Modular Motif, Structural Folds and Affinity Profiles of the PEVK Segment of Human Fetal Skeletal Muscle Titin

Abstract

The extension of the PEVK segment of the giant elastic protein titin is a key event in the elastic response of striated muscle to passive stretch. PEVK behaves mechanically as an entropic spring and is thought to be a random coil. cDNA sequencing of human fetal skeletal PEVK reveals a modular motif with tandem repeats of modules averaging 28 residues and with superrepeats of seven modules. Conformational studies of bacterially expressed 53-kDa fragment (TP1) by circular dichroism suggest that this soluble protein contains substantial polyproline II (PPII) type left-handed helices. Urea and thermal titrations cause gradual and reversible decrease in PPII content. The absence of sharp melting in urea and thermal titrations suggests that there is no long range cooperativity among the PPII helices. Studies with solid phase and surface plasmon resonance assays indicate that TP1 interacts with actin and some but not all cloned nebulin fragments with high affinity. Interestingly, Ca 2؉ /calmodulin and Ca 2؉ /S100 abolish nebulin/PEVK interaction. We suggest that in aqueous solution, PEVK is an open and flexible chain of relatively stable structural folds of the polyproline II type. PEVK region of titin may be involved in interfilament association with thin filaments in a calcium/calmodulinsensitive manner. This adhesion may modulate titin extensibility and elasticity.The monumental sequencing work of Labeit and Kolmerer (1) has revealed the complete domain organization of the giant elastic protein titin. The bulk of cardiac titin consists of predominately two types of sequence motifs: immunoglobulin (Ig) and fibronectin arranged in three levels of motifs (repeats, superrepeats, and segments). In addition to these well characterized domains, a novel motif consisting of mainly four amino acid residues, Pro, Glu, Val, and Lys, is discovered in the elastic I band region of titin. The length of this PEVK segment varies among muscles, ranging from 183 residues in the human cardiac titin to 2174 residues in human soleus skeletal muscle. Differential splicing of the titin transcripts in the PEVK region as well as in an adjacent tandem Ig segment near the A band produce these size isoforms of titin (1, 2). Since the selective expression of titin size isoforms imparts distinct elasticity to skeletal and cardiac muscles, with the muscle expressing longer titin being more compliant (3), the observed length variation of PEVK and tandem Igs in titin isoforms immediately suggests the possibility that either or both segments may be the elastic elements. Labeit and Kolmerer (1) speculated that the PEVK region, with a predicted nonfolded polypeptide, is the key elastic element of titin. The concept of reversible unfolding and folding of Ig domains was considered unlikely, on the ground of the thermodynamic stability of Ig domains (4, 5).Recent works on the elasticity of single titin molecules (6), single myofibrils (7), and single fibers (8, 9) revealed that, stretched modestly, sarcomere elasticity can be explained by the straightening...

Studies on titin PEVK peptides and their interaction

“…Other work focusing on the conformation of PEVK peptides has suggested that this region may contain some secondary structure [14][15][16][17][18]. Two types of repeat domains have been identified in the PEVK region [17].…”

Titin as a Giant Scaffold for Integrating Stress and Src Homology Domain 3-mediated Signaling Pathways

“…The segment appears to be involved in interfilament adhesion between thin filaments in both skeletal and cardiac muscles. In skeletal muscle, PEVK binds actin and nebulin, with only the PEVK/nebulin interaction being inhibited by Ca 2ϩ /S100A (25,29). This S100 sensitivity of the adhesion between PEVK and thin filaments was later observed in the cardiac muscle, resulting instead from its effect on PEVK/actin interaction (30).…”

“…Pro is thus the second residue of the VPE consensus sequence. We have previously designated PE as the start of each module based on fetal PEVK exons (25). Other investigators have phased the PEVK module in the middle of the exons (54).…”

“…Because the 4-residue core motif is too short to be useful for identifying ligands, we have focused our analysis on the search for class I and II motifs, two well established SH3-binding motifs of 7 residues each. There are different copies of class I and II motifs in human fetal skeletal PEVK (25), human soleus PEVK (51), cardiac N2B PEVK (2), cardiac N2BA PEVK (19), I-connectin PEVK-1 and PEVK-2 (42), D-titin PEVK-1 and PEVK-2 (17), and C. elegans titin PEVT (0), respectively (note that the designation 1 and 2 refer to distinct PEVK segments and not SH3 ligand motifs).…”

“…Our recently detailed analysis of the conformational states of a representative PEVK module (human exon 172) and a 16-PEVK module (473-residue) protein fragment of human fetal titin (TP1) has clearly demonstrated the presence of PPII, unordered coil, and ␤-turn structure motifs in the largely unstructured PEVK segment (23)(24)(25). The facile interconversion of these conformational states under subtle environmental conditions such as temperature, ionic strength, and solvent polarity has revealed that the titin PEVK segment is conformationally malleable, without involving proline trans/cis isomerization.…”