Generation of novel long-acting GLP-1R agonists using DARPins as a scaffold.

Glucagon-like peptide-1 (GLP-1) has been considered to be a promising peptide for treatment of type 2 diabetes mellitus (T2DM). However, the extremely short half-life (minutes) of native GLP-1 limits its clinical application potential. Here, we designed two GLP-1 analogues by genetic fusion of GLP-1 to one or two tandem human serum albumin-binding designed ankyrin repeat proteins (DARPins), denoted as GLP-DARPin or GLP-2DARPin. The two DARPin-fusion GLP-1 proteins were expressed in E. coli and purified, followed by measurements of their bioactivities and half-lives in mice. The results revealed that the half-life of GLP-2DARPin, binding two HSA molecules, was approximately 3-fold longer than GLP-DARPin (52.3 h versus 18.0 h). In contrast, the bioactivity results demonstrated that the blood glucose-lowering effect of GLP-DARPin was more potent than that of GLP-2DARPin. The oral glucose tolerance tests indicated that blood glucose levels were significantly reduced for at least 48 h by GLP-DARPin, but were reduced for only 24 h by GLP-2DARPin. Injected once every two days, GLP-DARPin substantially reduced blood glucose levels in streptozotocin (STZ)-induced diabetic mice to the same levels as normal mice. During the treatment course, GLP-DARPin significantly reduced the food intake and body weight of diabetic mice up to approximately 17% compared with the control group. A histological analysis revealed that GLP-DARPin alleviated islet loss in diabetic mice. These findings suggest that long-acting GLP-DARPin holds great potential for further development into drugs for the treatment of T2DM and obesity. Meanwhile, our data indicate that albumin-binding DARPins can be used as a universal scaffold to improve the pharmacokinetic profiles and pharmacological activities of therapeutic peptides and proteins.

Membrane Interactions of the Peroxisomal Proteins PEX5 and PEX14.

Human PEX5 and PEX14 are essential components of the peroxisomal translocon, which mediates import of cargo enzymes into peroxisomes. PEX5 is a soluble receptor for cargo enzymes comprised of an N-terminal intrinsically disordered domain (NTD) and a C-terminal tetratricopeptide (TPR) domain, which recognizes peroxisomal targeting signal 1 (PTS1) peptide motif in cargo proteins. The PEX5 NTD harbors multiple WF peptide motifs (WxxxF/Y or related motifs) that are recognized by a small globular domain in the NTD of the membrane-associated protein PEX14. How the PEX5 or PEX14 NTDs bind to the peroxisomal membrane and how the interaction between the two proteins is modulated at the membrane is unknown. Here, we characterize the membrane interactions of the PEX5 NTD and PEX14 NTD in vitro by membrane mimicking bicelles and nanodiscs using NMR spectroscopy and isothermal titration calorimetry. The PEX14 NTD weakly interacts with membrane mimicking bicelles with a surface that partially overlaps with the WxxxF/Y binding site. The PEX5 NTD harbors multiple interaction sites with the membrane that involve a number of amphipathic α-helical regions, which include some of the WxxxF/Y-motifs. The partially formed α-helical conformation of these regions is stabilized in the presence of bicelles. Notably, ITC data show that the interaction between the PEX5 and PEX14 NTDs is largely unaffected by the presence of the membrane. The PEX5/PEX14 interaction exhibits similar free binding enthalpies, where reduced binding enthalpy in the presence of bicelles is compensated by a reduced entropy loss. This demonstrates that docking of PEX5 to PEX14 at the membrane does not reduce the overall binding affinity between the two proteins, providing insights into the initial phase of PEX5-PEX14 docking in the assembly of the peroxisome translocon.

Albumin-binding domain extends half-life of glucagon-like peptide-1.

Glucagon-like peptide-1 (GLP-1) is considered to be a promising peptide for the treatment of type 2 diabetes mellitus (T2DM). However, the extremely short half-life of GLP-1 limits its clinical application. Albumin-binding domain (ABD) with high affinity for human serum albumin (HSA) has been used widely for half-life extension of therapeutic peptides and proteins. In the present study, novel GLP-1 receptor agonists were designed by genetic fusion of GLP-1 to three kinds of ABDs with different affinities for HSA: GA3, ABD035 and ABDCon. The bioactivities and half-lives of ABD-fusion GLP-1 proteins with different types and lengths of linkers were investigated in vitro and in vivo. The results demonstrated that ABD-fusion GLP-1 proteins could bind to HSA with high affinity. The blood glucose-lowering effect of GLP-1 was significantly improved and sustained by fusion to ABD. Meanwhile, the fusion proteins significantly inhibited food intake, which was beneficial for T2DM and obesity treatment. The half-life of GLP-1 was substantially extended by virtue of ABD. The in vivo results also showed that a longer linker inserted between GLP-1 and ABD resulted in a higher blood glucose-lowering effect. The fusion proteins generated by fusion of GLP-1 to GA3, ABD035 and ABDCon exhibited similar bioactivities and pharmacokinetics in vivo. These findings demonstrate that ABD-fusion GLP-1 proteins retain the bioactivities of natural GLP-1 and can be further developed for T2DM treatment and weight loss. It also indicates that the ABD-fusion strategy can be generally applicable to any peptide or protein, to improve pharmacodynamic and pharmacokinetic properties.

Ferritin-Displayed GLP-1 with Improved Pharmacological Activities and Pharmacokinetics.

Glucagon-like peptide-1 (GLP-1) is an incretin (a type of metabolic hormone that stimulates a decrease in blood glucose levels), holding great potential for the treatment of type 2 diabetes mellitus (T2DM). However, its extremely short half-life of 1-2 min hampers any direct clinical application. Here, we describe the application of the heavy chain of human ferritin (HFt) nanocage as a carrier to improve the pharmacological properties of GLP-1. The GLP-HFt was designed by genetic fusion of GLP-1 to the N-terminus of HFt and was expressed in inclusion bodies in E. coli. The refolding process was developed to obtain a soluble GLP-HFt protein. The biophysical properties determined by size-exclusion chromatography (SEC), dynamic light scattering (DLS), circular dichroism (CD), transmission electron microscopy (TEM), and X-ray crystallography verified that the GLP-HFt successfully formed a 24-mer nanocage with GLP-1 displayed on the external surface of HFt. The in vivo pharmacodynamic results demonstrated that the GLP-HFt nanocage retained the bioactivity of natural GLP-1, significantly reduced the blood glucose levels for at least 24 h in a dose-dependent manner, and inhibited food intake for at least 8-10 h. The half-life of the GLP-HFt nanocage was approximately 52 h in mice after subcutaneous injection. The prolonged half-life and sustained control of blood glucose levels indicate that the GLP-HFt nanocage can be further developed for the treatment of T2DM. Meanwhile, the HFt nanocage proves its great potential as a universal carrier that improves the pharmacodynamic and pharmacokinetic properties of a wide range of therapeutic peptides and proteins.

Recent Advances in Half-life Extension Strategies for Therapeutic Peptides and Proteins.

Peptides and proteins are two classes of molecules with attractive possibilities for therapeutic applications. However, the bottleneck for the therapeutic application of many peptides and proteins is their short halflives in vivo, typically just a few minutes to hours. Half-life extension strategies have been extensively studied and many of them have been proven to be effective in the generation of long-acting therapeutics with improved pharmacokinetic and pharmacodynamic properties. In this review, we summarize the recent advances in half-life extension strategies, illustrate their potential applications and give some examples, highlighting the strategies that have been used in approved drugs and for drugs in clinical trials. Meanwhile, several novel strategies that are still in the process of discovery or at a preclinical stage are also introduced. In these strategies, the two most frequently used half-life extension methods are the reduction in the rate of renal clearance or the exploitation of the recycling mechanism of FcRn by binding to the albumin or IgG-Fc. Here, we discuss half-life extension strategies of recombinant therapeutic protein via genetic fusion, rather than chemical conjugation such as PEGylation. With the rapid development of genetic engineering and protein engineering, novel strategies for half-life extension have been emerged consistently. Some of these will be evaluated in clinical trials and may become viable alternatives to current strategies for making next-generation biodrugs.

Expression and purification of a difficult sarcomeric protein: Telethonin.

Telethonin anchors the N-terminal region of titin in the Z-disk of the sarcomere by binding to two immunoglobulin-like (Ig) domains (Z1 and Z2) of titin (Z1Z2). Thereby telethonin plays an important role in myofibril assembly and in muscle development and functional regulation. The expression and purification of recombinant telethonin is very challenging. In previous studies, recombinant telethonin expressed from E. coli was refolded in the presence of Z1Z2. Here, we report various strategies to establish a reliable and efficient protocol for the preparation of telethonin and titin Z1Z2 protein. First, a co-expression strategy was designed to obtain soluble Z1Z2/telethonin complexes. The concentration of antibiotics and the type of expression vector were found to be important for achieving high yields of purified complex. Second, the five cysteine residues of telethonin were mutated to serine to avoid severe problems with cysteine oxidation. Third, a short version of telethonin (telethonin1-90) was designed to avoid the proteolytic degradation observed for longer constructs of the protein. The short telethonin formed a highly stable complex with Z1Z2 with no degradation being observed for 30 days at 4 °C. Fourth, an improved refolding protocol was developed to achieve high yields of Z1Z2/telethonin complex. Finally, based on the crystal structure in which Z1Z2 and telethonin1-90 assemble into a 2:1 complex, a single chain fusion protein was designed, comprising two Z1Z2 modules that are connected by flexible linkers N- and C-terminally of the telethonin1-90. Expression of this fusion protein, named ZTZ, affords high yields of soluble expressed and purified protein.

[Analysis of immobilized L-glutamate oxidase fused with cellulose binding domain on microcrystalline cellulose].

Immobilization of enzymes is important and widely applied in biocatalysis. Streptomyces platensis gene gox, encoding an extracellular L-glutamate oxidase (Gox), was fused to cellulose binding domain (CBDcex) from Cellulomonas fimi and the recombinant protein Gox-CBD was expressed in Escherichia coli. The fusion protein (Gox-CBD) was immobilized onto microcrystalline cellulose. The preparation conditions, binding capacity, properties and stability of the immobilized enzyme were studied. Under the condition of 4 ℃, for 1 hour, the fusion protein Gox-CBD was able to bind microcrystalline cellulose at a ratio of 9.0 mg of protein per gram of microcrystalline cellulose. Enzymatic properties of free and immobilized L-glutamic oxidase (Gox-CBD) were compared. The specific activity of the immobilized enzyme decreased, but its thermal stability increased a lot compared with that of the free Gox-CBD. After incubation at 60 ℃ for 30 min, 70% of the total activity remained whereas the free recombinant Gox completely lost its activity. The immobilized protein was tightly bound to microcrystalline cellulose at pH below 10 or more than 5 mmol/L NaCl. The fusion protein of Gox-CBD can be specifically immobilized on the microcrystalline cellulose on a single step. Therefore, our findings can provide a novel strategy for protein purification and enzyme immobilization.

Efficient segmental isotope labeling of multi-domain proteins using Sortase A.

NMR studies of multi-domain protein complexes provide unique insight into their molecular interactions and dynamics in solution. For large proteins domain-selective isotope labeling is desired to reduce signal overlap, but available methods require extensive optimization and often give poor ligation yields. We present an optimized strategy for segmental labeling of multi-domain proteins using the S. aureus transpeptidase Sortase A. Critical improvements compared to existing protocols are (1) the efficient removal of cleaved peptide fragments by centrifugal filtration and (2) a strategic design of cleavable and non-cleavable affinity tags for purification. Our approach enables routine production of milligram amounts of purified segmentally labeled protein for NMR and other biophysical studies.

The EBNA-2 N-Terminal Transactivation Domain Folds into a Dimeric Structure Required for Target Gene Activation.

Epstein-Barr virus (EBV) is a γ-herpesvirus that may cause infectious mononucleosis in young adults. In addition, epidemiological and molecular evidence links EBV to the pathogenesis of lymphoid and epithelial malignancies. EBV has the unique ability to transform resting B cells into permanently proliferating, latently infected lymphoblastoid cell lines. Epstein-Barr virus nuclear antigen 2 (EBNA-2) is a key regulator of viral and cellular gene expression for this transformation process. The N-terminal region of EBNA-2 comprising residues 1-58 appears to mediate multiple molecular functions including self-association and transactivation. However, it remains to be determined if the N-terminus of EBNA-2 directly provides these functions or if these activities merely depend on the dimerization involving the N-terminal domain. To address this issue, we determined the three-dimensional structure of the EBNA-2 N-terminal dimerization (END) domain by heteronuclear NMR-spectroscopy. The END domain monomer comprises a small fold of four ß-strands and an α-helix which form a parallel dimer by interaction of two ß-strands from each protomer. A structure-guided mutational analysis showed that hydrophobic residues in the dimer interface are required for self-association in vitro. Importantly, these interface mutants also displayed severely impaired self-association and transactivation in vivo. Moreover, mutations of solvent-exposed residues or deletion of the α-helix do not impair dimerization but strongly affect the functional activity, suggesting that the EBNA-2 dimer presents a surface that mediates functionally important intra- and/or intermolecular interactions. Our study shows that the END domain is a novel dimerization fold that is essential for functional activity. Since this specific fold is a unique feature of EBNA-2 it might provide a novel target for anti-viral therapeutics.

[A novel reporter system monitoring sortase A catalyzed protein ligation efficiency].

Efforts on directed evolution of sortase A to optimize its catalytic properties have been undertaken and shown the promise. To facilitate screening of sortase A mutants with expected catalytic properties, a novel ligation efficiency monitoring system, including reporter substrates GFP-LPETG and GGGYK-Biotin, was developed. GFP-LPETG, wild type sortase A, and a recently reported high activity sortase A mutant were prepared recombinantly from Escherichia coli BL21 (DE3). Taking advantage of the newly designed reporter system, the ligation efficiency catalyzed by wild type and mutant form of sortase A could be sensitively monitored via SDS-PAGE directly. Consistent with previous report, the mutant sortase A displayed much higher catalytic activity compared to wild type enzyme, indicating the new reporter system is easily and fast handled and sensitive. The application of this reporter system into systemic screening will facilitate future directed optimization of sortase A.

Structural and functional analysis of the DEAF-1 and BS69 MYND domains.

DEAF-1 is an important transcriptional regulator that is required for embryonic development and is linked to clinical depression and suicidal behavior in humans. It comprises various structural domains, including a SAND domain that mediates DNA binding and a MYND domain, a cysteine-rich module organized in a Cys(4)-Cys(2)-His-Cys (C4-C2HC) tandem zinc binding motif. DEAF-1 transcription regulation activity is mediated through interactions with cofactors such as NCoR and SMRT. Despite the important biological role of the DEAF-1 protein, little is known regarding the structure and binding properties of its MYND domain.Here, we report the solution structure, dynamics and ligand binding of the human DEAF-1 MYND domain encompassing residues 501-544 determined by NMR spectroscopy. The structure adopts a ßßα fold that exhibits tandem zinc-binding sites with a cross-brace topology, similar to the MYND domains in AML1/ETO and other proteins. We show that the DEAF-1 MYND domain binds to peptides derived from SMRT and NCoR corepressors. The binding surface mapped by NMR titrations is similar to the one previously reported for AML1/ETO. The ligand binding and molecular functions of the related BS69 MYND domain were studied based on a homology model and mutational analysis. Interestingly, the interaction between BS69 and its binding partners (viral and cellular proteins) seems to require distinct charged residues flanking the predicted MYND domain fold, suggesting a different binding mode. Our findings demonstrate that the MYND domain is a conserved zinc binding fold that plays important roles in transcriptional regulation by mediating distinct molecular interactions with viral and cellular proteins.

Structure, phosphorylation and U2AF65 binding of the N-terminal domain of splicing factor 1 during 3'-splice site recognition.

Recognition of the 3'-splice site is a key step in pre-mRNA splicing and accomplished by a dynamic complex comprising splicing factor 1 (SF1) and the U2 snRNP auxiliary factor 65-kDa subunit (U2AF65). Both proteins mediate protein-protein and protein-RNA interactions for cooperative RNA-binding during spliceosome assembly. Here, we report the solution structure of a novel helix-hairpin domain in the N-terminal region of SF1 (SF1(NTD)). The nuclear magnetic resonance- and small-angle X-ray scattering-derived structure of a complex of the SF1(NTD) with the C-terminal U2AF homology motif domain of U2AF65 (U2AF65(UHM)) reveals that, in addition to the known U2AF65(UHM)-SF1 interaction, the helix-hairpin domain forms a secondary, hydrophobic interface with U2AF65(UHM), which locks the orientation of the two subunits. Mutational analysis shows that the helix hairpin is essential for cooperative formation of the ternary SF1-U2AF65-RNA complex. We further show that tandem serine phosphorylation of a conserved Ser80-Pro81-Ser82-Pro83 motif rigidifies a long unstructured linker in the SF1 helix hairpin. Phosphorylation does not significantly alter the overall conformations of SF1, SF1-U2AF65 or the SF1-U2AF65-RNA complexes, but slightly enhances RNA binding. Our results indicate that the helix-hairpin domain of SF1 is required for cooperative 3'-splice site recognition presumably by stabilizing a unique quaternary arrangement of the SF1-U2AF65-RNA complex.

Rational design to improve thermostability and specific activity of the truncated Fibrobacter succinogenes 1,3-1,4-ß-D-glucanase.

1,3-1,4-ß-D-Glucanase has been widely used as a feed additive to help non-ruminant animals digest plant fibers, with potential in increasing nutrition turnover rate and reducing sanitary problems. Engineering of enzymes for better thermostability is of great importance because it not only can broaden their industrial applications, but also facilitate exploring the mechanism of enzyme stability from structural point of view. To obtain enzyme with higher thermostability and specific activity, structure-based rational design was carried out in this study. Eleven mutants of Fibrobacter succinogenes 1,3-1,4-ß-D-glucanase were constructed in attempt to improve the enzyme properties. In particular, the crude proteins expressed in Pichia pastoris were examined firstly to ensure that the protein productions meet the need for industrial fermentation. The crude protein of V18Y mutant showed a 2 °C increment of Tm and W203Y showed ∼30% increment of the specific activity. To further investigate the structure-function relationship, some mutants were expressed and purified from P. pastoris and Escherichia coli. Notably, the specific activity of purified W203Y which was expressed in E. coli was 63% higher than the wild-type protein. The double mutant V18Y/W203Y showed the same increments of Tm and specific activity as the single mutants did. When expressed and purified from E. coli, V18Y/W203Y showed similar pattern of thermostability increment and 75% higher specific activity. Furthermore, the apo-form and substrate complex structures of V18Y/W203Y were solved by X-ray crystallography. Analyzing protein structure of V18Y/W203Y helps elucidate how the mutations could enhance the protein stability and enzyme activity.

Telethonin deficiency is associated with maladaptation to biomechanical stress in the mammalian heart.

RATIONALE: Telethonin (also known as titin-cap or t-cap) is a 19-kDa Z-disk protein with a unique ß-sheet structure, hypothesized to assemble in a palindromic way with the N-terminal portion of titin and to constitute a signalosome participating in the process of cardiomechanosensing. In addition, a variety of telethonin mutations are associated with the development of several different diseases; however, little is known about the underlying molecular mechanisms and telethonin's in vivo function. OBJECTIVE: Here we aim to investigate the role of telethonin in vivo and to identify molecular mechanisms underlying disease as a result of its mutation. METHODS AND RESULTS: By using a variety of different genetically altered animal models and biophysical experiments we show that contrary to previous views, telethonin is not an indispensable component of the titin-anchoring system, nor is deletion of the gene or cardiac specific overexpression associated with a spontaneous cardiac phenotype. Rather, additional titin-anchorage sites, such as actin-titin cross-links via α-actinin, are sufficient to maintain Z-disk stability despite the loss of telethonin. We demonstrate that a main novel function of telethonin is to modulate the turnover of the proapoptotic tumor suppressor p53 after biomechanical stress in the nuclear compartment, thus linking telethonin, a protein well known to be present at the Z-disk, directly to apoptosis ("mechanoptosis"). In addition, loss of telethonin mRNA and nuclear accumulation of this protein is associated with human heart failure, an effect that may contribute to enhanced rates of apoptosis found in these hearts. CONCLUSIONS: Telethonin knockout mice do not reveal defective heart development or heart function under basal conditions, but develop heart failure following biomechanical stress, owing at least in part to apoptosis of cardiomyocytes, an effect that may also play a role in human heart failure.

The oligomeric assembly of the novel haem-degrading protein HbpS is essential for interaction with its cognate two-component sensor kinase.

HbpS, a novel protein of previously unknown function from Streptomyces reticuli, is up-regulated in response to haemin- and peroxide-based oxidative stress and interacts with the SenS/SenR two-component signal transduction system. In this study, we report the high-resolution crystal structures (2.2 and 1.6 A) of octomeric HbpS crystallized in the presence and in the absence of haem and demonstrate that iron binds to surface-exposed lysine residues of an octomeric assembly. Based on an analysis of the crystal structures, we propose that the iron atom originates from the haem group and report subsequent biochemical experiments that demonstrate that HbpS possesses haem-degrading activity in vitro. Further examination of the crystal structures has identified amino acids that are essential for assembly of the octomer. The role of these residues is confirmed by biophysical experiments. Additionally, we show that while the octomeric assembly state of HbpS is not essential for haem-degrading activity, the assembly of HbpS is required for its interaction with the cognate sensor kinase, SenS. Homologs of HbpS and SenS/SenR have been identified in a number of medically and ecologically relevant bacterial species (including Vibrio cholerae, Klebsiella pneumoniae, Corynebacterium diphtheriae, Arthrobacter aurescens and Pseudomonas putida), suggesting the existence of a previously undescribed bacterial oxidative stress-response pathway common to Gram-negative and Gram-positive bacteria. Thus, the data presented provide the first insight into the function of a novel protein family and an example of an iron-mediated interaction between an accessory protein and its cognate two-component sensor kinase.

Crystallization and preliminary characterization of a novel haem-binding protein of Streptomyces reticuli.

Streptomyces reticuli is a soil-growing Gram-positive bacteria that has been shown to secrete a novel haem-binding protein known as HbpS. Sequence analysis reveals that homologues of HbpS are found in a wide variety of bacteria, including different Actinobacteria and the Gram-negative Vibrio cholera and Klebsiella pneumoniae. The in vivo production of HbpS is greatly increased when S. reticuli is cultured in the presence of the natural antibiotic haemin (Fe3+ oxidized form of haem). Mutational analysis demonstrated that HbpS significantly increases the resistance of S. reticuli to toxic concentrations of haemin. Previous data show that the presence of the newly identified two-component sensor system SenS-SenR also considerably enhances the resistance of S. reticuli to haemin and the redox-cycling compound plumbagin, suggesting a role in the sensing of redox changes. Specific interaction between HbpS and SenS-SenR, which regulates the expression of the catalase-peroxidase CpeB, as well as HbpS, has been demonstrated in vitro. HbpS has been recombinantly overexpressed, purified and crystallized in space group P2(1)3, with a cell edge of 152.5 A. Diffraction data were recorded to a maximal resolution of 2.25 A and phases were obtained using the SAD method from crystals briefly soaked in high concentrations of sodium bromide.

The Ig doublet Z1Z2: a model system for the hybrid analysis of conformational dynamics in Ig tandems from titin.

Titin is a gigantic elastic filament that determines sarcomere ultrastructure and stretch response in vertebrate muscle. It folds into numerous Ig and FnIII domains connected in tandem. Data on interdomain arrangements and dynamics are essential for understanding the function of this filament. Here, we report a mechanistic analysis of the conformational dynamics of two Ig domains from the N terminus of titin, Z1Z2, by using X-ray crystallography, SAXS, NMR relaxation data, and residual dipolar couplings in combination. Z1Z2 preferentially adopts semiextended conformations in solution, with close-hinge arrangements representing low-probability states. Although interdomain contacts are not observed, the linker appears to acquire moderate rigidity via small, local hydrophobic interactions. Thus, Z1Z2 constitutes an adaptable modular system with restricted dynamics. We speculate that its preexistent conformation contributes to the selective recruitment of the binding partner telethonin onto the repetitive surface of the filament. The structural interconversion of four Z1Z2 conformers is analyzed.

Evidence for a dimeric assembly of two titin/telethonin complexes induced by the telethonin C-terminus.

The Z-disk region defines the lateral boundary of the sarcomere and requires a high level of mechanical strength to provide a stable framework for large filamentous muscle proteins. The level of complexity at this area is reflected by a large number of protein-protein interactions. Recently, we unraveled how the N-terminus of the longest filament component, the giant muscle protein titin, is assembled into an antiparallel (2:1) sandwich complex by the N-terminal titin-binding segment of the Z-disk ligand telethonin/T-cap [Zou, P., Pinotsis, N., Lange, S., Song, Y.H., Popov, A., Mavridis, I., Mayans, O.M., Gautel, M., Wilmanns, M., 2006. Palindromic assembly of the giant muscle protein titin in the sarcomeric Z-disk. Nature 439, 229-233]. In this contribution, we present structural data of a related complex of the titin N-terminus with full-length telethonin. The C-terminus of telethonin remains invisible, suggesting that it does not fold into a defined structure even in the presence of titin. In contrast to the structure with truncated telethonin, a dimer of two titin/telethonin complexes is formed within the crystal environment, potentially indicating the formation of higher oligomers. We further investigated the structure and dynamics of this assembly by small-angle X-ray scattering, circular dichroism, and in vivo complementation data. The data consistently indicate the involvement of the C-terminal part of telethonin into the assembly of two titin/telethonin complexes.

Palindromic assembly of the giant muscle protein titin in the sarcomeric Z-disk.

The Z-disk of striated and cardiac muscle sarcomeres is one of the most densely packed cellular structures in eukaryotic cells. It provides the architectural framework for assembling and anchoring the largest known muscle filament systems by an extensive network of protein-protein interactions, requiring an extraordinary level of mechanical stability. Here we show, using X-ray crystallography, how the amino terminus of the longest filament component, the giant muscle protein titin, is assembled into an antiparallel (2:1) sandwich complex by the Z-disk ligand telethonin. The pseudosymmetric structure of telethonin mediates a unique palindromic arrangement of two titin filaments, a type of molecular assembly previously found only in protein-DNA complexes. We have confirmed its unique architecture in vivo by protein complementation assays, and in vitro by experiments using fluorescence resonance energy transfer. The model proposed may provide a molecular paradigm of how major sarcomeric filaments are crosslinked, anchored and aligned within complex cytoskeletal networks.

Crystal structure of the PB1 domain of NBR1.

The scaffold protein NBR1 is involved in signal transmission downstream of the serine/protein kinase from the giant muscle protein titin. Its N-terminal Phox and Bem1p (PB1) domain plays a critical role in mediating protein-protein interactions with both titin kinase and with another scaffold protein, p62. We have determined the crystal structure of the PB1 domain of NBR1 at 1.55A resolution. It reveals a type-A PB1 domain with two negatively charged residue clusters. We provide a structural perspective on the involvement of NBR1 in the titin kinase signalling pathway.