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J. Biol. Chem., Vol. 281, Issue 37, 27539-27556, September 15, 2006
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From the Muscle Proteomics and Nanotechnology Section, Laboratory of Muscle Biology, NIAMS, National Institutes of Health, Bethesda, Maryland 20892
Received for publication, May 11, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Rapid progress is being made in the roles of titin in several distinct signaling pathways (11). The kinase domain near the titin carboxyl terminus at the M-line region of the sarcomere is dually regulated by calcium/calmodulin and tyrosine phosphorylation. The agonist-sensitive phosphorylation of titin fore-casted its involvement in signaling mechanisms (12, 13). The stress-sensitive titin kinase interacts with the zinc finger protein NBR1 through a force-induced conformation change and eventually controls muscle gene expression and protein turn-over via p62, MuRF2, and serum-responsive transcription factor (14-16). At its amino terminus near the Z-line, repeats Z1-Z2 interact with telethonin (T-cap), which in turn interacts with a potassium channel subunit, myostatin, a muscle growth factor (17), and the muscle LIM protein, MLP (18). Z-line repeat Z4 and the 700-kDa titin isoform "novex-3 titin" interact with obscurin, an 
700-kDa protein that is involved in regulating Rho-like GTPases and A-band assembly (19, 20). In the I-band region, N2B domain and PEVK segment interact with crystallin that is known to be a chaperonin in assisting protein folding (21). The interaction of the polyproline type II helices (PPII) in the PEVK segment with Src homology domain 3 (SH3) invokes SH3 signaling pathways and possibly in the assembly and targeting and orientation of the nebulin during sarcomere assembly (22).
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 (23-25). 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. The elasticity of PEVK also varies with ionic strength, where the elasticity has been measured to either decrease (26, 27) or increase (28) with increasing ionic strength. This behavior implies that ionic interactions within the PEVK segment play an important role in the ensemble average of the polypeptide configuration and therefore the relative entropic and enthalpic contributions to molecular elasticity. We have proposed that the titin PEVK segment is an open and flexible poly-ampholyte with its elasticity driven and modulated by the number and location of internal salt bridges (26). The elasticity of adult soleus titin PEVK appears to vary along its length, with the amino-terminal region being more rigid than its carboxyl-terminal region (28).
The elastic PEVK segment is also a major binding region for protein interactions. 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 Ca2+/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). The interaction of titin PEVK with the thin filament proteins thus may be manifested as a calcium-sensitive and reversible resistance to stretching (i.e. viscous drag) of the muscle fiber (31).
Proline-rich peptide sequences in intrinsically disordered proteins or protein domains are hallmarks of protein-protein recognition sites in signaling pathways, by acting as ligands that bind modular adapter domains such as SH3, WW, and EVH1 (32). The SH3 domain, a family of small globular proteins of 60 amino acids found in more than 1500 eukaryotic proteins (33), has a hydrophobic cleft formed by two orthogonal -sheets that bind to 7-10-residue polyproline II helices with a canonical ligand motif PXXP. The peripheral sequences around the core-binding motif of SH3 determine which of the two possible orientations each ligand takes in the binding cleft. Two classes of ligands, class I, with the consensus sequence R(K)XXPXXP, and class II, with the consensus sequence XPXXPXR(K) (34), bind to the surface cleft of SH3 domains in opposite orientations. Recently, several SH3 domains were found to recognize non-PXXP motifs: PXXDY (by EPS) (35); WXXQF (by Pex13p) (36); VPMRLR (by p53BP-2) (37); RKXXYXXY (by SKAP55) (38); PXXXRXXKP (by Grb2 SH3 (C)) (39); PX(V/I)(D/N)RXXKP (by Hbp) (40); and RXXK (by Gads SH3 (C)) (41). Moreover, a few SH3 domains are known to bind ligands outside of the conventional hydrophobic cleft (42). Further insights of the determinants of ligand orientation have been provided by the "tryptophan switch" concept proposed by Fernandez-Ballester et al. (43). The authors classified the SH3-ligand complexes according to two distinct angles between the conserved Trp indole ring and Pro residues at the YPW triad in the binding site of the SH3, each correlating with one of the two ligand orientations (43).
The richness of natural proline sequences in the titin family qualifies titin as one of the largest intrinsically disordered proteins in nature and provides a unique opportunity to understand the biological function and structural basis of protein recognition based on natural selection. This search complements the powerful screening approaches based on combinatorial phage or bacterial display libraries. Our recent demonstration of nebulin SH3 domain interaction with the central PPII helix of a representative PEVK repeating module (22) raised the intriguing possibility that titin PEVK may contain multiple SH3-binding sites and even as tandem repeats in some titin isoforms. In this study, we address this question by carrying out an extensive search and analysis for SH3-binding motifs in the protein sequences of titin and titin-like proteins, including human titin, invertebrate connectin (titin), Drosophila titin PEVK and Caenorhabditis elegans titin PEVT/K segments. Besides classical class I and II motifs, novel motifs consisting of overlapping motifs with opposite orientation are abundant. Experimentally, we evaluated the binding affinity of these motifs toward nebulin SH3 domain and other SH3-containing proteins in muscle and non-muscle cells with the peptide array technology. The feasibility of binding the bipolar motifs to SH3 in opposite orientations was also demonstrated by molecular modeling. These findings have important implications for the signaling roles, mechanical sensing of and inter-receptor interactions of intrinsically disordered proline-rich domains in signaling proteins.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Preparation of nSH3, Muscle, and HeLa Extracts—The soluble nSH3 (AAB02622 [GenBank] ) was expressed using Bl21(DE3)pLysS host cells transfected with a pET3d plasmid (Novagen, Madison, WI) in LB medium and purified as described previously (44). For muscle extracts, an 8-pound New Zealand White rabbit was injected intramuscularly in the haunch with 3 ml of anesthetic (Ketaset/Xyla-Jet 9:1). After confirming euthanasia, the rabbit was exsanguinated followed by dissection. Each of the four striated muscles were dissected: longissimus dorsi (6 g), psoas (5.7 g), soleus (2.3 g), and heart (5.6 g). Each of the four rabbit muscles was individually homogenized three times, 15 s each, with a Polytron homogenizer (Brinkmann Instruments) in 5-8 ml/g of muscle (v/w) in pyrophosphate relaxing buffer (0.1 M KCl, 2 mM MgCl2, 2 mM EGTA, 0.01 M Tris, pH 6.7, 2 mM Na4P2O7, pH 7.2, 0.5 mM dithiothreitol, 0.1 mM phenylmethyl-sulfonyl fluoride, 1 mM diisopropyl fluorophosphate, 0.5% Triton X-100, v/v). Small aliquots of the homogenized material (whole muscle) were saved for further Western blotting analysis. The rest of the homogenates were centrifuged at 1500 x g for 15 min (Beckman, JA 25.50 rotor). The supernatants (S1) were then further centrifuged at 27,000 x g for 30 min using the same rotor and finally a high speed centrifugation at 65,000 x g for 15 min (Beckman TLA 100.4) to prepare soluble muscle extracts free of any traces of insoluble components. The pellets (P1) were resuspended by vortexing into 35 ml of low salt buffer (0.1 M KCl, 2 mM MgCl2, 2 mM EGTA, 0.01 M Tris, pH 6.7, 0.1% Triton X-100, v/v), followed by pelleting at 1500 x g for 15 min and washing nine times. These highly washed myofibrils were for Western blotting analysis. HeLa cell nuclear extract and HeLa cell cytoplasmic extract were generous gifts from Drs. Louis Schiltz and Vittorio Sartorelli of Laboratory of Muscle Biology, NIAMS, National Institutes of Health, which were prepared as described previously (45).
Western Blotting Analysis—The monoclonal anti-amphiphysin, anti-BMX, anti-BRAMP2, anti-Btk, anti-CASK, anti-CDC42GAP, anti-cortactin, anti-Crk, anti-Dlg, anti-Eps8, anti-Fyn, anti-GRB2, anti-GRB14, anti-HS1, anti-Lck, anti-Lyn, anti-Nck, anti-Ntk, anti-p130cas, anti-PI3-K, anti-PSD-95, anti-Ras-GAP, and anti-Csk were obtained from BD Biosciences. Polyclonal anti-Abl SH3 domain and anti-JIP
(SH3) were from Upstate%20Biotechnology">Upstate Biotechnology, Inc., and Zymed Laboratories Inc., respectively. Monoclonal anti-
-I spectrin and anti--II spectrin were purchased from Signet Laboratories, Inc. Polyclonal rabbit anti-nebulin SH3 was prepared against the human nebulin (P20929
[GenBank]
) carboxyl-terminal peptide 6652VQRTGRTGMLPANYVE6667C in our laboratory and was affinity-purified by binding to peptide sulfolink-coupled agarose gel (Pierce), followed by elution using 100 mM glycine-HCl, pH 2.8, and 8 M urea in PBS.
Protein loading per lane was adjusted according to the intensity of myosin for whole muscles and myofibrils with roughly 30 µg of total proteins per lane. Whole muscle tissues, myofibrils, and muscle extracts were resolved on either 2-well NuPAGE BisTris 4-12% gel (MOPS running buffer) or 2-well NuPAGE Tris acetate 4-12% (TA running buffer) in order to resolve giant muscle proteins. Proteins were electrophoretically transferred to PVDF membrane by 51 mA for 3 h in NuPAGE transfer buffer (Invitrogen), 10% methanol. Membrane was blocked using 1% alkali-soluble casein in PBS (Novagen), washed by TBS-T (10 mM Tris, pH 7.2, 150 mM NaCl, 0.05% Tween 20, v/v) for 10 min, and cut into 28 3-mm wide slices. Each slice was incubated with appropriate dilution of first antibodies as recommended by the manufacturers (supplemental Table ST1) and detected by either anti-mouse horseradish peroxidase (HRP) or anti-rabbit HRP (Zymed Laboratories Inc.), followed by chemiluminescence as described in the manufacturer's protocol (Amersham Biosciences). Several different exposures on film for each blot were made routinely to detect weaker bands. The apparent molecular weight for each positive band was determined by the standard curve derived from See Blue Plus2 Pre-stained Standard (Invitrogen) in either MOPS buffer or TA buffer.
Peptides and Ligand Arrays of SH3-binding Motifs—A 28-residue titin PEVK (GenBankTM accession number AF321609
[GenBank]
) module, PEPPKEVVPEKKAPVAPPKKPEVPPVKV, was synthesized and purified as described elsewhere (23). The purity of the peptides (>98%) was confirmed by analytical high pressure liquid chromatography and matrix-assisted laser desorption ionization time-of-flight. Peptide arrays containing peptide covalently attached to Whatman 50 cellulose membrane supports were prepared by Sigma Genosys using the in situ synthesis technique of SPOTs synthesis technology (46). Each peptide spot contains 5-10 nmol of 12-mer peptide with the carboxyl terminus attached to a diglycyl peptide spacer via an ester bond to the hydroxyls of cellulose. As controls, each membrane contains the same set of positive control peptides that are known to bind SH3 domains (Fig. 3B). A total of 229 (10-13)-mer peptides of class I, class II, and non-PXXP SH3-binding motifs were synthesized based on the entire human exons, as well as the open reading frames of D-titin, I-connectin, and Ce titin (supplemental Table ST1).
nSH3 Binding to Ligand Arrays—The dry SPOTs membranes were first rinsed with methanol for 2 min and three times for 10 min in TBS-T (10 mM Tris, pH 7.2, 150 mM NaCl, 0.05% Tween 20, v/v) and then blocked overnight in the blocking buffer (1% alkali-soluble casein, Novagen) and then shaken at room temperature. The blocked membranes were then incubated with nSH3 (2-5 µM) in blocking solution for 4 h at 4 °C with gentle shaking. Unbound nSH3 was removed by washing with TBS-T (three times for 10 min), plus a final wash with H2O (5 min) at 4 °C, and the bound nSH3 was electrotransferred in a semi-dry blotter (Bio-Rad) at a constant power of 0.8 mA/cm2 for 20 min between 50 mM sodium phosphate, pH 7.0, soaked blotting papers onto activated PVDF membrane (pretreated with 1% glutaraldehyde (Electron Microscopy Sciences, Ft. Washington, PA) for 30 min, followed by extensive washing with H2O (five times for 6 min)). Electrotransfer was conducted in buffer at pH 7 to allow the dissociated nSH3 (pI 4.3) to migrate toward the anode and be captured by the activated PVDF membrane. (The nonactivated PVDF membrane failed to capture the SH3 molecules.) After electrotransfer, the membrane was quenched in 0.1 M glycine, pH 7.0, for 30 min, followed by washing with H2O (5 min). The bound nSH3 was detected using anti-nSH3 rabbit IgG primary antibody (0.11 µg/ml) followed by a secondary goat anti-rabbit HRP-conjugated antibody and chemiluminescence measurement (ECL kit, Amersham Biosciences). The intensity of each positive spot was quantified by integrating the entire spot using ImageJ. Immediately after use, SPOTs membrane was regenerated according to the manufacturer's instructions (Sigma). Briefly, the membrane was first washed three times with 20 ml of water for 10 min each time at room temperature, followed by three times incubation with 20 ml of buffer A (8 M urea, 1% SDS, 0.1% 2-mercaptoethanol), 30 min each time at 40 °C, and three times washing with 20 ml of buffer B (50% ethanol, 10% acetic acid) for 30 min each time at 20 °C. Finally, the membrane was washed with methanol (20 ml, 10 min, two times) and TBS-T (20 ml, 10 min, three times). The regenerated membrane was routinely tested for complete removal of bound SH3 by anti-SH3 Western blotting before reuse.
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-II spectrin), followed by a secondary rabbit anti-mouse HRP-conjugated antibody and then a goat anti-rabbit HRP-conjugated antibody and chemiluminescence measurement (ECL kit, Amersham Biosciences). Films were quantitated with a Kodak 2000A system and Scion Image software.
Immunofluorescent Localization of SH3-containing Proteins in Split Rabbit Soleus Muscle Fibers—Single soleus muscle fibers were dissected from rabbit tissue essentially as described previously (47). Single muscle fibers were dissected in a relaxing buffer (150 mM potassium propionate, 5 mM potassium phosphate, 3 mM magnesium acetate, 5 mM potassium EGTA, 5 mM NaN3, 3 mM ATP, 20 µg/ml leupeptin, 5 µg/ml aprotinin, 1 mM dithiothreitol, 50 mM 2,3-butane-dione monoxime). They were then stretched to varying degrees (0, 50, and 100% resting length) and mounted onto one-hole gold EM grids and then blocked with 0.5% bovine serum albumin (globulin-free), 0.02% normal goat serum in a buffer (10 mM Tris maleate, 10 mM KCl, 2 mM MgCl2, 2 mM EGTA, pH 7.0) for 1 h. The grids were then incubated overnight with two dilutions (5 and 10 µg/ml) of each of the following antibodies: anti-ABl SH3, anti-JIP SH3, anti-spectrin II SH3, anti-nebulin SH3, anti-cortactin, anti-GRB2, anti-p130cas, anti-DLG, anti-titin RT11, anti--actinin, and mouse IgG1 as a negative control (supplemental Table ST2). Each grid was rinsed four times, 10 min each, with 0.5 ml of blocking solution, followed by a 1-h incubation with Alexa 568-labeled secondary antibody and Alexa 488-labeled phalloidin. Mounted fibers were washed as before and then mounted between a glass slide and coverslip using mounting media with antifade (20% w/v polyvinyl alcohol, 20% v/v glycerol in PBS, with freshly added 0.1 g of 1,4-diazobicyclo(2,2,2)octane per ml prior to use). Immunofluorescence microscopy was performed on a Zeiss LSM 50 confocal microscope.
Molecular Dynamics of nSH3 and PR Peptide—The atomic model of nebulin SH3 (Protein Data Bank file 1NEB) (48) was first put into a TIP3 water box allowing for at least5Åof water in all directions, and ions were added to neutralize the system. The structure was manipulated and visualized with VMD (49). This hydrated system was minimized for 10,000 steps at 0 K; the energy levels were reinitialized to 300 K, and the system was subjected to 1 ns of molecular dynamics using NAMD (50). The relaxed structure of nSH3 at 1 ns of simulation was used in subsequent docking simulations.
The atomic model of the 30-mer PR peptide of Ma and Wang (23) KVPEPPKEVVPEKKAPVAPPKKPEVPPVKV was obtained from the NMR structures of overlapping peptides that were derived from human exon 172 module by adding an extra Lys and Val at the amino and carboxyl termini, respectively, for a total length of 30. This NMR structural model was hydrated in a similar manner as with nSH3 and subjected to MD to fully relax the structure. The system was first minimized for 5000 steps, and the energy levels were then reinitialized to 300 K and then subjected to 30 ns of MD using NAMD. Fifty structures over the 30 ns of simulation were used for subsequent docking simulations. In the earlier work on PR peptide by Ma and Wang (23), the third residue (Pro) was identified as the start of the exon and the peptide sequence (i.e. residue 1). In this work, residue 1 is the first residue (Lys) to be consistent with the structural data file.
Molecular Dynamics and Docking of nSH3 to Peptides in Two Orientations—The HADDOCK protocol (51) consists of the following four stages: topology and structure generation; starting orientation randomization and (semi) rigid body energy minimization; semiflexible simulated annealing (SA); and flexible refinement in a water shell. For the PR peptide, the SH3-binding motif KKAPVAPPK (residues 13-21) was assumed to be the binding site for SH3. Nebulin SH3 residues involved in the docking of PPII peptides were those identified with HSQC by Ma and Wang (22) and tabulated in Fig. 4. The nSH3 construct used by Ma and Wang (22) lacked two amino-terminal residues in the 1NEB Protein Data Bank structure (48), and the indices have been changed accordingly. During the course of semi-rigid docking (1000 structures for each of the 50 starting structures), the side chains of residues 9-22 of PR and residues 11-18, 35-40, and 51-56 of nSH3 were allowed to move. Because the SH3-binding groove is rather shallow as compared with other protein/protein interactions, a large number (50,000) of semi-rigid docking structures were generated to increase the chance of obtaining an optimal configuration. The 1000 lowest energy structures were then used in the SA part of the protocol. The SA has four stages as follows: high temperature rigid body search; rigid body SA; semi-flexible SA with side chains at the interface; and semi-flexible SA with flexible side chains and backbone at the interface. To allow for motion of the portions of PR away from the interface to move and interact with the nSH3, residues 1-8 and 23-30 of PR were made flexible throughout the SA protocol. Allowing more of PR or parts of nSH3 to be flexible during the SA protocol leads to poor results with distortion of the SH3 fold or kinks in PR at the SH3-binding site. The 1000 structures were then hydrated in an 8-Å water shell and subjected to SA from 100 to 300 K and back to 100 K. The hydrated structures with the lowest interaction energies were compared with known structures of class I peptide bound to SH3 (1ABO) and class II peptide bound to SH3 (1SEM). For comparison of the structures, the conserved YPW triad in each SH3 was aligned, and the r.m.s.d. of the portion of the PR peptide in the binding groove was calculated. The structures with the lowest energy, the most similar conformation to the prototype structures, the greatest buried surface area, and a hydrogen bond to the conserved tryptophan in nSH3 were taken as the best fits.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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28 residues (Fig. 1A), (designated as PAKK by Greaser (54)), and group E exons include 14 glutamate-rich modules of more variable size (25-135 residues) (Fig. 1A), designated as poly(E) by Greaser (54). A search for the SH3-binding motifs in the PEVK modules revealed that there are 94 class I (Fig. 1A, green) or class II motifs (Fig. 1A, red). As indicated in the color-coded motifs within the aligned amino acid sequences of PEVK exons (designated as PEVK modules), there was no evidence of additional motifs when all PEVK exons are spliced together pairwise and translated to protein sequences. There are only five pure class I motifs scattered at different positions within the P group PEVK modules and none in the glutamate-rich group E modules. Twenty seven of 60 class II motifs are found in the first 7 residues of two blocks of PEVK exons from 121 to 162 and from 209 to 214. The remaining motifs are mostly found in the central region of the modules 118, 131, 171-203, and 217-218 and at the end of modules 122, 148b, and 224.
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Further analysis indicated that class I and II SH3-binding motifs are highly enriched in most PEVKs of titin orthologs, except C. elegans titin. Because some of the PEVK sequences are still incomplete, a density index (based on the copy number per 1000 residues) was used to normalize the values for comparison. There are 23-33 copies per 1000 residues in PEVK of human skeletal muscle titins, 11 and 20 in human cardiac N2B and N2BA PEVKs, and 12 and 5 in I-connectin and D-titin PEVK, respectively. In contrast, an average index of six is found in the group of 28 known SH3-binding proteins. Therefore, the density of these two types of SH3-binding motifs is two to six times higher in PEVK regions than other well characterized receptor proteins.
The distribution of class I and II motifs along the length of the available titin isoform/ortholog sequences in the corresponding sarcomere locations are plotted in Fig. 2B. Overall, there is a striking enrichment of these motifs in the PEVK segments of all titins, except C. elegans titin and I-connectin PEVK-2. It is interesting that the motifs in the I-band are nearly exclusively found in the PEVK region, with the remainder of the I-band and the Z-lines (titin amino terminus) almost free of such motifs. The only exception is the C. elegans titin, with motifs scattered along the I-band, with the PEVT region unexpectedly void of such motifs. This latter observation is paradoxical, because PEVT region is homologous to vertebrate PEVK (55). It is worth noting that even the shortest PEVK in N2B human heart isoform (186 residues) contains one class I motif in exon 223 and one class II motif in exon 224 with the N2BA isoform containing more than 19 motifs (Table 1).
Beyond the PEVK region, a large number of SH3 motifs are also found in the A-band titin exons where 54 motifs (Fig. 1, B and C) are evenly and more sparsely distributed (on average one per 300 residues) along the A-band region of the human titin (Fig. 2B). Significantly, most motifs (28 class II and 4 overlapping) are located in the amino terminus of the fibronectin type 3 (Fn3) domains, whereas 10 class I and II motifs (5 each) are located at the carboxyl terminus of the Fn3 domains (Fig. 1B). Interestingly, there are six and three motifs sitting in between Fn3-Fn3 and Ig-Ig domains, respectively (Fig. 1C and supplemental Table ST1). The distribution patterns are nearly identical between human skeletal and heart titin, because of the utilization of the same set of conserved exons. Because the Fn3 and Ig domains are -barrels of 100 residues each, these class I and II motifs in the A-band are located at the junctions of about one per three globular domains (Fig. 2).
In summary, the SH3-binding motifs are widespread in titin and titin-like proteins in nature. PEVK segments represent one of the richest sources of all types of SH3-binding motifs with the remaining portions of the I-band nearly void of such motifs. The A-band titin of vertebrates contain, in contrast, motifs dispersed throughout the A-band. The sarcomere distribution pattern for each ligand motif is unique for each species and each PEVK segment in titin orthologs.
Overlapping Ligand Motifs with Uni-, Bi-, and Mixed Orientations—Closer examination of the two classes of motifs in the PEVK modules revealed a novel class of overlapping motifs (Fig. 1A and Fig. 2A); 10 pairs of the 35 copies in fetal PEVK and 7 pairs of the 48 copies in human soleus PEVK total class I and II motifs are overlapping pairs that give rise to a 9-residue motif, KXXPXXPXK. Such a motif, being much shorter than the sum of two motifs (14 residues), may potentially allow SH3 to bind in either of the two orientations. Because class I and class II motifs bind to SH3 domains in the opposite orientation, these overlapping motifs can be considered as bipolar SH3-binding motifs. Significantly, overlapping motifs with different degrees of overlap and mixed polarity are also found in I-connectin PEVK-1 (9-residue) and in human soleus PEVK and cardiac PEVK (10-residue).
We designate these ligand motifs as class Ou,b,mn (for overlap), with the subscript n for the total length and the superscripts for the potential polarity relative to the SH3 domain (u for unipolar, b for bipolarity, and m for mixed polarity) (Fig. 2A). Most of the 24 class Ob9 in human titin are found in the middle of a cluster of exons 171-203 (Fig. 1A). Among the human titin isoforms, these novel overlapping motifs are present with the highest density in the human fetal PEVK sequence because of the selective utilization of exons 171-183 in fetal muscle. A search for a more generalized class O overlapping motifs with shorter overlap led to the detection of one 11-residue motif in exons 179, 188, and 197 (Ou11, KXXPKXPPXXP), two 12-residue motifs in exon 138 (Ou12, KXXPXKPXPXXP) and exon 218 (Ou12, KXXPXXPXXPXK), and one 13-residue motif (Ou13, KXXPXX- PPXXPXK) in exon 217. Of these motifs, exons 138-, 217-, and 218-based ones are expressed in human soleus titin PEVK.
Binding of nSH3 to Ligand Motifs in Human PEVK, A-band, and Titin Orthologs—To evaluate experimentally whether these titin SH3-binding motifs do indeed bind to SH3 domains, we first screened the interaction of nebulin SH3 domain with an array of these motif peptides covalently synthesized onto cellulose membrane by the SPOTs technology (46). SPOTs technology, originally designed for mapping epitopes, is being applied increasingly as a high throughput method for screening protein/peptide interactions (56). As shown in Fig. 3 and supplemental Table ST1, these titin peptide arrays include class I, class II, and non-PXXP motifs contained in PEVK exons (array P), titin isoforms (array M), and A-band (array A). Several SH3-binding peptides with known affinities are included as positive controls. The high density of the peptides on the membrane thus allows for the detection of even moderate binding affinity as frequently reported for such proline-rich peptides to SH3 domains (57). Incubation of these arrays with nebulin SH3 protein was performed under conditions that favor binding of PEVK modules to nSH3, including low temperature, moderate ionic strength (22), and the avoidance of prior exposure of nSH3 to low ionic strength buffer.6 In order to ease the stripping of the costly SPOTs membrane for repeated probing, we have chosen to electrophoretically dissociate and transfer the bound nSH3 to a glutaraldehyde-activated PVDF membrane, rather than the direct enzyme linked detection on the SPOTs membrane. The electrophoretic transfer was performed at pH 7.0, so the acidic nSH3 (pI 4.6) would migrate toward the PVDF membrane near the anode. Glutaraldehyde activation of PVDF was necessary, because nSH3 has very low affinity for PVDF alone. After transfer, the PVDF membrane was subject to peroxidase-based ECL detection using anti-nebulin SH3 antibody. The SPOTs membrane was then regenerated without having been subjected to oxidizing conditions of the peroxidase development (Fig. 3A). In our hands, this technique was very sensitive and could detect the signal of a peptide with a dissociation constant of 100 µM, as indicated by the spot intensity versus solution binding constants of the eight standard peptides toward SH3 domains (Fig. 3B and supplemental Table ST1). For this study, we classified the positive spots into two categories: strong (++) for intensity higher than 120 (corresponding roughly to a dissociation constant of 80 µM) and weak (+) for spot intensity between 10 and 120, and (-) for those below 10.
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Besides these strong binding motifs, the weak binding motifs contain class O overlapping motif (P7d), class I (Fig. 3B, spots P9d, P2f, and P6f), class II (Fig. 3B, spots P6b, P5c, P7c, P5e, P8e, P9e, P1f, and P5g), and unconventional motifs (Fig. 3B, spots P8f, KXXPXX-PXXPXK, P7g, VPKKREPVPVPV). Interestingly, peptides at P5g and P7g are based on exon 151 and exon 148 of the group E exons. It is noteworthy that in the I-band region, most class O overlapping motifs interact with nSH3 with higher affinity than other motifs. nSH3 also interacts with 45% of the predicted SH3 ligand motifs along the A-band (Fig. 3B, array A). Among these binding motifs, five show strong binding (four class II and one class I), and the remaining 16 are all weak binding motifs (12 class II, two class I, and two non-PXXP motifs). These nSH3-binding sites are evenly distributed, one site per 1250 residues along human titin A-band in the sarcomere (Fig. 2B).
In addition to human titin, nebulin SH3 also interacts with multiple SH3-binding motifs along the sequences of D-titin, I-connectin, and Ce titin. Twenty four of 30 predicted SH3-binding motifs in D-titin show positive binding to nSH3, and two-thirds of all confirmed nSH3-binding sites are located in its two PEVK regions, with the remainder located outside the PEVK region in the I-band (Fig. 3B, array E). I-connectin shows a similar SH3-binding site distribution pattern to that of human titin with class I and II nearly exclusively enriched in the PEVK-1 region (Fig. 2B). Although there is no class I and II motifs in I-connectin PEVK-2, six non-PXXP-binding motifs in PEVK-2 were found to interact with nSH3 (Fig. 3B, array E). In C. elegans titin, the 18 predicted SH3 ligand motifs are scattered along the sequence except in the PEVT region (Fig. 2B), with the identified nSH3-binding sites (Fig. 3B, array E) located either amino- or carboxyl-terminal to the C. elegans titin. It is noted that nearly all nSH3-binding sites in human titin PEVK were class II or overlapping motifs. In contrast, binding sites from other titin PEVKs involve both class I and class II motifs.
NMR Titration and Molecular Dynamics of PEVK Ligand and Docking an Ob9 Overlap Ligand to nSH3 in Opposite Orientations—To investigate the conformational basis of the strength, stereospecificity, and polarity of ligand/nSH3 interactions, we carried out NMR titration experiments on nSH3 with a wild type PEVK module (28-mer) containing an Ob9 overlapping motif and applied molecular dynamics simulations to model its binding to nSH3 in opposite orientations. The NMR titration was performed essentially as described previously (22). The titration of isotope-tagged nSH3 with increasing concentrations of PR resulted in the perturbation of chemical shifts of selected residues very similar to those titrated with a shorter ligand peptide (12-mer, VPEKKAPVAPPK) of the same module (22). To identify which of these chemical shifts arose from the binding of ligand in either class I or the class II orientation or both (as a mixture of class I and II orientations), molecular dynamics modeling and ligand docking simulation were then carried out. Because the flexibility and the ensemble conformations of SH3 and the ligands are likely to play significant roles in determining the specificity and affinity of interaction, docking simulations with molecular dynamics are more insightful because they can be performed using fully hydrated, relaxed structures. The available structures of both the PR peptide and nSH3, derived from NMR data, were first hydrated and then subjected to MD simulation to optimize the conformations before docking.
Portions of the 30-mer PR peptide underwent large fluctuations, but the center of the SH3-binding region (PVAP, residues 16-19) had the smallest r.m.s.d. of any four sequential residues during the 30-ns simulation. This core peptide varied less than 1 Å and was used as a reference state to align the ensemble conformations of the peptides. Overall, the fluctuations of the carboxyl-terminal portion of the wild type peptide were less than that of the amino-terminal portion as visualized by the superimposed conformations around the fixed reference core peptide (Fig. 4A).
Molecular dynamics simulation of the starting structure of nSH3 (reported by Politou et al. (48) as 1NEB in Protein Data Bank) at 300 K in water resulted in significant improvement over the starting structure that was optimized originally in vacuo (Fig. 4B). In their starting in vacuo structure, 64.7% of the non-glycine and non-proline residues were in the most favored region, and none were in the disallowed regions of the Ramachandran plot (48). In our final structure, 90.2% of the non-proline residues were in the most favored region, and none were in the disallowed regions. In particular, the 
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angles of the conserved Trp (Trp-38 in nebulin SH3, code 1NEB), an important residue in the tryptophan switch that governs the class I and class II binding configurations (43), changes considerably from 69.05, -166.80, for the first in vacuo structure to -132.44, 132.32, after 1 ns of MD. During the first 32 ps of the MD simulation at 300 K, the - angles of Trp-38, moved up into the -sheet region. Also, the C-C-C
angle of Trp-38 changes from 109.5° in the NMR structure to 117.3° following MD in this work.
The newly calculated structure of nSH3 was used to dock with the ensemble of peptide conformations to arrive at the complexes with the lowest interaction energy (van der Waals and electro-static) in both class I and class II binding orientations by an information-driven docking approach (HADDOCK program) (51). This program uses binding interactions, such as the residues identified via NMR titration, sequence homology, or other biophysical/biophysical means to limit the conformational searching problem inherent in docking simulations. For the PR peptide, the SH3-binding motif KKAPVAPPK (residues 13-21) was assumed to be the binding site for SH3 (22). Our strategy was to use only a subset of the nSH3 contact regions, as determined by HSQC NMR experiments (22), as docking inputs and then evaluate whether contact regions of the docked complexes in both orientations predict the entire set of experimental contacts of the 30-mer PR peptide (summarized in Fig. 4, nSH3 sequence annotation).
To exclude those structures in the docking simulations that were dominated by electrostatic interactions and mostly showed no stereo-specific binding to the hydrophobic cleft on SH3, the 1000 docked structures were first screened by examining those with an interaction energy function Evdw + 0.1 x Eelec less than -99 kcal/mol. The docked structures were screened further by comparison of the peptide orientation with the Protein Data Bank 1ABO structure for class I and with the 1SEM structure for class II binding. The 1ABO and 1SEM crystal structures were assumed to represent the canonical binding configurations for the two types of ligand binding. The structures with the lowest energy, the most similar conformation to the prototype structures, the greatest buried surface area, as well as a hydrogen bond to the conserved tryptophan in nSH3 were taken as the best fits. For class I structures, the configuration with the lowest r.m.s.d. from the peptide in the 1ABO structure (3.47 Å) was also within the Evdw + 0.1 x Eelec cutoff (-100.9 kcal/mol) with a buried surface area of 1974.3 Å2 (Fig. 4D). Three class II docked structures with an r.m.s.d. of less than 3.5 Å from the peptide in the 1SEM structure were identified within the Evdw + 0.1 x Eelec cutoff, -102.3, -100.9, and -99.9 kcal/mol, with interaction energies of -220.4, -260.3, and -286.6 kcal/mol, respectively. The structure with the lowest interaction energy also had the largest buried surface area at 2126.4 Å2 and is shown in Fig. 4. The tryptophan indole ring is hydrogen-bonded to the peptide Pro-16 and Val-17 carbonyls in the class I structure and to the peptide Ala-18 carbonyl in the class II structure.
Our modeling results indicate that the 30-mer PEVK module that contains the Ob9 motif can indeed bind in a stereospecific fashion in both directions with low and comparable net interaction energy. Moreover, the ligand makes substantially more contact with nSH3 beyond the canonical PPII-binding sequences in the binding groove. It is interesting that a comparison of the optimal docked peptide configurations for class I and II orientations with the starting ensemble configurations (Fig. 4C, where the configuration ensemble of free peptide is superimposed on the docked class I (purple) and class II (green) configurations) shows that the complexed configurations resemble closely some of the free ligand configurations. These pre-existing configurations may well be sampled preferentially by nSH3, resulting in a minimal loss of entropy upon binding and a higher binding affinity.
Orientation-specific Molecular Contacts of an Overlap Ligand—Several other features of the docked complexes of nSH3 and PR peptide in two orientations are worth noting. First, there is substantial difference in the conformation of SH3 with the ligand bound in two opposite orientations. Overall, the negative surface potentials are more compact in the class II complexes, especially the ones in both RT loop and nSrc loop and those lining up the edge of the binding cleft (in red, Fig. 4, D and E). Second, the surface negative potentials indicated by the asterisks in Fig. 4D (near residues D11, D16, D35, and E58) are involved in the charge interactions with the ligand lysine pairs that flank the sides of the PXXP core motif. Close examination of the ensembles of these complexes during MD indicated that in class II complexes, the 
-amino groups of both 21KK22 of the ligand flip-flop around these two negative charge centers between the RT and nSrc loops (Asp-16 and Asp-35). In addition, the 13KK14 from across the core PXXP flip-flop between the negative centers near residues Asp-11 and Asp-16 on the other side of the nSH3. Interestingly, class I ligands showed a similar charge interaction with the same pairs of negatively charge centers, but in the opposite orientation. These quadruple charge interactions undoubtedly reflect the symmetrical disposition of KK around the core motif and the presence of two symmetrically placed negative charge centers on both sides of the cleft of nSH3. Our initial mutagenesis studies of ligands confirmed the importance of these flanking double positive charges in binding affinity.6 Third, Tyr-10 at one end of the cleft appears to have flipped 35° between class I and class II complexes. The phenyl ring of Tyr-10 is rotated toward the protein core in class I binding and away from the protein core in class II binding. The phenyl rings in the free SH3 and in the class I binding are in the same plane, but in the class II binding the phenyl ring is rotated away from the PR peptide. These changes in the orientation of the Tyr-10 phenyl ring are due in part to hydrogen bonding of the phenyl OH to PR backbone carbonyls.
Overall, the PR peptide wraps around the nSH3 hemisphere in both orientations and forms contacts with nSH3 residues more broadly distributed than those in the hydrophobic cleft (Fig. 5, A and B). The validity of the modeled complexes is supported by the fact that the contact residues on nSH3 agree well with those determined by our NMR HSQC experiments, including several that were purposely ignored in the original inputs for the docking and molecular dynamic calculations. The modeling also revealed orientation-specific contacts: Thr-1, Val-31, and Gln-32 for class I complex and Arg-7, Thr-42, Gly-47, Arg-48, and Thr-49 for class II complex. Closer examination of the complexes and additional modeling indicated that these residues are orientation-specific. The fact that these orientation- or polarity-specific residues are indeed per-turbed by binding of an overlap ligand in NMR titration experiments strongly suggests that both complexes are present in an appreciable amount and in slow exchange on the NMR time scale. However, the observation that no separate signal was observed for free nSH3 indicates that the complexed nSH3 and nSH3 are in rapid exchange.
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-C-C angle in nSH3) to 113.8° for both class I and class II complexes (with the ligand in opposite orientations). The lack of a pronounced difference in tilt angles between the two orientations (by as much as 15° as reported by Fernandez-Ballester et al. (43)) raises the question as to whether the tryptophan switch is operative in the current ligand/SH3 pair. It is noted, however, that the Pro-53 is puckered differently as two rotamers (58), from DOWN (
2 = -28.1) in unbound nSH3 to DOWN (2 = -31.2) in class I binding and to UP (2 = 27.2) in class II binding. Thus, the prolines are puckered differently in these two ligand orientations. A difference in proline puckering was also observed between the ligand complexes of Abl SH3 (class I SH3, code 1ABO (59)) and of Sem SH3 (class II SH3, code 1SEM (60)). In the class I ligand, PR residue Pro-19 projects into the pocket formed by the YPW triad, and both Ala-17 and Pro-16 are in a position to hydrogen-bond with the Trp indole ring. In class II ligand, PR residue Val-17 projects into the binding pocket formed by the YPW triad, and the Ala-18 carbonyl forms a hydrogen bond with the SH3 Trp indole ring. The difference in the sizes of the side chains projecting into the pocket near Pro-53 may have caused the change in puckering of the Pro-53 to accommodate the side chains. As expected, Trp-38 is hydrogen-bonded to a carbonyl in both orientations, as dictated in the original selection criteria.
Binding of SH3 Proteins and Signaling Complexes in Muscle and Non-muscle Cell Extracts to Ligand Arrays—The experiments described above established the binding characteristics of the abundant nSH3-binding motifs in proline-rich sequences of titin and orthologs. Whether these motifs bind to SH3-containing proteins was then addressed by investigating the interaction between these SPOT sequences and total proteins in cell extracts of skeletal muscle (longissimus dorsi, soleus, and psoas), heart muscle, and nuclear and cytoplasmic extracts of HeLa cells. To maximize the scope of screening, two antibody mixtures consisting of either four anti-SH3 antibodies or a mixture of 13 antibodies to the SH3-containing signaling proteins (with antigens outside the SH3 domains) were used in Western blots (supplemental Table ST2). Major SH3-containing proteins in these cellular extracts were first identified by Western blotting with each of the antibodies against either SH3 domains or SH3-containing proteins. Additionally, to increase sensitivity, the SPOTS membranes were stained directly, without transfers, as was done with nSH3 screen.
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It is striking that the two sets based on nSH3 screening and cell extract mixture screening do not completely overlap as follows: 39 of 109 nSH3 positive spots were negative in the cell extracts screen and 15 cell extracts spots were negative for nSH3 screen (Fig. 3C and supplemental Table ST1), perhaps because of either distinct binding specificity of distinct SH3 domains and/or low SH3-containing protein concentrations in the extracts. Among the 39 extra signals detected in the nSH3 screen, five were strong binding (four class I and one class II SH3-binding motifs) (Fig. 3B, supplemental Table ST1), and the other 34 spots showed weak binding. Among the 15 extra signals detected in the extracts screen, one strong binding motif (PERKPEPKEEVV) derived from PEVK exon 219 was obtained. This difference again suggested that the cell extract mixture might contain different types of SH3 domains.
Immunolocalization of SH3-containing Proteins in Myofibrils—To demonstrate that these SH3-containing proteins do indeed bind to the muscle sarcomere, immunofluorescent localization on mechanically split single fibers from rabbit soleus muscle was performed with this set of antibodies, where they labeled to various degrees. Anti-titin RT11 (PEVK-specific (25)) labeled the AI junctions as a doublet per sarcomere (Fig. 6A), as expected from their known sarcomere location. The anti-Abl SH3, which detected nearly 20 bands on Western blots of rabbit soleus muscle, stained across the sarcomeres, intensely and in a granular form on the A-band and weakly at the Z-lines and occasionally on the I-band (Fig. 6C). Anti-nebulin SH3 (and anti--actinin) stained the Z-line (Fig. 6C). Anti-JIP SH3, anti-p130, and anti-cortactin all gave similar staining patterns as the anti-Abl SH3 (not shown). The Alexa-phalloidin labeled the actin in the Z-line and the I-band green (Fig. 6) and was used as an internal standard for all experiments. Even though the inherently low resolution of immunofluorescence did not allow us to resolve PEVK regions near the AI junctions from the A-bands, the staining patterns of rabbit soleus muscle sarcomere with anti-SH3 domains were strikingly similar to the distribution of SH3-binding motifs along the length of the human titin molecule (Fig. 2). We believe that the staining patterns reflect, at least in part, the presence of bound SH3-containing proteins on the titin in situ.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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