Characterizing interaction forces between actin and proteins of the tropomodulin family reveals the presence of the N-terminal actin-binding site in leiomodin

Highlights

• Unbinding forces between G-actin and Tmod1, Tmod2, Tmod3 or Lmod2 were quantified.

• Tmod1 and Tmod3 have unimodal unbinding force distributions.

• Tmod2 has a bimodal force distribution and Lmod2 has a trimodal distribution.

• Specific unbinding forces were assigned to actin-binding sites of Tmod2 and Lmod2.

• The existence of the N-terminal actin-binding site in Lmod2 was confirmed.

Abstract

Tropomodulin family of proteins includes several isoforms of tropomodulins (Tmod) and leiomodins (Lmod). These proteins can sequester actin monomers or nucleate actin polymerization. Although it is known that their actin-binding properties are isoform-dependent, knowledge on how they vary in strengths of interactions with G-actin is missing. While it is confirmed in many studies that Tmods have two actin-binding sites, information on number and location of actin-binding sites in Lmod2 is controversial. We used atomic force microscopy to study interactions between G-actin and proteins of the tropomodulin family. Unbinding forces between G-actin and Tmod1, Tmod2, Tmod3, or Lmod2 were quantified. Our results indicated that Tmod1 and Tmod3 had unimodal force distributions, Tmod2 had a bimodal distribution and Lmod2 had a trimodal distribution. The number of force distributions correlates with the proteins' abilities to sequester actin or to nucleate actin polymerization. We assigned specific unbinding forces to the individual actin-binding sites of Tmod2 and Lmod2 using mutations that destroy actin-binding sites of Tmod2 and truncated Lmod2. Our results confirm the existence of the N-terminal actin-binding site in Lmod2. Altogether, our data demonstrate how the differences between the number and the strength of actin-binding sites of Tmod or Lmod translate to their functional abilities.

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.