1H and 15N NMR resonance assignments and secondary structure of titin type I domains

Titin/connectin is a giant muscle protein with a highly modular architecture consisting ofmultiple repeats of two sequence motifs, named type I and type II. Type I modules have beensuggested to be intracellular members of the fibronectin type III (Fn3) domain family. Alongthe titin sequence they are exclusively present in the region of the molecule located in thesarcomere A-band. This region has been shown to interact with myosin and C-protein. Oneof the most noticeable features of type I modules is that they are particularly rich insemiconserved prolines, since these residues account for about 8% of their sequence. We havedetermined the secondary structure of a representative type I domain (A71) by 15N and 1HNMR. We show that the type I domains of titin have the Fn3 fold as proposed, consisting ofa three- and a four-stranded -sheet. When the two sheets are placed on top of each other toform the -sandwich characteristic of the Fn3 fold, 8 out of 10 prolines are found on the sameside of the molecule and form an exposed hydrophobic patch. This suggests that thesemiconserved prolines might be relevant for the function of type I modules, providing asurface for binding to other A-band proteins. The secondary structure of A71 was structurallyaligned to other extracellular Fn3 modules of known 3D structure. The alignment shows thattitin type I modules have closest similarity to the first Fn3 domain of Drosophila neuroglian.     

Modularity and homology: modelling of the type II module family from titin.

We report the homology modelling of the structures of the 162 type II modules from the giant multi-domain protein titin (also known as connectin). The package MODELLER was used and implemented in an automated fashion using four experimentally determined structures as templates. Validation of the models was assessed in terms of divergence from the templates and consensus of the alignments. The homology within the whole family of type II modules as well as with the templates is relatively high (20-35% identity and ca 50% similarity). Comparison between the models of domains for which an NMR structure has been solved and the experimental solution gives an estimate of the quality of the modelling. Our results allow us to distinguish between a set of structurally relevant residues, which are conserved throughout the whole family and buried in the hydrophobic core, from the residues that are conserved and exposed. These latter residues are potentially functionally important. Comparison of exposed conserved patches for modules in different regions of the titin molecule suggests potential interaction surfaces. Our results may be tested directly for those modules whose binding partner is known.     

The mechanical stability of immunoglobulin and fibronectin III domains in the muscle protein titin measured by atomic force microscopy.

The domains of the giant muscle protein titin (connectin) provide interaction sites for other sarcomeric proteins and fulfill mechanical functions. In this paper we compare the unfolding forces of defined regions of different titin isoforms by single-molecule force spectroscopy. Constructs comprising six to eight immunoglobulin (Ig) domains located in the mechanically active I-band part of titin are compared to those containing fibronectin III (Fn3) and Ig domains from the A-band part. The high spatial resolution of the atomic force microscope allows us to detect differences in length as low as a few amino acids. Thus constructs of different lengths may be used as molecular rulers for structural comparisons with other modular proteins. The unfolding forces range between 150 and 300 pN and differ systematically between the constructs. Fn3 domains in titin exhibit 20% lower unfolding forces than Ig domains. Fn3 domains from tenascin, however, unfold at forces only half those of titin Fn3 domains. This indicates that the tightly folded titin domains are designed to maintain their structural integrity, even under the influence of stretching forces. Hence, at physiological forces, unfolding is unlikely unless the forces are applied for a long time (longer than minutes).     

Titins in C.elegans with unusual features: coiled-coil domains,novel regulation of kinase activity and two new possible elastic regions

We report that there are previously unrecognized proteins in Caenorhabditis elegans that are similar to the giant muscle proteins called titins, and these are encoded by a single approximately 90kb gene. The gene structure was predicted by GeneMark.hmm and then experimentally verified. The Ce titin gene encodes polypeptides of 2.2MDa, 1.2MDa and 301kDa. The 2.2MDa isoform resembles twitchin and UNC-89 in that it contains multiple Ig (56) and FnIII (11) domains, and a single protein kinase domain. In addition, however, the 2.2MDa isoform contains four classes of short, 14-51 residue, repeat motifs arranged mostly in many tandem copies. One of these tandem repeat regions is similar to the PEVK regions of vertebrate and fly titins. As the PEVK region is one of the main elastic elements of the titins and is also composed of short tandem repeats, this suggests that the repeat motifs in the Ce titins may have a similar elastic function. An interesting aspect of the two largest Ce titin isoforms, is that in contrast to other members of the twitchin/titin family, there are multiple regions which are likely to form coiled-coil structure. In transgenic animals, the first approximately 100 residues of the largest isoforms targets to dense bodies, the worm analogs of Z-discs. Anti-Ce titin antibodies show localization to muscle I-bands beginning at the L2-L3 larval stages and this pattern continues into adult muscle. Ce titins may not have a role in early myofibril assembly: (1) Ce titins are too short to span half a sarcomere, and the onset of their expression is well after the initial assembly of thick filaments. (2) Ce titins are not localized to I-bands in embryonic or L1 larval muscle. The Ce titin protein kinase domain is most similar to the kinase domains of the twitchins and projectin. The Ce titin kinase has protein kinase activity in vitro, and this activity is regulated by a novel mechanism.     

Specific interactions between F1 adhesin of Streptococcus pyogenes and N-terminal modules of fibronectin

Protein F1 is a surface protein of Streptococcus pyogenes that mediates high affinity binding to fibronectin (Fn) and facilitates S. pyogenes adherence and penetration into cells. The smallest portion of F1 known to retain the full binding potential of the intact protein is a stretch of 49 amino acids known as the functional upstream domain (FUD). Synthetic and recombinant versions of FUD were labeled with fluorescein isothiocyanate and used in fluorescence anisotropy experiments. These probes bound to Fn or the 70-kDa fragment of Fn with dissociation constants of 8-30 nm. Removal of the N-terminal seven residues of FUD did not cause a change in binding affinity. Further N- or C-terminal truncations resulted in complete loss of binding activity. Analysis of recombinant versions of the 70-kDa fragment that lacked one or several type I modules indicates that residues 1-7 of the 49-mer bind to type I modules I1 and I2 of the 27-kDa subfragment and the C-terminal residues bind to modules I4 and I5. Fluorescein isothiocyanate-labeled 49-mer also bound with lower affinity to large Fn fragments that lack the five type I modules of the 27-kDa fragment but contain the other seven type 1 modules of Fn. These results indicate that, although FUD has a general affinity for type I modules, high affinity binding of FUD to Fn is mediated by specific interactions with N-terminal type I modules.     

Specific titin and myomesin domains stimulate myoblast proliferation

Myofibrillar proteins titin and myomesin stimulated myoblast proliferation as determined by MTT-test and labelled thymidine incorporation in the DNA. Specific Fn type III and Ig-like domains of these proteins were able to exert mitogenic effects as well. Proliferative effect of Fn type III domains was highly sensitive to inhibition of Ca²⁺/calmodulin dependent protein kinase, whereas the effect of Ig-like domains showed greater sensitivity to the inhibition of adenylyl cyclase – cAMP – PKA pathway. IGF-1 autocrine signalling inhibition partially suppressed mitogenic effects revealed by both domain types.     

Mechanical stability and differentially conserved physical–chemical properties of titin Ig‐domains

The mechanisms that determine mechanical stabilities of protein folds remain elusive. Our understanding of these mechanisms is vital to both bioengineering efforts and to the better understanding and eventual treatment of pathogenic mutations affecting mechanically important proteins such as titin. We present a new approach to analyze data from single‐molecule force spectroscopy for different domains of the giant muscle protein titin. The region of titin found in the I‐band of a sarcomere is composed of about 40 Ig‐domains and is exposed to force under normal physiological conditions and connects the free‐hanging ends of the myosin filaments to the Z‐disc. Recent single‐molecule force spectroscopy data show a mechanical hierarchy in the I‐band domains. Domains near the C‐terminus in this region unfold at forces two to three times greater than domains near the beginning of the I‐band. Though all of these Ig‐domains are thought to share a fold and topology common to members of the Ig‐like fold family, the sequences of neighboring domains vary greatly with an average sequence identity of only 25%. We examine in this study the relation of these unique mechanical stabilities of each I‐band Ig domain to specific, conserved physical–chemical properties of amino acid sequences in related Ig domains. We find that the sequences of each individual titin Ig domain are very highly conserved, with an average sequence identity of 79% across species that are divergent as humans, chickens, and zebra fish. This indicates that the mechanical properties of each domain are well conserved and tailored to its unique position in the titin molecule. We used the PCPMer software to determine the conservation of amino acid properties in titin Ig domains grouped by unfolding forces into “strong” and “weak” families. We found two motifs unique to each family that may have some role in determining the mechanical properties of these Ig domains. A detailed statistical analysis of properties of individual residues revealed several positions that displayed differentially conserved properties in strong and weak families. In contrast to previous studies, we find evidence that suggests that the mechanical stability of Ig domains is determined by several residues scattered across the β‐sandwich fold, and force sensitive residues are not only confined to the A′‐G region. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Domain Expansion and Functional Diversification in Vertebrate Reproductive Proteins

The rapid evolution of fertilization proteins has generated remarkable diversity in molecular structure and function. Glycoproteins of vertebrate egg coats contain multiple zona pellucida (ZP)-N domains (1–6 copies) that facilitate multiple reproductive functions, including species-specific sperm recognition. In this report, we integrate phylogenetics and machine learning to investigate how ZP-N domains diversify in structure and function. The most C-terminal ZP-N domain of each paralog is associated with another domain type (ZP-C), which together form a “ZP module.” All modular ZP-N domains are phylogenetically distinct from nonmodular or free ZP-N domains. Machine learning–based classification identifies eight residues that form a stabilizing network in modular ZP-N domains that is absent in free domains. Positive selection is identified in some free ZP-N domains. Our findings support that strong purifying selection has conserved an essential structural core in modular ZP-N domains, with the relaxation of this structural constraint allowing free N-terminal domains to functionally diversify.     

BRCT domains: A little more than kin, and less than kind

BRCT domains are versatile protein modular domains found as single units or as multiple copies in more than 20 different proteins in the human genome. Interestingly, most BRCT-containing proteins function in the same biological process, the DNA damage response network, but show specificity in their molecular interactions. BRCT domains have been found to bind a wide array of ligands from proteins, phosphorylated linear motifs, and DNA. Here we discuss the biology of BRCT domains and how a domain-centric analysis can aid in the understanding of signal transduction events in the DNA damage response network.     

RNA-binding proteins: modular design for efficient function

Many RNA-binding proteins have modular structures and are composed of multiple repeats of just a few basic domains that are arranged in various ways to satisfy their diverse functional requirements. Recent studies have investigated how different modules cooperate in regulating the RNA-binding specificity and the biological activity of these proteins. They have also investigated how multiple modules cooperate with enzymatic domains to regulate the catalytic activity of enzymes that act on RNA. These studies have shown how, for many RNA-binding proteins, multiple modules define the fundamental structural unit that is responsible for biological function.     

Titin as a giant scaffold for integrating stress and Src homology domain 3-mediated signaling pathways: the clustering of novel overlap ligand motifs in the elastic PEVK segment

The richness of proline sequences in titins qualifies these giant proteins as the largest source of intrinsically disordered structures in nature. An extensive search and analysis for Src homology domain 3 (SH3) ligand motifs revealed a myriad of broadly distributed SH3 ligand motifs, with the highest density in the PEVK segments of human titin. Besides the canonical class I and II motifs with opposite orientations, novel overlapping motifs consisting of one or more of each canonical motif are abundant. Experimentally, the binding affinity and critical residues of these putative titin-based SH3 ligands toward nebulin SH3 and other SH3-containing proteins in muscle and non-muscle cell extracts were validated with peptide array technology and by the sarcomere distribution of SH3-containing proteins. A 28-mer overlapping motif-containing PEVK module binds to nebulin SH3 in and around the canonical cleft, especially to the acidic residues in the loops, as revealed by NMR titration. Molecular dynamics and molecular docking studies indicated that the overlapping motif can bind in opposite orientations with comparable energy and contact areas and predicts correctly orientation-specific contacts in NMR data. We propose that the overlap ligand motifs are a new class of ligands with innate ability to dictate SH3 domain orientation and to facilitate the rate, strength, and stereospecificity of receptor interactions. Proline-rich sequences of titins are candidates as major hubs of SH3-dependent signaling pathways. The interplay of elasticity and dense clustering of mixed receptor orientations in titin PEVK segment have important implications for the mechanical sensing, force sensitivity, and inter-adapter interactions in signaling pathways.     

Extensive and Modular Intrinsically Disordered Segments in C. elegans TTN-1 and Implications in Filament Binding, Elasticity and Oblique Striation

TTN-1, a titin like protein in Caenorhabditis elegans, is encoded by a single gene and consists of multiple Ig and fibronectin 3 domains, a protein kinase domain and several regions containing tandem short repeat sequences. We have characterized TTN-1's sarcomere distribution, protein interaction with key myofibrillar proteins as well as the conformation malleability of representative motifs of five classes of short repeats. We report that two antibodies developed to portions of TTN-1 detect an ∼ 2-MDa polypeptide on Western blots. In addition, by immunofluorescence staining, both of these antibodies localize to the I-band and may extend into the outer edge of the A-band in the obliquely striated muscle of the nematode. Six different 300-residue segments of TTN-1 were shown to variously interact with actin and/or myosin in vitro. Conformations of synthetic peptides of representative copies of each of the five classes of repeats—39-mer PEVT, 51-mer CEEEI, 42-mer AAPLE, 32-mer BLUE and 30-mer DispRep—were investigated by circular dichroism at different temperatures, ionic strengths and solvent polarities. The PEVT, CEEEI, DispRep and AAPLE peptides display a combination of a polyproline II helix and an unordered structure in aqueous solution and convert in trifluoroethanol to α-helix (PEVT, CEEEI, DispRep) and β-turn (AAPLE) structures, respectively. The octads in BLUE motifs form unstable α-helix-like structures coils in aqueous solution and negligible heptad-based, α-helical coiled-coils. The α-helical structure, as modeled by threading and molecular dynamics simulations, tends to form helical bundles and crosses based on its 8-4-2-2 hydrophobic helical patterns and charge arrays on its surface. Our finding indicates that APPLE, PEVT, CEEEI and DispRep regions are all intrinsically disordered and highly reminiscent of the conformational malleability and elasticity of vertebrate titin PEVK segments. The proposed presence of long, modular and unstable α-helical oligomerization domains in the BLUE region of TTN-1 could bundle TTN-1 and stabilize oblique striation of the sarcomere.     

Phosphoserine/threonine-binding domains

Phosphorylation of proteins on serine and threonine residues has traditionally been viewed as a means to allosterically regulate catalytic activity. Research within the past five years, however, has revealed that serine/threonine phosphorylation can also directly result in the formation of multimolecular signaling complexes through specific interactions between phosphoserine/threonine (pSer/Thr)-binding modules and phosphorylated sequence motifs. pSer/Thr-binding proteins and domains currently include 14-3-3, WW domains, forkhead-associated domains, and, tentatively, WD40 repeats and leucine-rich regions. It seems likely that additional modules will be found in the future. The amino acid sequences recognized by these pSer/Thr-binding modules show partial overlap with the optimal phosphorylation motifs for different protein kinase subfamilies, allowing the formation of specific signaling complexes to be controlled through combinatorial interactions between particular upstream kinases and a particular binding module. The structural basis for pSer/Thr binding differs dramatically between 14-3-3 proteins, WW domains and forkhead-associated domains, suggesting that their pSer/Thr binding function was acquired through convergent evolution.     

Single molecule measurements of titin elasticity

Titin, with a massive single chain of 3--4MDa and multiple modular motifs, spans the half-sarcomere of skeletal and cardiac muscles and serves important, multifaceted functions. In recent years, titin has become a favored subject of single molecule observations by atomic force microscopy (AFM) and laser optical trap (LOT). Here we review these single titin molecule extension studies with an emphasis on understanding their relevance to titin elasticity in muscle function. Some fundamental aspects of the methods for single titin molecule investigations, including the application of dynamic force, the elasticity models for filamentous titin motifs, the technical foundations and calibrations of AFM and LOT, and titin sample preparations are provided. A chronological review of major publications on recent single titin extension observations is presented. This is followed by summary evaluations of titin domain folding/unfolding results and of elastic properties of filamentous titin motifs. Implications of these single titin measurements for muscle physiology/pathology are discussed and forthcoming advances in single titin studies are anticipated.     

Towards a complete atomic structure of spectrin family proteins

The spectrin family of proteins represents a discrete group of cytoskeletal proteins comprising principally alpha-actinin, spectrin, dystrophin, and homologues and isoforms. They all share three main structural and functional motifs, namely, the spectrin repeat, EF-hands, and a CH domain-containing actin-binding domain. These proteins are variously involved in organisation of the actin cytoskeleton, membrane cytoskeleton architecture, cell adhesion, and contractile apparatus. The highly modular nature of these molecules has been a hindrance to the determination of their complete structures due to the inherent flexibility imparted on the proteins, but has also been an asset, inasmuch as the individual modules were of a size amenable to structural analysis by both crystallographic and NMR approaches. Representative structures of all the major domains shared by spectrin family proteins have now been solved at atomic resolution, including in some cases multiple domains from several family members. High-resolution structures, coupled with lower resolution methods to determine the overall molecular shape of these proteins, allow us for the first time to build complete atomic structures of the spectrin family of proteins.     

A Novel Fibronectin Binding Motif in MSCRAMMs Targets F3 Modules

Background

BBK32 is a surface expressed lipoprotein and fibronectin (Fn)-binding microbial surface component recognizing adhesive matrix molecule (MSCRAMM) of Borrelia burgdorferi, the causative agent of Lyme disease. Previous studies from our group showed that BBK32 is a virulence factor in experimental Lyme disease and located the Fn-binding region to residues 21–205 of the lipoprotein.

Methodology/Principal Findings

Studies aimed at identifying interacting sites between BBK32 and Fn revealed an interaction between the MSCRAMM and the Fn F3 modules. Further analysis of this interaction showed that BBK32 can cause the aggregation of human plasma Fn in a similar concentration-dependent manner to that of anastellin, the superfibronectin (sFn) inducing agent. The resulting Fn aggregates are conformationally distinct from plasma Fn as indicated by a change in available thermolysin cleavage sites. Recombinant BBK32 and anastellin affect the structure of Fn matrices formed by cultured fibroblasts and inhibit endothelial cell proliferation similarly. Within BBK32, we have located the sFn-forming activity to a region between residues 160 and 175 which contains two sequence motifs that are also found in anastellin. Synthetic peptides mimicking these motifs induce Fn aggregation, whereas a peptide with a scrambled sequence motif was inactive, suggesting that these motifs represent the sFn-inducing sequence.

Conclusions/Significance

We conclude that BBK32 induces the formation of Fn aggregates that are indistinguishable from those formed by anastellin. The results of this study provide evidence for how bacteria can target host proteins to manipulate host cell activities.     

Ca2+-independent binding of an EF-hand domain to a novel motif in the alpha-actinin-titin complex

The interaction between alpha-actinin and titin, two modular muscle proteins, is essential for sarcomere assembly. We have solved the solution structure of a complex between the calcium-insensitive C-terminal EF-hand domain of alpha-actinin-2 and the seventh Z-repeat of titin. The structure of the complex is in a semi-open conformation and closely resembles that of myosin light chains in their complexes with heavy chain IQ motifs. However, no IQ motif is present in the Z-repeat, suggesting that the semi-open conformation is a general structural solution for calcium-independent recognition of EF-hand domains.     

Obscurin, a giant sarcomeric Rho guanine nucleotide exchange factor protein involved in sarcomere assembly.

Vertebrate-striated muscle is assumed to owe its remarkable order to the molecular ruler functions of the giant modular signaling proteins, titin and nebulin. It was believed that these two proteins represented unique results of protein evolution in vertebrate muscle. In this paper we report the identification of a third giant protein from vertebrate muscle, obscurin, encoded on chromosome 1q42. Obscurin is approximately 800 kD and is expressed specifically in skeletal and cardiac muscle. The complete cDNA sequence of obscurin reveals a modular architecture, consisting of >67 intracellular immunoglobulin (Ig)- or fibronectin-3-like domains with multiple splice variants. A large region of obscurin shows a modular architecture of tandem Ig domains reminiscent of the elastic region of titin. The COOH-terminal region of obscurin interacts via two specific Ig-like domains with the NH(2)-terminal Z-disk region of titin. Both proteins coassemble during myofibrillogenesis. During the progression of myofibrillogenesis, all obscurin epitopes become detectable at the M band. The presence of a calmodulin-binding IQ motif, and a Rho guanine nucleotide exchange factor domain in the COOH-terminal region suggest that obscurin is involved in Ca(2+)/calmodulin, as well as G protein-coupled signal transduction in the sarcomere.     

Localization of a cryptic binding site for tenascin on fibronectin

Fibronectin and tenascin are large extracellular matrix proteins that interact with each other and with integrin receptors to regulate cell growth and movement. They are both modular proteins composed of independently folded domains (modules) that are arranged in linear fashion. Fibronectin is a covalent dimer and tenascin is a hexamer. The site on tenascin to which fibronectin binds has been localized to type III modules 3-5. In this study we use surface plasmon resonance to examine the interaction between various fragments of fibronectin and tenascin to further characterize and localize the binding sites. We found that tenascin fragments that contain type III modules 3-5 bind primarily to the N-terminal 29-kDa hep-1/fib-1 domain, which contains the first five type I modules of fibronectin. The dissociation constant, K(d), is approximately 1 microm. The binding site on fibronectin appears to be cryptic in the whole molecule in solution but is exposed on the proteolytic fragments and probably when fibronectin is in the extended conformation.     

Molecular evolution of immunoglobulin and fibronectin domains in titin and related muscle proteins.

The family of regulatory and structural muscle proteins, which includes the giant kinases titin, twitchin and projectin, has sequences composed predominantly of serially linked immunoglobulin I set (Ig) and fibronectin type III (FN3) domains. This paper explores the evolutionary relationships between 16 members of this family. In titin, groups of Ig and FN3 domains are arranged in a regularly repeating pattern of seven and 11 domains. The 11-domain super-repeat has its origins in the seven-domain super-repeat and a model for the duplications which gave rise to this super-repeat is proposed. A super-repeat composed solely of immunoglobulin domains is found in the skeletal muscle isoform of titin. Twitchin and projectin, which are presumed to be orthologs, have undergone significant insertion/deletion of domains since their divergence. The common ancestry of myomesin, skelemin and M-protein is shown. The relationship between myosin binding proteins (MyBPs) C and H is confirmed, and MyBP-H is proposed to have given rise to MyBP-C by the acquisition of some titin domains.