Structural basis of fast- and slow-severing actin–cofilactin boundaries
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100337
ISSN1083-351X
AutoresGlen M. Hocky, Charles V. Sindelar, Wenxiang Cao, Gregory A. Voth, Enrique M. De La Cruz,
Tópico(s)Force Microscopy Techniques and Applications
ResumoMembers of the ADF/cofilin family of regulatory proteins bind actin filaments cooperatively, locally change actin subunit conformation and orientation, and sever filaments at "boundaries" between bare and cofilin-occupied segments. A cluster of bound cofilin introduces two distinct classes of boundaries due to the intrinsic polarity of actin filaments, one at the "pointed" end side and the other at the "barbed" end-side of the cluster; severing occurs more readily at the pointed end side of the cluster ("fast-severing" boundary) than the barbed end side ("slow-severing" boundary). A recent electron-cryomicroscopy (cryo-EM) model of the slow-severing boundary revealed structural "defects" at the interface that potentially contribute to severing. However, the structure of the fast-severing boundary remains uncertain. Here, we use extensive molecular dynamics simulations to produce atomic resolution models of both severing boundaries. Our equilibrated simulation model of the slow-severing boundary is consistent with the cryo-EM structural model. Simulations indicate that actin subunits at both boundaries adopt structures intermediate between those of bare and cofilin-bound actin subunits. These "intermediate" states have compromised intersubunit contacts, but those at the slow-severing boundary are stabilized by cofilin bridging interactions, accounting for its lower fragmentation probability. Simulations where cofilin proteins are removed from cofilactin filaments favor a mechanism in which a cluster of two contiguously bound cofilins is needed to fully stabilize the cofilactin conformation, promote cooperative binding interactions, and accelerate filament severing. Together, these studies provide a molecular-scale foundation for developing coarse-grained and theoretical descriptions of cofilin-mediated actin filament severing. Members of the ADF/cofilin family of regulatory proteins bind actin filaments cooperatively, locally change actin subunit conformation and orientation, and sever filaments at "boundaries" between bare and cofilin-occupied segments. A cluster of bound cofilin introduces two distinct classes of boundaries due to the intrinsic polarity of actin filaments, one at the "pointed" end side and the other at the "barbed" end-side of the cluster; severing occurs more readily at the pointed end side of the cluster ("fast-severing" boundary) than the barbed end side ("slow-severing" boundary). A recent electron-cryomicroscopy (cryo-EM) model of the slow-severing boundary revealed structural "defects" at the interface that potentially contribute to severing. However, the structure of the fast-severing boundary remains uncertain. Here, we use extensive molecular dynamics simulations to produce atomic resolution models of both severing boundaries. Our equilibrated simulation model of the slow-severing boundary is consistent with the cryo-EM structural model. Simulations indicate that actin subunits at both boundaries adopt structures intermediate between those of bare and cofilin-bound actin subunits. These "intermediate" states have compromised intersubunit contacts, but those at the slow-severing boundary are stabilized by cofilin bridging interactions, accounting for its lower fragmentation probability. Simulations where cofilin proteins are removed from cofilactin filaments favor a mechanism in which a cluster of two contiguously bound cofilins is needed to fully stabilize the cofilactin conformation, promote cooperative binding interactions, and accelerate filament severing. Together, these studies provide a molecular-scale foundation for developing coarse-grained and theoretical descriptions of cofilin-mediated actin filament severing. The actin cytoskeleton is a dynamic biopolymer network that powers cell motility and division (1Pollard T.D. Earnshaw W.C. Lippincott-Schwartz J. Johnson G. Cell Biology. Saunders (Elsevier), Philadelphia, PA2016Google Scholar). The primary component of this network is the actin filament—a linear, helical, and polar polymer formed from the head-to-tail assembly of actin monomers. Actin filament assembly dynamics are controlled by a wide variety of regulatory proteins, among which are filament severing proteins that accelerate network turnover by increasing the concentration of polymer ends where subunits can add and dissociate (1Pollard T.D. Earnshaw W.C. Lippincott-Schwartz J. Johnson G. Cell Biology. Saunders (Elsevier), Philadelphia, PA2016Google Scholar). Filament severing proteins in the cofilin/ADF family (herein referred to as cofilin) bind actin filaments between longitudinally adjacent actin subunits (2Bamburg J.R. Proteins of the ADF/cofilin family: Essential regulators of actin dynamics.Annu. Rev. Cell Dev. Biol. 1999; 15: 185-230Crossref PubMed Scopus (822) Google Scholar). A fully decorated 'cofilactin" filament (having a stoichiometry of one cofilin per actin subunit (3De La Cruz E.M. Cofilin binding to muscle and non-muscle actin filaments: Isoform-dependent cooperative interactions.J. Mol. Biol. 2005; 346: 557-564Crossref PubMed Scopus (115) Google Scholar)) is more compliant in bending (4McCullough B.R. Blanchoin L. Martiel J.L. De La Cruz E.M. Cofilin increases the bending flexibility of actin filaments: Implications for severing and cell mechanics.J. Mol. Biol. 2008; 381: 550-558Crossref PubMed Scopus (147) Google Scholar, 5Pfaendtner J. De La Cruz E.M. Voth G.A. Actin filament remodeling by actin depolymerization factor/cofilin.Proc. Natl. Acad. Sci. 2010; 107: 7299-7304Crossref PubMed Scopus (75) Google Scholar, 6McCullough B.R. Grintsevich E.E. Chen C.K. Kang H.R. Hutchison A.L. 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Weeds A. Cofilin changes the twist of F-actin: Implications for actin filament dynamics and cellular function.J. Cell Biol. 1997; 138: 771-781Crossref PubMed Scopus (554) Google Scholar, 10Galkin V.E. Orlova A. Lukoyanova N. Wriggers W. Egelman E.H. Actin depolymerizing factor stabilizes an existing state of F-actin and can change the tilt of F-actin subunits.J. Cell Biol. 2001; 153: 75-86Crossref PubMed Scopus (200) Google Scholar) compared with a bare actin filament. Cofilin binding displaces a stabilizing intersubunit contact formed by the actin "D-loop" of one subunit and the "target binding cleft" of its longitudinally adjacent, pointed end neighbor (Fig. 1A) (11Galkin V.E. Orlova A. Kudryashov D.S. Solodukhin A. Reisler E. Schroder G.F. Egelman E.H. Remodeling of actin filaments by ADF/cofilin proteins.Proc. Natl. Acad. Sci. 2011; 108: 20568-20572Crossref PubMed Scopus (151) Google Scholar, 12Wong D.Y. Sept D. The interaction of cofilin with the actin filament.J. Mol. 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Rev. 2009; 1: 51-59Crossref PubMed Scopus (88) Google Scholar, 16De La Cruz E.M. Sept D. The kinetics of cooperative cofilin binding reveals two states of the cofilin-actin filament.Biophys. J. 2010; 98: 1893-1901Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 17Suarez C. Roland J. Boujemaa-Paterski R. Kang H. McCullough B.R. Reymann A.C. Guerin C. Martiel J.L. De la Cruz E.M. Blanchoin L. Cofilin tunes the nucleotide state of actin filaments and severs at bare and decorated segment boundaries.Curr. Biol. 2011; 21: 862-868Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 18Bibeau J.P. Gray S. De La Cruz E.M. Clusters of a few bound cofilins sever actin filament.J. Mol. Biol. 2021; 433: 166833Crossref PubMed Scopus (4) Google Scholar, 19Elam W.A. Kang H. De La Cruz E.M. Biophysics of actin filament severing by cofilin.FEBS Lett. 2013; 587: 1215-1219Crossref PubMed Scopus (68) Google Scholar, 20De La Cruz E.M. Gardel M.L. Actin mechanics and fragmentation.J. Biol. Chem. 2015; 290: 17137-17144Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), thereby explaining why fully decorated filaments are more stable than partially decorated ones (3De La Cruz E.M. Cofilin binding to muscle and non-muscle actin filaments: Isoform-dependent cooperative interactions.J. Mol. Biol. 2005; 346: 557-564Crossref PubMed Scopus (115) Google Scholar, 15De La Cruz E.M. How cofilin severs an actin filament.Biophys. Rev. 2009; 1: 51-59Crossref PubMed Scopus (88) Google Scholar, 21Andrianantoandro E. Pollard T.D. Mechanism of actin filament turnover by severing and nucleation at different concentrations of ADF/cofilin.Mol. Cell. 2006; 24: 13-23Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar, 22Dedova I.V. Nikolaeva O.P. Mikhailova V.V. dos Remedios C.G. Levitsky D.I. Two opposite effects of cofilin on the thermal unfolding of F-actin: A differential scanning calorimetric study.Biophys. 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J. 2017; 112: 2624-2633Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 27Schramm A.C. Hocky G.M. Voth G.A. Martiel J.L. De La Cruz E.M. Plastic Deformation and fragmentation of strained actin filaments.Biophys. J. 2019; 117: 453-463Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar). However, filaments in solution that are partially decorated with cofilin also spontaneously fragment due to thermal fluctuations, indicating that the boundary interface is less stable than actin–actin or cofilactin–cofilactin interfaces (6McCullough B.R. Grintsevich E.E. Chen C.K. Kang H.R. Hutchison A.L. Henn A. Cao W.X. Suarez C. Martie J.L. Blanchoin L. Reisler E. De La Cruz E.M. Cofilin-linked changes in actin filament flexibility promote severing.Biophys. J. 2011; 101: 151-159Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). The two boundaries of a cofilin cluster are not structurally identical, and it has been shown that the barbed end side severs at a lower rate than the pointed end side (18Bibeau J.P. Gray S. De La Cruz E.M. Clusters of a few bound cofilins sever actin filament.J. Mol. Biol. 2021; 433: 166833Crossref PubMed Scopus (4) Google Scholar, 28Wioland H. Guichard B. Senju Y. Myram S. Lappalainen P. Jegou A. Romet-Lemonne G. ADF/Cofilin accelerates actin dynamics by severing filaments and promoting their depolymerization at both ends.Curr. Biol. 2017; 27: 1956-1967.e7Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). An intermediate resolution structure (subnanometer) of the slow-severing cofilactin/actin boundary was recently solved by electron cryo-microscopy (cryo-EM), but even this level of detail has not been achieved for the fast-severing boundary (29Huehn A.R. Bibeau J.P. Schramm A.C. Cao W. De La Cruz E.M. Sindelar C.V. Structures of cofilin-induced structural changes reveal local and asymmetric perturbations of actin filaments.Proc. Natl. Acad. Sci. 2020; 117: 1478-1484Crossref PubMed Scopus (17) Google Scholar). Molecular dynamics (MD) simulations have been successful in capturing the molecular details and dynamics of actin filaments, including cofilin-linked changes to structure and filament rigidity (5Pfaendtner J. De La Cruz E.M. Voth G.A. Actin filament remodeling by actin depolymerization factor/cofilin.Proc. Natl. Acad. Sci. 2010; 107: 7299-7304Crossref PubMed Scopus (75) Google Scholar, 7Fan J. Saunders M.G. Haddadian E.J. Freed K.F. De La Cruz E.M. Voth G.A. Molecular origins of cofilin-linked changes in actin filament mechanics.J. Mol. Biol. 2013; 425: 1225-1240Crossref PubMed Scopus (36) Google Scholar, 12Wong D.Y. Sept D. The interaction of cofilin with the actin filament.J. Mol. Biol. 2011; 413: 97-105Crossref PubMed Scopus (20) Google Scholar, 13Elam W.A. Cao W. Kang H. Huehn A. Hocky G.M. Prochniewicz E. Schramm A.C. Negron K. Garcia J. Bonello T.T. Gunning P.W. Thomas D.D. Voth G.A. Sindelar C.V. De La Cruz E.M. Phosphomimetic S3D cofilin binds but only weakly severs actin filaments.J. Biol. Chem. 2017; 292: 19565-19579Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 30Hocky G.M. Baker J.L. Bradley M.J. Sinitskiy A.V. De La Cruz E.M. Voth G.A. Cations Stiffen actin filaments by Adhering a Key structural Element to adjacent subunits.J. Phys. Chem. B. 2016; 120: 4558-4567Crossref PubMed Scopus (24) Google Scholar). Here, we employ MD simulations to predict structures of these two boundaries starting from the bare actin and cofilactin filament structures. Our simulations are consistent with known data for the slow-severing boundary, predict an intermediate state of actin subunits within both interfaces, and provide a structural basis for the asymmetric severing of filaments by cofilin. Further simulations of synthetic boundaries generated by removal of cofilin from cofilactin structures allow us to assess stability of cofilin clusters of varying size. Initial periodic structures for bare and cofilin-decorated filaments (herein referred to as cofilactin) were generated from EM structures of ADP-actin (PDB 2ZWH) and cofilactin (PDB 3J0S) (11Galkin V.E. Orlova A. Kudryashov D.S. Solodukhin A. Reisler E. Schroder G.F. Egelman E.H. Remodeling of actin filaments by ADF/cofilin proteins.Proc. Natl. Acad. Sci. 2011; 108: 20568-20572Crossref PubMed Scopus (151) Google Scholar, 31Oda T. Iwasa M. Aihara T. Maeda Y. Narita A. The nature of the globular- to fibrous-actin transition.Nature. 2009; 457: 441-445Crossref PubMed Scopus (434) Google Scholar) (see Simulation Methods), followed by all-atom MD. The resulting, equilibrated structures (Fig. 1A) were joined end to end in a head-to-tail manner (e.g., the barbed end of one filament was placed adjacent to the pointed end of the other) through alignment of two subunits from each structure (details in Simulation Methods), yielding starting models for the slow- and fast-severing boundaries (Fig. 1A). Extensive additional MD simulations were then performed and analyzed as described below. The resulting systems contain cofilactin segments with bare actin at either the pointed end (herein referred to as the "fast-severing" boundary) or barbed end (herein referred to as the "slow-severing" boundary) of the bound cofilin cluster (Fig. 1, A and B). Both fast-severing and slow-severing boundaries of modeled filaments appear to stabilize within the first 25–30 ns of the MD simulations, as indicated by the root-mean-square-deviation (RMSD) of the actin subunits at the boundary (subunits i-2: i+1 in Fig. 1B); filaments remain stable and do not undergo significant further structural rearrangements for >150 ns, after which slower-timescale rearrangements occur on the <2 Å length scale (Fig. S1). A fast-severing boundary model lacking intersubunit D-loop contacts was not stable, resulting in partial filament rupture (Fig. S1; described in Simulation Methods). The atomic structure of our slow-severing boundary model is consistent with a previously reported ∼9 Å resolution cryo-EM map (Fig. 2) (29Huehn A.R. Bibeau J.P. Schramm A.C. Cao W. De La Cruz E.M. Sindelar C.V. Structures of cofilin-induced structural changes reveal local and asymmetric perturbations of actin filaments.Proc. Natl. Acad. Sci. 2020; 117: 1478-1484Crossref PubMed Scopus (17) Google Scholar). A boundary model had been generated from that map by rigid body docking of prior actin and cofilactin subunit structures into the electron density. Our MD model is in reasonable overall agreement with this structure (Fig. 2), as quantified by either the RMSD of "boundary subunits" (i-2: i+1) or the "interfacial subunits" (i and i+1) (Fig. S2). The "flatness" of these subunits also agrees well with the cryo-EM model (see next section; Fig. S3). The data from Ref. (29Huehn A.R. Bibeau J.P. Schramm A.C. Cao W. De La Cruz E.M. Sindelar C.V. Structures of cofilin-induced structural changes reveal local and asymmetric perturbations of actin filaments.Proc. Natl. Acad. Sci. 2020; 117: 1478-1484Crossref PubMed Scopus (17) Google Scholar) is not of high enough resolution to evaluate the precise details of our simulation model beyond these comparisons, but the level of agreement between our simulations and the assumed molecular structure lends confidence to our approach and supports the utility of MD to predict features of the fast-severing interface, for which no high resolution structure is available. Cofilin binding changes the helical twist of actin filaments by ∼5 degrees (on average) per subunit (167° → 162° as measured by cryo-EM (10Galkin V.E. Orlova A. Lukoyanova N. Wriggers W. Egelman E.H. Actin depolymerizing factor stabilizes an existing state of F-actin and can change the tilt of F-actin subunits.J. Cell Biol. 2001; 153: 75-86Crossref PubMed Scopus (200) Google Scholar) or 166° →161° by MD (7Fan J. Saunders M.G. Haddadian E.J. Freed K.F. De La Cruz E.M. Voth G.A. Molecular origins of cofilin-linked changes in actin filament mechanics.J. Mol. Biol. 2013; 425: 1225-1240Crossref PubMed Scopus (36) Google Scholar). In our simulations, the filament twist changes from the cofilactin value to the bare value (and vice versa) over the course of 1–3 subunits at both the fast- and slow-severing boundaries, with a slightly shorter crossover length at the slow-severing boundary than the fast-severing boundary (Fig. 1C, Table 1), consistent with recent cryo-EM analysis (34Huehn A. Cao W.X. Elam W.A. Liu X.Q. De La Cruz E.M. Sindelar C.V. The actin filament twist changes abruptly at boundaries between bare and cofilin-decorated segments.J. Biol. Chem. 2018; 293: 5377-5383Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar).Table 1Twist angles (Fig. 1C) and internal flattening angles (Fig. 3) are fit to a transition model as in Ref (33Huehn A.R. Cao W.X. Elam W.A. De La Cruz E. Sindelar C.V. Cofilin Induces a local change in the twist of actin filaments.Biophys. J. 2018; 114: 145aAbstract Full Text Full Text PDF Google Scholar, 34Huehn A. Cao W.X. Elam W.A. Liu X.Q. De La Cruz E.M. Sindelar C.V. The actin filament twist changes abruptly at boundaries between bare and cofilin-decorated segments.J. Biol. Chem. 2018; 293: 5377-5383Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar).System-observableA1 (barbed side angle, °)A2 (pointed side angle, °)n0 (central position)N (crossover length)Slow-twist165.1155.11.41.1Fast-twist150.0169.40.12.9Slow-ϕ−9.0−28.9−0.31.1Fast-ϕ−29.4−4.22.22.8The fit function used for the angle as a function of position n is θ(n)=A2−A2−A11+exp(n−n0)/N where n refers to subunit position i+n (n from –8 to 9 in Fig. 1B), N is the exponential decay length (crossover length), n0 is the center of exponential decay or crossover, and A1 and A2 are limits of the function for either twist or flattening angles across a boundary. The result of the fit for the dihedral angles is shown in Fig. S3. Open table in a new tab The fit function used for the angle as a function of position n is θ(n)=A2−A2−A11+exp(n−n0)/N where n refers to subunit position i+n (n from –8 to 9 in Fig. 1B), N is the exponential decay length (crossover length), n0 is the center of exponential decay or crossover, and A1 and A2 are limits of the function for either twist or flattening angles across a boundary. The result of the fit for the dihedral angles is shown in Fig. S3. In addition to altering the helical twist, cofilin binding also tilts the outer domain of filament subunits such that bare actin subunits are "flatter" than those in cofilactin (11Galkin V.E. Orlova A. Kudryashov D.S. Solodukhin A. Reisler E. Schroder G.F. Egelman E.H. Remodeling of actin filaments by ADF/cofilin proteins.Proc. Natl. Acad. Sci. 2011; 108: 20568-20572Crossref PubMed Scopus (151) Google Scholar). Therefore, actin subunit "flatness" serves an additional proxy for assessing cofilin-induced structural changes, quantified here by the dihedral angle ϕ of the four major actin subdomains (SDs; Fig. 3, inset). Actin subunits within bare and cofilin-decorated regions maintain their initial subunit flatness, as indicated by ϕ values that remain near the canonical values (horizontal dashed lines in Fig. 3). In contrast, "interfacial subunits" i, i+1 adopt a ϕ value intermediate between that of bare actin and cofilactin (Fig. 3). The abrupt change in twist and subunit conformation at cofilactin boundaries observed in our MD models provide further evidence that actin structural changes linked to cofilin binding are local, propagating only to nearest neighbors directly in contact with cofilin (3De La Cruz E.M. Cofilin binding to muscle and non-muscle actin filaments: Isoform-dependent cooperative interactions.J. Mol. Biol. 2005; 346: 557-564Crossref PubMed Scopus (115) Google Scholar, 15De La Cruz E.M. How cofilin severs an actin filament.Biophys. Rev. 2009; 1: 51-59Crossref PubMed Scopus (88) Google Scholar, 16De La Cruz E.M. Sept D. The kinetics of cooperative cofilin binding reveals two states of the cofilin-actin filament.Biophys. J. 2010; 98: 1893-1901Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 19Elam W.A. Kang H. De La Cruz E.M. Biophysics of actin filament severing by cofilin.FEBS Lett. 2013; 587: 1215-1219Crossref PubMed Scopus (68) Google Scholar, 29Huehn A.R. Bibeau J.P. Schramm A.C. Cao W. De La Cruz E.M. Sindelar C.V. Structures of cofilin-induced structural changes reveal local and asymmetric perturbations of actin filaments.Proc. Natl. Acad. Sci. 2020; 117: 1478-1484Crossref PubMed Scopus (17) Google Scholar, 34Huehn A. Cao W.X. Elam W.A. Liu X.Q. De La Cruz E.M. Sindelar C.V. The actin filament twist changes abruptly at boundaries between bare and cofilin-decorated segments.J. Biol. Chem. 2018; 293: 5377-5383Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Interfacial actin subunits (subunits i and i+1) adopt unique structures intermediate between that of subunits in bare actin and cofilactin. As a consequence of this intermediate structure induced by cofilin binding at either the pointed end (slow-severing) or barbed end (fast-severing) of the actin subunits, we expect a change in the nature of the interactions between subunits at the boundary. In Figure 4A, we show the distribution of the number of D-loop contacts in an actin subunit with its longitudinal neighbor at the pointed (right) end. As a consequence of the intermediate configurations adopted at the interfacial subunits, intersubunit D-loop contacts and other longitudinal contacts at the pointed end side (Fig. S4A) are compromised for both boundary models (contacts between subunits i: i+2, i+1: i+3). This disruption occurs without formation of additional lateral contacts (Fig. S4B). However, in the case of the slow-severing model, the reduction in longitudinal filament contacts is compensated by the stabilizing cofilactin "bridge" interactions (Fig. S4C), with a cofilin nestled between SD2 of the actin subunit at its barbed end side and SD1 of the actin subunit at its pointed end side (see ribbon diagram in Fig. 1A) (24Pavlov D. Muhlrad A. Cooper J. Wear M. Reisler E. Actin filament severing by cofilin.J. Mol. Biol. 2007; 365: 1350-1358Crossref PubMed Scopus (136) Google Scholar). Hence, the pointed end of subunits i,i+1 is less likely to be a locus of severing at the slow-severing boundary than at the fast-severing one. Indeed, our prediction is that filament fragmentation is most likely to occur where bare actin-like subunits contact the interfacial subunits ("putative severing interface" in Figs. 1B and 4A). This location is asymmetric with respect to the fast- and slow-severing models, since it occurs at the barbed end of the interfacial actin subunits in the slow-severing case (interface i-2:i, i-1:i+1) and at the pointed end in the fast-severing case (interface i:i+2, i+1:i+3). For the fast-severing model, this putative severing interface is coincident with the location of a pronounced reduction in D-loop contacts (Fig. 4A), consistent with our hypothesis that this interface is structurally weak and more prone to failure than those between other subunits. In contrast, we do not observe a substantial reduction in D-loop contacts at our predicted slow-severing location (Fig. 4A); this greater total number of contacts at the D-loops of actin subunits i-2, i-1 is consistent with the slower severing observed experimentally (18Bibeau J.P. Gray S. De La Cruz E.M. Clusters of a few bound cofilins sever actin filament.J. Mol. Biol. 2021; 433: 166833Crossref PubMed Scopus (4) Google Scholar, 28Wioland H. Guichard B. Senju Y. Myram S. Lappalainen P. Jegou A. Romet-Lemonne G. ADF/Cofilin accelerates actin dynamics by severing filaments and promoting their depolymerization at both ends.Curr. Biol. 2017; 27: 1956-1967.e7Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). However, the interface between bare actin and cofilactin is still the most plausible location for severing due to (1Pollard T.D. Earnshaw W.C. Lippincott-Schwartz J. Johnson G. Cell Biology. Saunders (Elsevier), Philadelphia, PA2016Google Scholar) the abrupt change in structure of the subunits, (2Bamburg J.R. Proteins of the ADF/cofilin family: Essential regulators of actin dynamics.Annu. Rev. Cell Dev. Biol. 1999; 15: 185-230Crossref PubMed Scopus (822) Google Scholar) a reduction in lateral contacts (subunit i-2, Fig. S4B), and (3De La Cruz E.M. Cofilin binding to muscle and non-muscle actin filaments: Isoform-dependent cooperative interactions.J. Mol. Biol. 2005; 346: 557-564Crossref PubMed Scopus (115) Google Scholar) the intermediate flattening observed at subunits i-2, i-1 (Fig. 3) (29Huehn A.R. Bibeau J.P. Schramm A.C. Cao W. De La Cruz E.M. Sindelar C.V. Structures of cofilin-induced structural changes reveal local and asymmetric perturbations of actin filaments.Proc. Natl. Acad. Sci. 2020; 117: 1478-1484Crossref PubMed Scopus (17) Google Scholar). Finally, we note that while the reduction in D-loop contacts at the fast-severing interface could be a consequence of model construction, these D-loop interactions were required for a stable model (see Simulation Methods). As noted in describing model construction, the filament partially ruptured unless a biasing potential was used to drive interfacial D-loops within interacting distance of their longitudinal neighbors. Despite this biasing potential, D-loops of interfacial subunits at the fast-severing boundary failed to fully restore actin-like interactions with their longitudinal neighbors—for example, not wrapping around the Y169 as seen in experimentally determined actin filament structures (36von der Ecken J. Muller M. Lehman W. Manstein D.J. Penczek P.A. Raunser S. Structure of the F-actin-tropomyosin complex.Nature. 2015; 519: 114-117Crossref PubMed Scopus (238) Google Scholar, 37Chou S.Z. Pollard T.D. Mechanism of actin polymerization revealed by cryo-EM structures of actin filaments with three different bound nucleotides.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 4265-4274Crossref PubMed Scopus (72) Google Scholar). Our data suggest that bridging cofilin interactions at the barbed end of interfacial subunits at the fast-severing boundary allosterically restrict the ability to adopt actin-like conformations and corresponding D-loop contacts at the putative fast-severing interface (pointed end of the interfacial subunits). Simulations of "cofilin-ablated" filaments further support the notion that a reduction in intersubunit D-loop contacts contributes to changes in filament compliance and severing. We developed an alternative strategy to examine boundaries whereby we simulated a cofilactin filament after computationally "ablating" a fraction of initially bound cofilins, resolvating and equilibrating (Fig. 5A; see also Simulation Methods). The segments with cofilin removed, as initialized, have a drastically reduced number of D-loop contacts relative to a bare actin filament and undergo thermally driven, nonequilibrium bending fluctuations that far exceed those of fully occupied cofilactin filaments (Movie M.1–Movie M.3). Further, the reduction of intersubunit contacts in the cofilin-ablated region led to hinge-like bending adjacent to the interfacial subunits where the contacts are only partially compromised (Movie M.1 and Movie M.2). The flexible zone in both slow- and fast-severing cases extends beyond the "hinge" region to include all actin subunits in the cofilin-ablated segment. In both simulations, a partial rupture occurred in the high-flexibility (cofilin-ablated) zone, severing the protofilament at one place (marked by "∗" in Fig. 5A). In contrast, cofilin-bound subunits (including the interfacial subunits) retain cofilactin conformations, as measured by their lower ϕ values, albeit with significantly larger variance (Fig. S5). Although the way in which these simulation models were created is not physiological, the observed hinge-like motion has been observed at boundaries and linked to a higher severing probability (6McCullough B.R. Grintsevich E.E. Chen C.K. Kang H.R. Hutchison A.L. Henn A. Cao W.X. Suarez C. Martie J.L. Blanchoin L. Reisler E. De La Cruz E.M. Cofilin-linked changes in actin filament flexibility promote severing.Biophys. J. 2011; 101: 151-159Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Small, bound cofilin clusters (n = 2 or 3; Fig. 5) formed by computational ablation were found to be stable, as shown by computing the RMSD of actin subunits within the cluster to the starting model (over the course of 160 ns of MD simulations following ablation; Fig. 5C). In contrast, ablation of all but a single cofilin does not retain the cofilactin structure (Fig. 5C). In the corresponding simulation (n = 1), the average RMSD to the starting model increases soon after the start and continues to increase for the duration of the simulation although the cofilin remains stably bound (Fig. 5C). This behavior suggests that two contiguously bound cofilin molecules, either longitudinal or lateral, are sufficient to retain the cofilactin conformation and are consistent with a cooperative binding nucleus size of two contiguously bound cofilins (29Huehn A.R. Bibeau J.P. Schramm A.C. Cao W. De La Cruz E.M. Sindelar C.V. Structures of cofilin-induced structural changes reveal local and asymmetric perturbations of actin filaments.Proc. Natl. Acad. Sci. 2020; 117: 1478-1484Crossref PubMed Scopus (17) Google Scholar). A model of the slow-severing actin–cofilactin boundary (barbed end of a cofilin domain) constructed and observed by MD captured critical structural features of the interface that was recently determined at intermediate resolution by cryo-EM. This consistency lends credence to a computational model of the fast-severing boundary (pointed end of a cofilactin domain), which has not been visualized by cryo-EM. The proposed severing location for the modeled fast-severing boundary (pointed end of the interfacial subunits) exhibits compromised D-loop and other longitudinal contacts without compensatory stabilizing cofilin interactions, commensurate with enhanced severing at that boundary. While the same location in the slow-severing boundary has a reduction in longitudinal contacts at interfacial subunits, similar to what is seen at the fast-severing boundary, this is compensated by contacts with a bound cofilin at each subunit and hence is not the most likely locus for severing. Instead, we propose that the severing interface for the slow-severing boundary is at the barbed end of the interfacial subunits due to the abrupt change in structure. However, the higher number of contacts at the position we deem most likely for severing is consistent with a slower-severing rate compared with the severing location (interface) for the fast-severing boundary (pointed end of interfacial subunits).
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