5 μm versus 3.2 μm). sub1/YPF interactions were entropically driven, whereas the sub12/YPF binding was enthalpically driven. Compared with WT yeast actin, YPF binding to sub1 occurred with a 5 times faster koff and a 2 times faster kon. sub12 bound with a 3 times faster koff and a 1.5 times slower kon. Profilin controls the energetics of its interaction with nonhybrid actin, but interactions between actin subdomains 1 and 2 affect the topography of the profilin binding site. The interaction of actin with the small protein profilin is central to the regulation of actin filament dynamics within the cell. Profilin was first identified as a protein that bound to G-actin and, through sequestration of actin monomers, inhibited filament formation (1Southwick F.S. Young C.L. J. Cell Biol. 1990; 110: 1965-1973Crossref PubMed Scopus (67) Google Scholar, 2Babcock G. Rubenstein P.A. Cell Motil. Cytoskeleton. 1993; 24: 179-188Crossref PubMed Scopus (22) Google Scholar). The majority of subsequent work demonstrated that profilin exhibited a preference for ATP versus ADP actin and catalyzed the exchange of actin-bound adenine nucleotide (3Romero S. Didry D. Larquet E. Boisset N. Pantaloni D. Carlier M.F. J. Biol. Chem. 2007; 282: 8435-8445Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 4Pantaloni D. Carlier M.F. Cell. 1993; 75: 1007-1014Abstract Full Text PDF PubMed Scopus (456) Google Scholar, 5Vinson V.K. De La Cruz E.M. Higgs H.N. Pollard T.D. Biochemistry. 1998; 37: 10871-10880Crossref PubMed Scopus (130) Google Scholar). However, a few studies have disputed this nucleotide preference (6Kinosian H.J. Selden L.A. Gershman L.C. Estes J.E. Biochemistry. 2002; 41: 6734-6743Crossref PubMed Scopus (44) Google Scholar, 7Selden L.A. Kinosian H.J. Estes J.E. Gershman L.C. Biochemistry. 1999; 38: 2769-2778Crossref PubMed Scopus (66) Google Scholar). Later work showed that profilin could also work with the actin filament nucleator, formin, to promote filament elongation by delivering actin monomers to the growing end of the formin-capped filament (8Vavylonis D. Kovar D.R. O'Shaughnessy B. Pollard T.D. Mol. Cell. 2006; 21: 455-466Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 9Kovar D.R. Harris E.S. Mahaffy R. Higgs H.N. Pollard T.D. Cell. 2006; 124: 423-435Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar, 10Romero S. Le Clainche C. Didry D. Egile C. Pantaloni D. Carlier M.F. Cell. 2004; 119: 419-429Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar).The ability of profilin to preferentially sequester ATP G-actin and to facilitate adenine nucleotide exchange from the actin is important, considering the role that an actin-dependent ATP hydrolysis cycle plays in actin dynamics. G-actin is a poor ATPase whose activity is stimulated by polymerization. Subsequent discharge of the Pi from ADP-Pi F-actin occurs at different rates, depending on the particular actin isoform involved, and results in the generation of ADP-F-actin (11Yao X. Rubenstein P.A. J. Biol. Chem. 2001; 276: 25598-25604Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 12Carlier M.F. Adv. Biophys. 1990; 26: 51-73Crossref PubMed Scopus (65) Google Scholar). In terms of filament stability, ATP and ADP-Pi actin are more stable than ADP-F-actin (13Carlier M.F. J. Biol. Chem. 1991; 266: 1-4Abstract Full Text PDF PubMed Google Scholar). After release of ADP-monomers from the end of the actin filament, the ADP exchanges for ATP, and the polymerization cycle starts again. Profilin may have a role in facilitating this exchange and thereby help to regulate the dynamics of actin filament formation (13Carlier M.F. J. Biol. Chem. 1991; 266: 1-4Abstract Full Text PDF PubMed Google Scholar). However, the necessity for this rate enhancement may not be universally applicable. For example, plant profilin does not catalyze nucleotide exchange from actin (14Perelroizen I. Didry D. Christensen H. Chua N.H. Carlier M.F. J. Biol. Chem. 1996; 271: 12302-12309Abstract Full Text PDF PubMed Scopus (123) Google Scholar). However, it can complement a profilin-deficient strain of Dictyostelium discoideum with little if any loss of normal cell behavior (15Arasada R. Gloss A. Tunggal B. Joseph J.M. Rieger D. Mondal S. Faix J. Schleicher M. Noegel A.A. Biochim. Biophys. Acta. 2007; 1773: 631-641Crossref PubMed Scopus (10) Google Scholar).Although profilin generally is not thought of as an enzyme, it actually acts as one in its facilitation of the actin nucleotide exchange reaction. It must first reversibly bind to the actin and then cause a conformational change that results in enhanced rates of release of the bound adenine nucleotide. Initial studies revealed that the extent of the enhancement is highly dependent on the type of profilin used. For example, under saturating conditions, with muscle actin, mammalian profilin enhances the rate of exchange 30-1000-fold (16Goldschmidt-Clermont P.J. Machesky L.M. Doberstein S.K. Pollard T.D. J. Cell Biol. 1991; 113: 1081-1089Crossref PubMed Scopus (181) Google Scholar), yeast profilin enhances it 3-fold (17Eads J.C. Mahoney N.M. Vorobiev S. Bresnick A.R. Wen K.K. Rubenstein P.A. Haarer B.K. Almo S.C. Biochemistry. 1998; 37: 11171-11181Crossref PubMed Scopus (69) Google Scholar), and profilin from the plant Arabidopsis shows no enhancement of exchange rate (14Perelroizen I. Didry D. Christensen H. Chua N.H. Carlier M.F. J. Biol. Chem. 1996; 271: 12302-12309Abstract Full Text PDF PubMed Scopus (123) Google Scholar).Initial studies also suggest that the nature of the binding of profilin to actin seems to depend on both the profilin and the actin involved. For example, human platelet profilin binds muscle actin about 50-fold more tightly than it binds yeast actin (17Eads J.C. Mahoney N.M. Vorobiev S. Bresnick A.R. Wen K.K. Rubenstein P.A. Haarer B.K. Almo S.C. Biochemistry. 1998; 37: 11171-11181Crossref PubMed Scopus (69) Google Scholar). The energetics that defines the actin-profilin interaction can be very different depending on the particular actin-profilin pair involved. Based on incomplete data obtained so far, change in enthalpy seems to control the interaction of yeast profilin with muscle actin, whereas change in entropy seems to drive the interaction of human profilin with muscle actin (17Eads J.C. Mahoney N.M. Vorobiev S. Bresnick A.R. Wen K.K. Rubenstein P.A. Haarer B.K. Almo S.C. Biochemistry. 1998; 37: 11171-11181Crossref PubMed Scopus (69) Google Scholar). Whether this difference applies only to these two actin-profilin pairs or is more general is not known. Insight into the molecular basis of the profilin-actin interaction came from the crystallographic studies of the profilin-actin complex carried out by Schutt and co-workers (18Schutt C.E. Myslik J.C. Rozycki M.D. Goonesekere N.C. Lindberg U. Nature. 1993; 365: 810-816Crossref PubMed Scopus (594) Google Scholar, 19Chik J.K. Lindberg U. Schutt C.E. J. Mol. Biol. 1996; 263: 607-623Crossref PubMed Scopus (184) Google Scholar). Profilin binds to actin across actin subdomains 1 and 3 at the barbed end of the actin monomer, and this interaction seems to result in an opening of the cleft separating the two domains of the actin molecule in which the ATP resides. This opening occurs by a pivoting motion of the two domains around a hinge region involving a helix containing residues 137-144 in the bridge between subdomains 1 and 3 (20Page R. Lindberg U. Schutt C.E. J. Mol. Biol. 1998; 280: 463-474Crossref PubMed Scopus (83) Google Scholar). However, the manner in which this movement is brought about is not understood.To gain more insight into the mechanism governing the profilin-dependent acceleration of the release of actin-bound nucleotide, we have carried out a detailed study of both the binding and exchange reactions involving the interaction of both yeast and human profilins with both muscle and yeast actins. We were especially interested in the yeast/muscle actin comparison, because yeast actin inherently exchanges its nucleotide 30 times faster than does muscle actin despite the 87% homology between the two proteins (17Eads J.C. Mahoney N.M. Vorobiev S. Bresnick A.R. Wen K.K. Rubenstein P.A. Haarer B.K. Almo S.C. Biochemistry. 1998; 37: 11171-11181Crossref PubMed Scopus (69) Google Scholar, 21Rubenstein P.A. BioEssays. 1990; 12: 309-315Crossref PubMed Scopus (196) Google Scholar). We also present work with a hybrid actin we have constructed in which subdomains 1 and 2 are from muscle actin and subdomains 3 and 4 are from yeast actin to better understand the relative importance of subdomains 1 and 3 in its interaction with profilin.MATERIALS AND METHODSProtein Preparations—Yeast hybrid and H372R mutant actins were generated as described previously (22McKane M. Wen K.K. Meyer A. Rubenstein P.A. J. Biol. Chem. 2006; 281: 29916-29928Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 23McKane M. Wen K.K. Boldogh I.R. Ramcharan S. Pon L.A. Rubenstein P.A. J. Biol. Chem. 2005; 280: 36494-36501Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Yeast wild type (WT) 2The abbreviations used are: WTwild typeITCisothermal titration calorimetryϵATPetheno-ATP. and mutant actins were purified by a combination of DNase I affinity chromatography and DEAE-cellulose chromatography as described by Cook et al. (24Cook R.K. Sheff D.R. Rubenstein P.A. J. Biol. Chem. 1991; 266: 16825-16833Abstract Full Text PDF PubMed Google Scholar). Globular actins (G-actins) were stored in G buffer (10 mm Tris-HCl, pH 7.5, 0.2 mm CaCl2, 0.2 mm ATP, and 0.1 mm dithiothreitol) at 4 °C and used within 5 days. The yeast profilin and human profilin I Escherichia coli expression plasmids were kindly provided by S. Almo (Albert Einstein College of Medicine) and D. Schafer (University of Virginia), respectively. The mutant human profilin I molecules were engineered using the QuikChange® site-directed mutagenesis kit from Stratagene (La Jolla, CA). All profilins were expressed by E. coli BL21 and purified with a procedure similar to that described by Eads et al. (17Eads J.C. Mahoney N.M. Vorobiev S. Bresnick A.R. Wen K.K. Rubenstein P.A. Haarer B.K. Almo S.C. Biochemistry. 1998; 37: 11171-11181Crossref PubMed Scopus (69) Google Scholar) with modifications. Briefly, the cells expressing the profilin were lysed by sonication in the presence of 1 unit/μl rLysozyme™ (Novagen, San Diego, CA) and 50 μg/ml DNase I (Worthington). The profilin in the cell supernatant obtained by centrifugation of the cell lysate was further purified by polyproline affinity chromatography and Q Sepharose fast flow chromatography. SDS-PAGE of the final material on 15% acrylamide gels revealed a single band. The concentration of either yeast profilin or human profilin was determined spectrophotometrically using an ϵ280 of 20,300 and 10,800 m-1 cm-1, respectively (17Eads J.C. Mahoney N.M. Vorobiev S. Bresnick A.R. Wen K.K. Rubenstein P.A. Haarer B.K. Almo S.C. Biochemistry. 1998; 37: 11171-11181Crossref PubMed Scopus (69) Google Scholar). Purified profilins were stored at 4 °C.Etheno-ATP (ϵATP)-bound Actin Preparation and Actin Binding Assay—ϵATP-bound G-actin was prepared as described by Wen (25Wen K.K. Yao X. Rubenstein P.A. J. Biol. Chem. 2002; 277: 41101-41109Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar) with modifications. Briefly, the free nucleotide was removed from G-actin stock solutions by centrifugation through Zeba Desalting Spin Columns (Pierce) preequilibrated with G0 buffer (10 mm Tris-HCl, pH 7.5, 0.4 mm CaCl2, and 1 mm dithiothreitol) at 4 °C as described by the manufacturer's protocol. A 20 μm G-actin solution in ATP-free G-buffer was incubated with ϵATP (Invitrogen) at a final concentration of 1 mm at 4 °C overnight. Unbound nucleotide was removed by desalting as above in the presence of 0.4 mm CaCl2, which was present throughout the remainder of the procedure. Thus, following the final desalting, the solution containing the 20 mm nucleotide-G-actin complex was in the presence of 0.4 mm CaCl2. Profilin at various concentrations was mixed at 25 °C in G0 buffer with 1 μm ϵATP G-actin to a final volume of 1.5 ml. Free ATP was added to the reaction mixture to a final concentration of 250 μm with constant stirring. Thus, at this time, for all experiments, the free Ca2+ was ∼150 mm for all reactions tested.The decrease in fluorescence due to the disassociation of actin-bound ϵATP was monitored over time with a Fluorolog-3 instrument containing a thermostatted sample holder (Spex, Edison, NJ) at an excitation wavelength of 340 nm and an emission wavelength of 410 nm. The net fluorescence change was determined at each time point and normalized against the total fluorescence change at the end of the exchange. The actin-bound ϵATP disassociation rate of each experiment was obtained by fitting each individual normalized data set to a single-exponential function by using Excel software (Microsoft).For assessing the effects of profilin on the nucleotide exchange rate, the data at different profilin concentrations were analyzed by Excel and fitted to Equation 1, kobs[A]T=ka[A]T+(kpa-ka)·(Kd+[P]+[A]T)-(Kd+[P]+[A]T)2-(4[P][A]T)2(Eq. 1) which is derived from Equation 2, kobs=ka[A][A]T+kpa[PA][A]T(Eq. 2) where kobs is the observed disassociation rate constant, ka is the disassociation rate constant of G-actin in the absence of profilin, kpa is the theoretical disassociation rate constant of the profilin-G-actin complex, [A]T is the concentration of total actin, [A] is the concentration of free G-actin, [P] is the concentration of free profilin, and Kd is the dissociation equilibrium constant of the complex. To obtain the best fit, kpa was subjected to the constraint that it is larger than ka.Isothermal Titration Calorimetry (ITC)—ITC measurements were performed using a VP-ITC calorimeter (MicroCal, Northampton, MA). The concentrations of the profilin and actin were measured by UV absorption at 280 or 290 nm as described above, and the proteins were degassed before each experiment. Titrations were performed in 20 mm PIPES, pH 7.5, 0.2 mm ATP, 0.2 mm CaCl2, and 1 mm dithiothreitol. The concentrations of profilin and the actin mutants varied among experiments, and all interactions were repeated two times. Heats of dilution were calculated by averaging the last 3-5 injections and then were subtracted from the raw data. The data sets were then analyzed individually using a single-site binding model from the ORIGIN ITC analysis software package provided by the VP-ITC calorimeter manufacturer. In this analysis, the values for stoichiometry (n), change in enthalpy (ΔH), and the affinity constant (Ka) were fit using nonlinear least squares analysis. The reported values for n, ΔH, and Ka are the average and S.D. of all injections for an individual interaction.Kinetics of Profilin Binding to G-actin—The kinetics of the binding of profilin to G-actin was monitored over time by the decrease in intrinsic tryptophan fluorescence caused by the interaction using a BioLogic SFM3 stopped-flow instrument (BioLogic). The data were further analyzed with Kinsim/Fitsim software (available on the World Wide Web) and fitted to the following model (Reaction 1),where A represents G-actin, P is profilin, and AP is the actin-profilin complex. The kinetic rate constants (kon and koff) of profilin binding to actin were obtained from the average of fitting data from at least three sets of experiments with different profilin concentrations. The Kd is calculated from the results of koff/kon.RESULTSOur goal was to determine the molecular basis underlying the ability of profilin to facilitate the exchange of nucleotide from actin. Toward this end, we analyzed the interaction of yeast and human profilins with yeast and muscle actin. These two actin isoforms are 87% identical in sequence (21Rubenstein P.A. BioEssays. 1990; 12: 309-315Crossref PubMed Scopus (196) Google Scholar), and they only vary in three residues in what is thought to constitute the profilin binding surface (17Eads J.C. Mahoney N.M. Vorobiev S. Bresnick A.R. Wen K.K. Rubenstein P.A. Haarer B.K. Almo S.C. Biochemistry. 1998; 37: 11171-11181Crossref PubMed Scopus (69) Google Scholar). Residues Glu167, Tyr169, and Arg372 in muscle actin are replaced by Ala167, Phe169, and His372 in yeast actin, respectively.We first assessed the rate of nucleotide exchange brought about by increasing concentrations of profilin in the presence of a constant amount of actin. In this analysis, the Kd for the actin-profilin interaction can be derived from the first-order rate constants for the decrease in fluorescence resulting from the exchange of bound ϵATP from the actin surface. This analysis has been used previously (5Vinson V.K. De La Cruz E.M. Higgs H.N. Pollard T.D. Biochemistry. 1998; 37: 10871-10880Crossref PubMed Scopus (130) Google Scholar) and is described under “Materials and Methods.” Fig. 1A shows the increase in nucleotide exchange rate from yeast actin caused by saturating concentrations of yeast profilin (YPF) and human profilin 1 (HPF1). HPF1 produces a 10-fold increase in this rate, whereas YPF produces only a 4-fold acceleration (Table 1). For muscle actin with YPF, there was 3-fold activation (17Eads J.C. Mahoney N.M. Vorobiev S. Bresnick A.R. Wen K.K. Rubenstein P.A. Haarer B.K. Almo S.C. Biochemistry. 1998; 37: 11171-11181Crossref PubMed Scopus (69) Google Scholar), and with HPF1 and muscle actin, there was a 35-fold enhancement (data not shown). Clearly, the small enhancement of nucleotide exchange previously observed with yeast actin and yeast profilin does not result from some maximal rate at which a yeast actin can exchange nucleotide due to its inherently more open conformation (17Eads J.C. Mahoney N.M. Vorobiev S. Bresnick A.R. Wen K.K. Rubenstein P.A. Haarer B.K. Almo S.C. Biochemistry. 1998; 37: 11171-11181Crossref PubMed Scopus (69) Google Scholar, 26Belmont L.D. Orlova A. Drubin D.G. Egelman E.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 29-34Crossref PubMed Scopus (138) Google Scholar). HPF1, at saturating conditions, is simply a better catalyst of nucleotide exchange than YPF, whether yeast or muscle actin is utilized.TABLE 1Experimental parameters for the actin-bound ∈ATP exchange for the profilin-actin isoform interactionYPFHPF1-Fold increasea-Fold increase: k (profilin at saturating concentration)/k (no profilin).KdbKd is obtained from fitting the data described in the legend to Fig. 1B with Equation 1 as described under “Materials and Methods.”EfficiencycEfficiency = -fold increase in exchange rate/Kd.-Fold increaseKdEfficiency-foldμmμm−1-foldμmμm−1Yeast actin40.6dIn our case, the binding of YPF to yeast actin is about 3-fold stronger than previously determined (17).6.7103.23a -Fold increase: k (profilin at saturating concentration)/k (no profilin).b Kd is obtained from fitting the data described in the legend to Fig. 1B with Equation 1 as described under “Materials and Methods.”c Efficiency = -fold increase in exchange rate/Kd.d In our case, the binding of YPF to yeast actin is about 3-fold stronger than previously determined (17Eads J.C. Mahoney N.M. Vorobiev S. Bresnick A.R. Wen K.K. Rubenstein P.A. Haarer B.K. Almo S.C. Biochemistry. 1998; 37: 11171-11181Crossref PubMed Scopus (69) Google Scholar). Open table in a new tab Fig. 1B shows the rate constants calculated from curves similar to those described in Fig. 1A for the interaction of different concentrations of YPF or HPF1 with yeast actin. Table 1 shows that like the case involving the two profilins with muscle actin (17Eads J.C. Mahoney N.M. Vorobiev S. Bresnick A.R. Wen K.K. Rubenstein P.A. Haarer B.K. Almo S.C. Biochemistry. 1998; 37: 11171-11181Crossref PubMed Scopus (69) Google Scholar), the homologous pair had a Kd about 5 times stronger than that exhibited by the heterologous pair. However, it is apparent from the data that Kd and rate catalytic ability do not strictly correlate.A better measure of catalytic activity with respect to enzyme function is the catalytic efficiency or kcat/Kd, since most enzymes do not work at saturating conditions. This may very well be the case in vivo for profilins (16Goldschmidt-Clermont P.J. Machesky L.M. Doberstein S.K. Pollard T.D. J. Cell Biol. 1991; 113: 1081-1089Crossref PubMed Scopus (181) Google Scholar, 27Magdolen V. Drubin D.G. Mages G. Bandlow W. FEBS Lett. 1993; 316: 41-47Crossref PubMed Scopus (60) Google Scholar). To apply this analysis to the profilin/actin system, for a given actin, we divided the -fold enhancement of rate exchange by the Kd for the particular actin-profilin pair. With yeast actin, this efficiency measure was 6.7 μm-1 for YPF and only 3 μm-1 for HPF1 (Table 1). Based on the data referred to above, the catalytic efficiency for the HPF1-muscle actin pair is 350 μm-1. From the work of Eads et al. (17Eads J.C. Mahoney N.M. Vorobiev S. Bresnick A.R. Wen K.K. Rubenstein P.A. Haarer B.K. Almo S.C. Biochemistry. 1998; 37: 11171-11181Crossref PubMed Scopus (69) Google Scholar), the catalytic efficiency of the YPF-muscle actin pair is about 1 μm-1. In summary, the homologous set of proteins was always more efficient than the heterologous pair.We next used ITC to examine the energetics that characterized the interaction of yeast actin with yeast and human profilins. Fig. 2A shows the experimental data for the repeated injection of YPF into a solution of yeast actin, and Fig. 2B presents the corrected integrated data with a curve fit for the data in Fig. 2A. Values for ΔH and TΔS were then extracted from these data as described under “Materials and Methods.” Table 2 shows these values along with the corresponding values for Ka or Kd for the interaction of yeast and human profilins with yeast actin. For the sake of comparison, values obtained previously for the interaction of these two profilins with muscle actin are shown (17Eads J.C. Mahoney N.M. Vorobiev S. Bresnick A.R. Wen K.K. Rubenstein P.A. Haarer B.K. Almo S.C. Biochemistry. 1998; 37: 11171-11181Crossref PubMed Scopus (69) Google Scholar). With both actins, the interaction with YPF is strongly enthalpically driven. The change in entropy is actually unfavorable. Conversely, for HPF1, although the change in enthalpy is favorable, it is much less so than for yeast actin. This lower ΔH, however, is offset by a favorable change in entropy. The data demonstrate that the nature of the profilin isoform and not the actin isoform appears to dictate the energetics that characterize the interaction of these two proteins. It is interesting that the nature of the interactions is so different, considering that the profilin binding surface on actin is very much alike for the different isoforms involved.FIGURE 2ITC analysis for the interaction of yeast WT actin with yeast profilin. A, the raw data for a total of 290 μl of 250 μm yeast profilin was injected in 20 aliquots into 2 ml of 20 μm yeast WT actin in ITC G buffer (2 mm PIPES, pH 7.5, 0.2 mm CaCl2, 0.2 mm ATP, and 1 mm dithiothreitol). B, the corrected integrated data with a curve fit for the data in A.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 2Thermodynamic parameters obtained from ITC for the interaction between different profilins and actinsYPFHPF1-yeast actinHuman platelet profilin-muscle actinaThe data for the binding of profilins with muscle actin as determined by Edas et al. (17).Yeast actinMuscle actinaThe data for the binding of profilins with muscle actin as determined by Edas et al. (17).ΔH (kcal/mol)−10 ± 0.5−12.1−4.4 ± 0.2−3.4TΔSbThe value is indirectly obtained for the computer simulation as described under “Materials and Methods.” (kcal/mol)−1.3−4.52.746.2Ka (μm−1)2.6 ± 0.30.2 ± 0.05Kd (μm)0.42.95.50.1a The data for the binding of profilins with muscle actin as determined by Edas et al. (17Eads J.C. Mahoney N.M. Vorobiev S. Bresnick A.R. Wen K.K. Rubenstein P.A. Haarer B.K. Almo S.C. Biochemistry. 1998; 37: 11171-11181Crossref PubMed Scopus (69) Google Scholar).b The value is indirectly obtained for the computer simulation as described under “Materials and Methods.” Open table in a new tab Role of Human Profilin Residue Glu82 in the Actin-bound Nucleotide Exchange—We wished to gain insight into the reason that HPF1 produced a faster nucleotide exchange rate than did YPF. In comparison with YPF, mammalian profilins contain an extra loop between Leu78 and Asp86. Based on the β-actin-bovine profilin co-crystal (18Schutt C.E. Myslik J.C. Rozycki M.D. Goonesekere N.C. Lindberg U. Nature. 1993; 365: 810-816Crossref PubMed Scopus (594) Google Scholar), Glu82 in this loop might form a hydrogen bond with actin Lys113 in subdomain 1, which is conserved in both yeast and muscle actins, as shown in Fig. 3. Lys113 is located on the back face of the actin monomer close to His73 and the hinge region. This extra actin Lys113-profilin Glu82 hydrogen bond interaction might enhance the ability of profilin to open the actin cleft, leading to the greater rate of exchange that is observed. To test this hypothesis, we mutated HPF1 Glu82 to Lys, Ser, or Ala and assessed the actin-bound ϵATP release rate in the presence of the mutant profilins. Fig. 4 demonstrates that at saturating concentrations, all three mutant profilins facilitate the yeast actin nucleotide exchange 9-13-fold, respectively, similar to the value obtained with WT HPF1. The mutations also cause little if any effect on the Kd for the actin-profilin interactions (data not shown). A similar enhancement for muscle actin-bound ATP was also observed (data not shown). Thus, the ability to form this postulated hydrogen bond is not critical for catalysis of nucleotide exchange or for the affinity of the interaction (data not shown).FIGURE 3Location of actin Lys113 and profilin Glu82 in the actin-profilin complex. Actin Lys113 (green) and bovine profilin Glu82 (blue) are highlighted in the co-crystal structure of β-actin (light red) and bovine profilin (light blue) (Protein Data Bank code 2BTF). The ribbon structure of the extra loop from Leu78 to Asp86 in bovine profilin, absent in YPF, is in red. His73 (purple) and the hinge region (brown ribbon) are highlighted.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4Time course of ϵATP exchange of yeast actin with mutant human profilin I. The release of actin-bound ϵATP bound from 1 μm yeast actin in G buffer without free ATP in the presence of 15 μm HPF1 E82S (□), E82A (○), or E82K (○) was triggered by the addition of 250 μm ATP. The decrease in fluorescence caused by ϵATP exchange was followed over time, and data were fit to a first order reaction mechanism (solid lines) as described under “Materials and Methods.” The exper" @default.
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• W2005473412 date "2008-04-01" @default.
• W2005473412 modified "2023-10-16" @default.
• W2005473412 title "Control of the Ability of Profilin to Bind and Facilitate Nucleotide Exchange from G-actin" @default.
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