The - complex is a directed, superstable SEE COMMENTARY molecular bond in the muscle Z-disk

Morten Bertza, Matthias Wilmannsb, and Matthias Riefa,c,1

aPhysik Department E22, Technische Universita¨t Mu¨ nchen, James-Franck-Strasse, 85748 Garching, Germany; and cCenter for Integrated Science Munich (CIPSM), 81377 Munich, Germany; bEuropean Molecular Biology Laboratory Hamburg, c/o DESY, Notkestrasse 85, 22603 Hamburg, Germany

Edited by James A. Spudich, Stanford University School of Medicine, Stanford, CA, and approved June 11, 2009 (received for review March 2, 2009)

Mechanical stability of bonds and protein interactions has recently producing regular sawtooth patterns as the chain of identical become accessible through single molecule mechanical experi- subunits unfolds sequentially (13). The dissociation of a protein ments. So far, mechanical information about molecular bond complex, which is connected by non-covalent bonds, however, mechanics has been largely limited to a single direction of force will yield an unfolding fingerprint that is indistinguishable from application. However, mechanical force acts as a vector in space the detachment of the polypeptide chain from the cantilever. and hence mechanical stability should depend on the direction of How can we detect an unambiguous mechanical fingerprint of force application. In , the giant protein titin is the rupture of the titin-telethonin complex? We used cysteine anchored in the Z-disk by telethonin. Much of the structural engineering to introduce disulfide bridges covalently connecting integrity of the Z-disk hinges upon the titin-telethonin bond. In this the 3 polypeptide chains of telethonin and the 2 titin fragments, paper we show that the complex between the muscle titin respectively (for details, see Methods and Fig. S1). The covalent and telethonin forms a highly directed molecular bond. It is crosslinks will prevent early detachment of the protein after designed to resist ultra-high forces if they are applied in the forced dissociation of the titin-telethonin assembly and allow direction along which it is loaded under physiological conditions, observation of the complete sequence of unbinding/unfolding while it breaks easily along other directions. Highly directed events of the whole complex. As handles for attachment of the molecular bonds match in an ideal way the requirements of tissues complex to tip and surface we fused domains to the subject to mechanical stress. titin fragment (compare construct in Fig. 1C). The small protein ubiquitin (76 amino acids) can be readily distinguished from the atomic force microscopy ͉ force spectroscopy ͉ protein engineering ͉ unfolding of the larger Ig-domains (94 residues) present in the protein folding titin-telethonin complex by their contour length increase. Since in vivo, most of the long titin polypeptide chain aligns he giant muscle protein titin (1, 2) spans the complete half with the principal filament direction of muscle Tsarcomere from the Z-disk to the M-line and is involved in (compare Fig. 1B), we postulate that mechanical forces gener- a multitude of functions including passive muscle elasticity (3, 4), ated by the /relaxation cycle translate into stress sensing (5, 6), regulation (7), and muscle assembly (8). pull/push forces that act on the C terminus of the N-terminal titin Rigid anchoring of titin within the Z-disk even under extreme fragment (Ig domains Z1 and Z2), from where the remaining titin filament extends. Therefore, we first studied the stability of mechanical loads is key for proper muscle function (9). Recently, the titin-telethonin complex by applying force at the titin C- Zou et al. have shown that 2 titin molecules are connected at termini. For this purpose, we designed the molecular construct their N termini in a palindromic arrangement through the shown in Fig. 1C (Z1Z2T ) where the ubiquitin handles were muscle protein telethonin (also known as T-Cap) into a 2:1 C-C fused to the C-termini of the titin fragments. assembly (see Fig. 1A and B) (10). Palindromic arrangements are Force extension traces at low extensions show the character- rare in protein-protein complexes (11) and may provide a unique istic unfolding patterns of the ubiquitin handles (14) (gray peaks interaction under specific physiological conditions such as me- in Fig. 1C). Since attachment to tip and surface occurs through chanical force. In this complex, 2 identical titin-telethonin-titin non-specific adsorption not necessarily at the termini of the ␤-sheets are formed that are in a diagonal orientation with protein, the number of ubiquitin unfolding events can vary. After respect to the principal axis of the complex that is defined by the complete unfolding of all , a marked fingerprint of the 2 telethonin ␤-turns (Fig. 1A). The 2 C-terminal Z2 Ig-domains stepwise breaking of the titin-telethonin complex can be ob- of titin are in distal positions of the overall complex. Since the served (colored section of the force curves in Fig. 1C). The most remaining titin fiber extends from the C terminus of this complex striking result is the extremely high force required to initiate the it is expected that any mechanical forces originating from unbinding process (green circle in Fig. 1C and histogram in Fig. BIOPHYSICS AND operating muscles would act on the Z2 domains, as opposed the 1D). An average force of 707 Ϯ 24 pN (average Ϯ SEM, n ϭ 34) COMPUTATIONAL BIOLOGY N-terminal titin Z1 domains. at a pulling speed of 1 ␮m/s exceeds all known measured Recent molecular dynamics simulations have suggested that a stabilities of protein structures or complexes (15). In fact, tight hydrogen bond network between titin and telethonin covalent bonds break at only twice this value (16, 17). We used conveys a mechanical stabilization to the complex (12). Direct the worm-like chain (WLC) model (18) to determine the in- experimental measurement of the mechanical stability of a crease in contour length that follows an unfolding event (dashed protein complex, however, has been missing to date. Therefore, a molecular basis of a force-resistant anchoring mechanism of the titin N terminus within the sarcomeric Z-disk remains yet Author contributions: M.B. and M.R. designed research; M.B. performed research; M.B. elusive. analyzed data, M.W. contributed new reagents/analytic tools; and M.B., M.W., and M.R. wrote the paper. Results The authors declare no conflict of interest. Single molecule mechanical experiments using AFM critically This article is a PNAS Direct Submission. rely on the identification of mechanical fingerprints that allow See Commentary on page 13149. distinguishing single molecule events from nonspecific interac- 1To whom correspondence should be addressed. E-mail: [email protected]. tions as well as multimolecule events. The engineering of mo- This article contains supporting information online at www.pnas.org/cgi/content/full/ lecular fingerprints has so far involved the use of polyproteins 0902312106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0902312106 PNAS ͉ August 11, 2009 ͉ vol. 106 ͉ no. 32 ͉ 13307–13310 Downloaded by guest on September 27, 2021 Fig. 1. (A) Structure of the antiparallel titin-telethonin complex (pdb: 1YA5) (10). The first 2 N-terminal domains [Z1 (dark) and Z2 (light)] of 2 titin molecules [chain A (green) and chain B (blue)] are assembled into a palindromic complex by telethonin (pink). (B) Schematic overview of the and its 3 major filaments: , , and titin. Only one-half of the sarcomere is shown. The titin-telethonin complex located in the Z-disk is shown as a space-filling model. (C) Typical force-extension traces of the titin-telethonin complex pulled at its C termini (Z1Z2TC-C): The structural model illustrates the pulling geometry and the location of the cysteine crosslinks (yellow bars, compare Fig. S1) necessary to obtain a clear fingerprint of the rupture of the complex. Unfolding events of the ubiquitin handles (gray filled circles in the structural model) are colored gray in the unfolding traces. WLC curves fit to the rupture of the complex (colored part of the traces) are shown as dashed lines with contour length increases indicated above the curves. (D) Force histogram of the initial rupture event of the complex (green circle in C).

curves in Fig. 1C). Analysis of these contour length increases To assess the amount of stabilization conveyed onto the titin observed during rupture of the complex allows drawing a domains by forming the titin-telethonin complex, we now inves- structural picture of the sequence of events in the rupture tigated the stability of the Z1 and Z2 domains in the absence of process (Fig. S2). The major rupture peak is associated with a telethonin. Ubiquitin handles were attached to the titin fragment length increase of 29.7 Ϯ 0.4 nm (Ϯ SEM). This length increase at the N terminus of Z1 and at the C terminus of Z2 (compare corresponds exactly to the length expected for unfolding of the construct in Fig. 2A). The unfolding events of the titin domains approximate 94 residues of titin domain Z2 (29.8 (green events in Fig. 2A) can be clearly distinguished from the nm). In the subsequent peak (pink in Fig. 1C), both Z1 domains ubiquitin handles (gray in Fig. 2A) by their larger contour length as well as telethonin unfold. It is important to note that a large increase. We find that the 2 Ig-domains Z1 and Z2 unfold with part of the Z1 domains and of telethonin are shut off from force an average contour length increase of 29.4 Ϯ 0.2 nm (Ϯ SEM, by the cysteine crosslinks and hence do not contribute to the n ϭ 205) at an average force of 168 Ϯ 2pN(Ϯ SEM) (compare length increase (see Fig. S2). The last peak (blue event) reflects histogram in Fig. 2B). Interestingly, most titin Ig domains unfolding of the now isolated remaining Z2 domain. investigated so far exhibit larger unfolding forces (4) and hence

Fig. 2. (A) Force-extension traces of the Z1Z2 fragment from titin flanked by ubiquitin domains (gray filled circles). Unfolding events of the ubiqutin handles are colored in gray in the traces. Unfolding events of the titin fragment are colored in green. Dashed lines indicate WLC fits with the contour length increases shown above the curves. (B) Force histogram of the mechanical unfolding of the Z1Z2 domain pair. (C) Force-extension traces of the inverted pulling geometry of the titin-telethonin complex (Z1Z2TN-N): The structural model illustrates the architecture of the construct. Cysteine crosslinks are shown as yellow bars (compare Fig. S1), ubiquitin handles as gray filled circles. The rupture of Z1Z2TN-N is indicated by the various colors in the traces. WLC fits to the data and contour length increases are show in the left panel. (D) Force histogram of the initial rupture event of Z1Z2TN-N.

13308 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0902312106 Bertz et al. Downloaded by guest on September 27, 2021 the domain pair Z1Z2 alone is a comparatively labile structure in vitro. The formation of the titin-telethonin sandwich complex increases the stability of this structure 4-fold, far beyond all other SEE COMMENTARY Ig domains present in the titin molecule examined so far by mechanical experiments. How is the mechanical superstability of the titin-telethonin complex tuned to its physiological function of forming a durable link in the mechanically strained Z-disk? To answer this ques- tion, we applied mechanical load to the titin-telethonin complex in a geometry reverse to the physiological C-terminal direction. We fused the ubiquitin handle domains to the N terminus of the titin Z1 domain (Z1Z2TN-N) and applied load in the single molecule experiment (compare structural model in Fig. 2C). Sample traces and a force histogram of this inverted pulling experiment are shown in Fig. 2C and D. The characteristic unfolding events of ubiquitin are shown in gray whereas the colored part of the curve corresponds to the rupture of the titin-telethonin complex when load is applied to the N termini. The difference in rupture force (green circle Fig. 2C)inthe inverted pulling geometry Z1Z2TN-N is indeed drastic if com- pared to the physiological pulling geometry Z1Z2TC-C:Inthe inverted pulling direction, the stability is almost indistinguish- able from the isolated domains Z1Z2 with only a slightly elevated average stability of 237 Ϯ 5pN(Ϯ SEM, n ϭ 47). The sequence of events leading to breakage of Z1Z2TN-N is illustrated in Fig. S3. The initial event is detachment and unfolding of a Z1 domain (dark green peak in Fig. 2C) from the complex. In the next step, telethonin detaches and unfolds (red peak). Finally, the remain- ing 3 Ig domains unfold subsequently (light green and blue peaks). Fig. 3. (A) Top: Structure of the titin-telethonin complex with part of the Discussion hydrogen bond network (black lines) stabilizing the distal Z2 domains when Our results reveal the titin-telethonin interaction as a strongly load is applied at the C termini. Bottom: Back view of the titin-telethonin directed molecular bond, optimized to resist loads applied in the complex. The N termini of the proximal Z1 domains do not form a tight C-terminal direction (see Fig. 3B, bottom), which presents the hydrogen bond network with telethonin. (B) The titin-telethonin complex is direction of the remaining titin fiber. While the titin-telethonin a directed, highly stable molecular bond. Load applied to the C termini, as is the case in the muscle during passive stretching, requires high forces to complex strongly resists unbinding when loaded in the naturally dissociate the complex. If loaded at the N termini, the complex unravels occurring pulling direction, it slips apart relatively easily when readily. pulled in the reverse geometry. Consistent with the strong mechanical directedness of the titin-telethonin complex, pulling the complex in an N-C-terminal direction (Z1Z2TN-C) (see Fig. function beyond mechanosensing. Our results support this: With S4) leads to only slight stabilization compared to the isolated a rupture force of 700 pN, exceeding all Ig domains from titin domains. A likely molecular explanation for this directedness is measured so far (4), the titin-telethonin complex is perfect for the tight network of hydrogen bonds established by telethonin firmly anchoring the giant muscle protein titin in the Z-disk. binding and stabilizing predominantly the C terminus of the Such a rigid molecular complex, however, is rather ill suited for distal Z2 domains (see Fig. 3A, top) (12), where load is applied contributing to a strain sensor in muscle. during passive muscle stretching. In contrast, the N terminus of Our data may thus provide a molecular rationale of an the proximal Z1 domains does not interact with telethonin and N-terminal titin assembly complex that allows stable anchoring only forms hydrogen bridges to a neighboring strand from the of this filament system in the sarcomeric Z-disk, while being same Ig domain (Fig. 3A, bottom); hence, a significant stabili- exposed to large mechanical forces during the muscle contrac-

zation of the complex if pulled from the N terminus cannot tion/relaxation cycle. We anticipate that directedness of molec- BIOPHYSICS AND

occur. Apparently, nature uses selective clustering of H-bonds to ular bonds will be a concept important for a variety of other COMPUTATIONAL BIOLOGY reinforce the C terminus of Z2 and thus prevent breaking of the molecular complexes subject to mechanical strain in living titin-telethonin complex under mechanical loads. A macroscopic organisms. mechanical analog of the directed titin-telethonin bond is a hook connecting the 2 Z2 domains thus preventing unbinding of the Methods complex in C-terminal direction but letting the complex slide Cloning and Protein Expression. The human titin N terminus covering domains apart if pulled from the N terminus (see Fig. 3B). It is important Z1 and Z2 (residues 1–195) was fused to the C terminus of 3 ubiquitin domains to note, that the concept of directedness of a molecular bond carrying an N-terminal His-Tag (3U-Z1Z2) or to the N terminus of 3 ubiquitin cannot be understood if considering only thermodynamic pa- subunits carrying a C-terminal His-tag (Z1Z2–3U) in pET28a (Novagen) by rameters like free energy of complex formation or off-rates of standard molecular biology techniques. Cysteine residues for disulfide the complex, since those parameters do not contain directional crosslinks (S86C in Z1Z2–3U and T188C in 3U-Z1Z2) were introduced using the information. Quikchange site-directed mutagenesis kit (Stratagene). Furthermore, Z1Z2 was sandwiched between subunits 3 and 4 in a construct containing 6 con- A complex between telethonin and the muscle LIM protein catenated ubiquitin domains in pET28a (3U-Z1Z2–3U,). The N-terminal trun- (MLP) has been suggested to be a key component of the muscle cation of telethonin (residues 1–90) with all cysteines mutated to has stretch sensing machinery (19). Recent findings, however, have been described previously (21). We constructed 2 variants for cysteine shown that MLP is a highly mobile component of muscle cells crosslinking (TeleAT: A20C/T50C and TeleEQ: E16C/Q46C) using the and not anchored to sarcomeric structures (20) suggesting a Quikchange Multi Site directed mutagenesis kit (Stratagene).

Bertz et al. PNAS ͉ August 11, 2009 ͉ vol. 106 ͉ no. 32 ͉ 13309 Downloaded by guest on September 27, 2021 The telethonin and titin constructs were expressed and purified as de- recorded, 47 of which contained the unfolding fingerprint of the titin- scribed previously (21). The titin-telethonin complexes were formed by adding telethonin in the N-terminal pulling geometry. For Z1Z2TC-C, 939 single mol- the appropriate purified titin-ubiquitin chimeras to telethonin solutions (3U- ecule traces were recorded, 34 of which showed the rupture of the titin- Z1Z2(T188C) ϩ TeleAT forming Z1Z2TN-N and Z1Z2(S86C)-3U ϩ TeleEQ forming telethonin complex. The low probability of obtaining single molecule events Z1Z2TC-C, compare Fig. S1) at a final concentration of 4 M urea with excess for Z1Z2TC-C is likely due to detachment of the protein from the cantilever telethonin followed by dialysis over night against PBS containing 10 mM DTT. before the high forces necessary to dissociate the complex could be reached After dialysis, the complexes were further purified by size-exclusion chroma- or incomplete disulfide bond formation. tography using a Superdex 200 column (GE Healthcare) in PBS. 3U-Z1Z2–3U Contour length increases (⌬L) were determined by fitting worm-like chain was purified by size exclusion chromatography after elution from the Ni-NTA curves calculated using the interpolation formula by Bustamante et al. (18) with matrix and used directly for measurements. a fixed persistence length of 0.5 nm for forces up to 250 pN. For the higher force regime in Z1Z2TC-C, 0.3 nm was used. Contour length increases determined at a Single-Molecule Force Spectroscopy. Single-molecule force spectroscopy was persistence length of 0.3 nm were corrected to a persistence length of 0.5 nm by performed on a custom-built atomic force microscope at ambient tempera- ⌬L0.5 ϭ⌬L0.3⅐␥ with ␥ ϭ 0.964 (23). Contour length increases at a persistence ture. Gold-coated cantilevers (Biolever Type A, Olympus) with a spring con- length of 0.5 nm were used to calculate the number of amino acids n involved in stant of 30 pN/nm were used in all experiments. In a typical experiment, an unfolding event via ⌬L ϭ n⅐daa Ϫ dintermediateϩ dfolded. Here, dintermediate and protein solution was applied to a freshly activated Ni-NTA glass slide and force dfolded denote the distance between the points of force application in the inter- ␮ traces were recorded at a pulling velocity of 1 m/s. All traces were inspected mediate and the native state, respectively, as determined from the crystal struc- upon recording and traces that showed no clear single molecule events (at ture of the titin-telethonin complex (1YA5) (10). daa is the contour length increase least 2 ubiquitin unfolding peaks) were discarded immediately. For Z1Z2 the per amino acid, which has been determined to be daa ϭ 0.365 Ϯ 0.002 nm for our probability to pick up a single molecule was approximately 5%, for Z1Z2TN-N instrument at a persistence length of 0.5 nm (23). and Z1Z2TC-C the probability was Ͻ2%. These probabilities are typical for single-molecule force spectroscopy experiments (22). All traces were then ACKNOWLEDGMENTS. We thank Jan-Philipp Junker for discussions and as- screened visually and by fitting WLC-curves as described below in Igor Pro sistance with cloning, Michael Schlierf for discussions, and Anja Gieseke for (Wavemetrics) for reproducible features that appeared in addition to the assistance with cloning. Financial support of Deutsche Forschungsgemein- regular sawtooth pattern of the ubiquitin handles. For Z1Z2, a total of 173 schaft Grant RI 990/3-1 (to M.R.) is gratefully acknowledged. M.W. acknowl- single molecule traces was recorded, 102 of which showed unfolding events of edges funding from Fonds zur Fo¨rderung der wissenschaftlichen Forschung/ both the Z1 and Z2 domains. For Z1Z2TN-N, 594 single-molecule traces were Deutsche Forschungsgemeinschaft (P1906).

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