The assembly of immunoglobulin-like modules in titin: implications for muscle elasticity¹

Abstract

Titin, a giant muscle protein, forms filaments that span half of the sarcomere and cover, along their length, quite diversified functions. The region of titin located in the sarcomere I-band is believed to play a major rôle in extensibility and passive elasticity of muscle. In the I-band, the titin sequence contains tandem immunoglobulin-like (Ig) modules intercalated by a potentially non-globular region. By a combined approach making use of small angle X-ray scattering and nuclear magnetic resonance techniques, we have addressed the questions of what are the average mutual orientation of poly-Igs and the degree of flexibility around the domain interfaces. Various recombinant fragments containing one, two and four titin I-band tandem domains were analysed. The small-angle scattering data provide a picture of the domains in a mostly extended configuration with their long axes aligned head-to-tail. There is a small degree of bending and twisting of the modules with respect to each other that results in an overall shortening in their maximum linear dimension compared with that expected for the fully extended, linear configurations. This shortening is greatest for the four module construct (≈15%). ¹⁵N NMR relaxation studies of one and two-domain constructs show that the motions around the interdomain connecting regions are restricted, suggesting that titin behaves as a row of beads connected by rigid hinges. The length of the residues in the interface seems to be the major determinant of the degree of flexibility. Possible implications of our results for the structure and function of titin in muscles are discussed.

Introduction

Recognizing that proteins are often assembled by smaller building blocks which form independently folded units or domains may be considered a major break-through in structural biology. This realization has allowed the study of proteins whose size would otherwise be well outside the range of even the most advanced high-resolution techniques. Multidomain (or modular) proteins may be approached by characterizing the structure of their isolated domains under the assumption that these will not, to a first approximation, be influenced by sequence or spatially contiguous regions. This approach has been successfully applied to the study of several protein fragments whose structures have been solved by crystallography and/or NMR (for reviews see Bork et al 1996, McEvoy et al 1997). Once the structures of individual modules have been obtained, we need to determine how they are assembled to reconstruct the shape and the structure of the whole protein, therefore characterizing possible interactions (or lack of them) between domains. Mutual domain orientations and dynamics are critical parts of this analysis. It is of particular interest to determine the degree of flexibility (or rigidity) of the domain interfaces, to understand whether they allow movement and, if so, how much. A particularly fruitful approach to acquiring this information is to combine high resolution techniques with lower resolution studies using, for instance, microscopy or spectroscopy. However, there are relatively few examples of such reconstructions and thus characterization of domain interfaces remains a subject requiring further investigation and the development of novel approaches to these studies.

The philosophy outlined above has inspired our work on titin, a giant muscle protein of a size (three million dalton) that prohibits direct study of the whole molecule by most high resolution techniques (for reviews, see Maruyama 1994, Maruyama 1997, Keller 1995, Trinick 1996). Single titin molecules form filaments 1 μm long and connect half of the sarcomere fibre from the Z-disk to the M-line, probably determining the length of the muscle fibre as a sort of “molecular ruler” Furst et al 1988, Nave et al 1989, Labeit 1990. The functions of titin vary along the sarcomere (Trinick, 1994), ranging from regulation of the assembly of the thick filaments in the A-band, to providing an anchoring point for the titin filaments at both ends in the Z-disc and in the M-line, through interactions with a number of other muscle proteins. In the I-band, titin acts as an elastic connector between the thick filaments and the Z-disk and prevents the thick filaments from moving from the centre of the sarcomere Horowits et al 1989, Horowits 1992, Funatsu 1993, Trombitas et al 1991, Wang et al 1991. This last feature has recently made titin the subject of particular interest both from the perspective of developing a more thorough understanding of muscle mechanical properties, and in the more general context of bioelastic materials Tskhovrebov et al 1997, Kellermayer et al 1997, Rief et al 1997.

Sequence determination has shown that titin is assembled by multiple copies of two types of sequence motifs, named type I and II, which have been shown to belong to the fibronectin type III (fn3) and the immunoglobulin (Ig) protein superfamily, respectively Labeit 1990, Labeit and Kolmerer 1995. In the elastic region of titin located in the sarcomere I-band, multiple copies of type II domains are arranged in tandem (Figure 1). This feature is specific to the titin I-band, since Ig domains in other regions are either flanked by type I domains or connected by linkers (by “linkers” we refer to a sequence which does not belong to either the fn3 or the Ig consensus sequences). In the various titin isoforms, such an arrangement is interrupted by only two major insertions, one of which is supposedly non-globular and contains a stretch rich in proline, glutamic acid, valine and lysine (PEVK-rich region). It has been shown by electron microscopy studies that these elastic properties are related to a subtle interplay between the PEVK-rich region and the poly-Ig stretch Gautel and Goulding 1996, Linke et al 1996, Linke et al 1998a, Linke et al 1998b. For a thorough understanding of how titin poly-Ig domains are assembled and the rôle they play in muscle elasticity, it is therefore important to determine the structure and dynamical properties of single and double modules which are the smallest units containing a domain interface.

Previously, we have reported the structure of a representative I-band titin Ig domain (I27) and used that structure to model the I27I28 pair (Improta et al., 1996). I27 belongs to the constitutively expressed tandem domains of the I-band (Labeit & Kolmerer, 1995). Spectroscopic data relating to thermodynamic stability upon thermal and chemical unfolding of isolated I27 and I28 in the context of a double module suggest that, when covalently linked, domains retain autonomous cooperative unfolding and stabilities that are indistinguishable from those of the corresponding isolated domains (Politou et al., 1996). These results prompted us to conclude that the interactions between titin Ig domains are weak, although such a statement has so far been quantified only in terms of thermodynamical (and therefore macroscopic) properties.

Here, we seek a detailed description of the interface and address the question of whether the interdomain mutual orientation is rigid or flexible. To this end, we have used two complementary approaches. Small-angle X-ray scattering (SAXS) was used to determine the average shape of titin fragments in solution, and hence the relative dispositions of individual modules with respect to each other. ¹⁵N relaxation NMR experiments provided information on the dynamical properties of residues as a function of their position in the sequence.