Characterizing interaction forces between actin and proteins of the tropomodulin family reveals the presence of the N-terminal actin-binding site in leiomodin
Introduction
Actin filaments drive many cellular processes such as cell motility, membrane transport, chemotaxis, cellular morphogenesis and force generation [1], [2]. The formation and determination of the correct lengths of actin filaments are essential for many cellular processes to take place in an orderly manner. Actin monomers (G-actin) polymerize to form arrays of actin filaments (F-actin). F-actin has two structurally and biochemically distinct ends: a barbed end and a pointed end. Polymerization and depolymerization occur at both ends but polymerization is faster at the barbed end. In the absence of actin-binding proteins, F-actin is said to “treadmill” when it reaches steady state; G-actin continuously polymerizes at the barbed end and depolymerizes from the pointed end [3], [4].
Actin is capable of polymerizing spontaneously. However, this process is relatively slow and kinetically unfavorable in cells. Formation of actin dimers and trimers, which can easily disassemble due to their instability, is a rate-determining step in actin polymerization [5], [6]. The shape and the length of F-actin are regulated by actin-binding proteins, which assist actin polymerization and depolymerization [1], [2], [7], [8], [9]. Some actin-binding proteins can also sequester G-actin and prevent it from being added to the filament.
Proteins from the tropomodulin family, tropomodulin (Tmod) and leiomodin (Lmod), can bind both G-actin and F-actin (see reviews [10], [11]). By binding G-actin, they can sequester actin monomers or nucleate actin polymerization. They bind at the pointed end of F-actin in a tropomyosin (Tpm)-dependent fashion. Tmod caps the pointed ends of F-actin to inhibit polymerization, whereas Lmod binds at the same end but still allows filament elongation although at lower rates than in the absence of Lmod [12], [13].
Tmod and Lmod have several isoforms that are differentially expressed in various cell types [14]. Tmod1 is expressed in, but not limited to, erythrocytes, cardiac and skeletal muscle cells [15], [16], [17]. Tmod2 is expressed only in the brain [18], [19]. Tmod3 is expressed in many cell types [14], [20] and Tmod4 is expressed in adult skeletal muscle cells [21]. Of the Tmod isoforms, Tmod3 is the best actin-sequestering isoform with weak nucleation ability, while Tmod2 is the best actin-nucleating isoform [22], [23], [24]. Of the three known Lmod isoforms, Lmod1 is expressed in smooth muscle cells, whereas Lmod2 and Lmod3 are expressed in cardiac and skeletal muscle cells [14], [25], [26]. Lmods were shown to be potent nucleators of actin polymerization [27], [28].
Both Tmod and Lmod are indispensable for cytoskeleton structure and function and vital for organisms. In mice, Tmod1 knockout leads to embryonic lethality due to cardiac defects [29], [30], [31], [32], Tmod2 knockout causes reduced sensorimotor gating, impaired learning and memory [33], and Tmod3 knockout is lethal due to anemia [34]. The knockout of Lmod2 in mice causes dilated cardiomyopathy, resulting in juvenile death [35]. Mutations in the LMOD3 gene found in human patients [28] or the knockout of Lmod3 in mice [36] were shown to cause severe nemaline myopathy. In addition, the knockout of Lmod3 or Tmod4 in frog disrupted the sarcomeric assembly [37]. These findings highlight the necessity of Tmod and Lmod isoforms in maintaining normal cellular properties in various type of tissues. Understanding the structure/function relationship for the members of the tropomodulin family is necessary to unravel their exact roles in cells.
Tmod and Lmod have similar domain structures, however, there are essential differences in the number of actin- and Tpm-binding sites (Fig. 1). Tmod has two Tpm-binding sites and two actin-binding sites [38], [39], [40]. Lmod, a bigger homolog of Tmod [14], [26], has the C-terminal extension that comprises a WH2 (Wiskott-Aldrich syndrome Homology 2) domain and a proline-rich region. Lmod2, the most studied Lmod isoform, contains a single Tpm-binding site [41], [42] and three actin-binding sites [12], [13], [27]. The presence of the first actin-binding site of Lmod2 is debatable. There are contradicting data obtained with Lmod2 fragments arguing the actin-binding ability of this site [12], [43], [44]. In a recent study, Boczkowska et al. [43] stated that Lmods lost pointed-end capping elements present in Tmods. This statement was based on isothermal titration calorimetry (ITC) experiments where Tmod1's N-terminal fragment A1 was shown to interact with actin while the corresponding N-terminal fragment of Lmod2 did not. The authors made the conclusion that Lmod2 lacks the N-terminal actin-binding site. On the other hand, using nuclear magnetic resonance (NMR), we showed that Lmod2's N-terminal fragment bind actin [12]. In a recent review, Fowler and Dominguez highlighted the need for additional experiments using full-length proteins to test the actin-binding function of the N-terminal region in Lmod2 [11] and to resolve the existing contradiction.
Until now, the molecular interactions of Tmod or Lmod with actin had been assessed in pyrene-actin polymerization assays [13], [23], [27], [28], [44], [45] or directly measured using non-denaturing polyacrylamide gel-electrophoresis (for Tmod isoforms only [23]), ITC (for Tmod1 and Lmod2) [43], [46], and for Lmod2 only, bioLayer interferometry [44] and NMR spectroscopy [12]. Although the use of these techniques has given valuable insight, there may be challenges in their interpretation. The latter three methods needed high concentrations and used Lmod2 fragments. Crystal structures of complexes between Lmod2 or Tmod1 and actin also were obtained using fragments [43], [44], [46]. The use of fragments is convenient for understanding the function of multidomain proteins, as well as for estimating the binding kinetics between Tmod or Lmod and their binding partners. However, they may not reflect the binding expected between full-length proteins. Additionally, the tendency of actin to polymerize rapidly under physiological conditions complicates the measurement of its binding constants with Tmod or Lmod, as well as prevents crystallization [44]. In order to overcome this problem, previous efforts for measuring the interactions of Tmod or Lmod with actin involved the use of latrunculin B [43] or mutated actin [44] to stabilize the monomeric state of actin and prevent polymerization.
Atomic force microscopy (AFM) with its ability to measure interactions between single molecules allows quantification of protein-protein interactions with high accuracy under native conditions and using full-length proteins [47]. This technique is an exciting complement to previous studies of the structure/function relationship between Tmods and actin. In this study, we utilized AFM to characterize the unbinding forces between G-actin and proteins of the tropomodulin family. By creating mutants or fragments of Tmod2 and Lmod2, we assigned specific unbinding forces to their individual actin-binding sites. Our findings demonstrated that the N-terminal domain of Lmod2 interacts with actin and confirmed the existence of the N-terminal actin-binding site.
Section snippets
Protein sequences
Sequences of Tmod1 (NP_990358.1), Tmod2 (NP_001033799.1), Tmod3 (NP_058659.1) and Lmod2 (NP_001186644.2) were downloaded from NCBI.
Plasmid construction
The plasmids for expression of Tmod1, Tmod11-344[L71D], Tmod2 and Tmod21-346 (pET(His)Tmod1, pET(His)Tmod11-344[L71D], pET(His)Tmod1 and pET(His)Tmod21-346, respectively) were generated previously [24], [39], [48]. pET(His)Tmod2[L73D] was generated by site-directed mutagenesis using pET(His)Tmod2 as a template by using Pfu Turbo DNA polymerase (Agilent Technologies,
Immobilization of G-actin on the mica surface shows uniform distribution
G-actin was immobilized on a mica disk prior to unbinding force measurements, and topography images were captured in order to verify the surface coverage with G-actin. Successful immobilization of G-actin on mica with almost a full coverage of both at 2 × 2 and 1 × 1 μm2 scan areas are shown in Fig. 2. Our images show globular particles on the mica surface and they correlate well in terms of shape with previous AFM images of G-actin [50].
Interactions of actin with Tmod2 have a bimodal unbinding force distribution, while interactions with other Tmod isoforms have unimodal distributions
In order to perform the force measurements between
Conclusions
The unbinding forces between G-actin and proteins from the tropomodulin family were quantified and characterized using single molecule force spectroscopy. By creating mutants or fragments of Tmod2 and Lmod2, we assigned specific unbinding forces to their individual actin-binding sites. Our results demonstrate how the differences between the number and the strength of the actin-binding sites translate to the actin-sequestering and -nucleating abilities of Lmod and Tmod. Finally, our results
Acknowledgements
The authors thank Christopher Keller for assistance in Tmod2[L73D] preparation. This work was supported by the National Institutes of Health grants GM081688 and GM120137 to ASK. We thank Dr. Carol Gregorio for providing WT-Lmod2, Lmod21-514 and Lmod21-201.
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