Structural and functional studies of CARP and titin N2A protein complexes involved in the stress-response of muscle to apoptotic stimuli

The cardiac ankyrin repeat protein (CARP) has been reported to bind to the N2A region of the giant sarcomeric protein titin. The interaction allegedly plays a role in preventing cardiomyocyte apoptosis, thereby protecting against heart failure (HF). The latter is now a leading cause of mortality in the developed world. In this work, we focus on CARP to guide future functional studies that can clarify its significance in heart disease

Molecular Characterisation of Titin N2A and Its Binding of CARP Reveals a Titin/Actin Cross-linking Mechanism

Striated muscle responds to mechanical overload by rapidly up-regulating the expression of the cardiac ankyrin repeat protein, CARP, which then targets the sarcomere by binding to titin N2A in the I-band region. To date, the role of this interaction in the stress response of muscle remains poorly understood. Here, we characterise the molecular structure of the CARP-receptor site in titin (UN2A) and its binding of CARP. We find that titin UN2A contains a central three-helix bundle fold (ca 45 residues in length) that is joined to N- and C-terminal flanking immunoglobulin domains by long, flexible linkers with partial helical content. CARP binds titin by engaging an α-hairpin in the three-helix fold of UN2A, the C-terminal linker sequence, and the BC loop in Ig81, which jointly form a broad binding interface. Mutagenesis showed that the CARP/N2A association withstands sequence variations in titin N2A and we use this information to evaluate 85 human single nucleotide variants. In addition, actin co-sedimentation, co-transfection in C2C12 cells, proteomics on heart lysates, and the mechanical response of CARP-soaked myofibrils imply that CARP induces the cross-linking of titin and actin myofilaments, thereby increasing myofibril stiffness. We conclude that CARP acts as a regulator of force output in the sarcomere that preserves muscle mechanical performance upon overload stress

Scalable, Non-denaturing Purification of Phosphoproteins Using Ga³⁺-IMAC: N2A and M1M2 Titin Components as Study case

The purification of phosphorylated proteins in a folded state and in large enough quantity for biochemical or biophysical analysis remains a challenging task. Here, we develop a new implementation of the method of gallium immobilized metal chromatography (Ga3+-IMAC) as to permit the selective enrichment of phosphoproteins in the milligram scale and under native conditions using automated FPLC instrumentation. We apply this method to the purification of the UN2A and M1M2 components of the muscle protein titin upon being monophosphorylated in vitro by cAMP-dependent protein kinase (PKA). We found that UN2A is phosphorylated by PKA at its C-terminus in residue S9578 and M1M2 is phosphorylated in its interdomain linker sequence at position T32607. We demonstrate that the Ga3+-IMAC method is efficient, economical and suitable for implementation in automated purification pipelines for recombinant proteins. The procedure can be applied both to the selective enrichment and to the removal of phosphoproteins from biochemical samples

Structural and functional studies of CARP/titin-N2A interaction

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What does fluorine do to a protein? : Thermodynamic, and highly-resolved structural insights into fluorine-labelled variants of the cold shock protein

Fluorine labelling represents one promising approach to study proteins in their native environment due to efficient suppressing of background signals. Here, we systematically probe inherent thermodynamic and structural characteristics of the Cold shock protein B from Bacillus subtilis (BsCspB) upon fluorine labelling. A sophisticated combination of fluorescence and NMR experiments has been applied to elucidate potential perturbations due to insertion of fluorine into the protein. We show that single fluorine labelling of phenylalanine or tryptophan residues has neither significant impact on thermodynamic stability nor on folding kinetics compared to wild type BsCspB. Structure determination of fluorinated phenylalanine and tryptophan labelled BsCspB using X-ray crystallography reveals no displacements even for the orientation of fluorinated aromatic side chains in comparison to wild type BsCspB. Hence we propose that single fluorinated phenylalanine and tryptophan residues used for protein labelling may serve as ideal probes to reliably characterize inherent features of proteins that are present in a highly biological context like the cell.publishe

Single-Molecule Force Spectroscopy on the N2A Element of Titin: Effects of Phosphorylation and CARP

Titin is a large filamentous protein that forms a sarcomeric myofilament with a molecular spring region that develops force in stretched sarcomeres. The molecular spring has a complex make-up that includes the N2A element. This element largely consists of a 104- residue unique sequence (N2A-Us) flanked by immunoglobulin domains (I80 and I81). The N2A element is of interest because it assembles a signalosome with CARP (Cardiac Ankyrin Repeat Protein) as an important component; CARP both interacts with the N2AUs and I81 and is highly upregulated in response to mechanical stress. The mechanical properties of the N2A element were studied using single-molecule force spectroscopy, including how these properties are affected by CARP and phosphorylation. Three protein constructs were made that consisted of 0, 1, or 2 N2A-Us elements with flanking I80 and I81 domains and with specific handles at their ends for study by atomic force microscopy (AFM). The N2A-Us behaved as an entropic spring with a persistence length (Lp) of 0.35 nm and contour length (Lc) of 39 nm. CARP increased the Lp of the N2A-Us and the unfolding force of the Ig domains; force clamp experiments showed that CARP reduced the Ig domain unfolding kinetics. These findings suggest that CARP might function as a molecular chaperone that protects I81 from unfolding when mechanical stress is high. The N2A-Us was found to be a PKA substrate, and phosphorylation was blocked by CARP. Mass spectrometry revealed a PKA phosphosite (Ser-9895 in NP_001254479.2) located at the border between the N2A-Us and I81. AFM studies showed that phosphorylation affected neither the Lp of the N2A-Us nor the Ig domain unfolding force (Funfold). Simulating the force-sarcomere length relation of a single titin molecule containing all spring elements showed that the compliance of the N2A-Us only slightly reduces passive force (1.4%) with an additional small reduction by CARP (0.3%). Thus, it is improbable that the compliance of the N2A element has a mechanical function per se. Instead, it is likely that this compliance has local effects on binding of signaling molecules and that it contributes thereby to strain- and phosphorylation- dependent mechano-signaling

CARP interacts with titin at a unique helical N2A sequence and at the domain Ig81 to form a structured complex

The cardiac ankyrin repeat protein (CARP) is up-regulated in the myocardium during cardiovascular disease and in response to mechanical or toxic stress. Stress-induced CARP interacts with the N2A spring region of the titin filament to modulate muscle compliance. We characterize the interaction between CARP and titin-N2A and show that the binding site in titin spans the dual domain UN2A-Ig81. We find that the unique sequence UN2A is not structurally disordered, but that it has a stable, elongated α-helical fold that possibly acts as a constant force spring. Our findings portray CARP/titin-N2A as a structured node and help to rationalize the molecular basis of CARP mechanosensing in the sarcomeric I-band.publishe

Segmental Motion Adjustment of the Polycarbonate Electrolyte for Lithium-Metal Batteries

Carbonyl oxygen atoms are the primary active sites to solvate Li salts that provide a migration site for Li ions conducting in a polycarbonate-based polymer electrolyte. We here exploit the conductivity of the polycarbonate electrolyte by tuning the segmental motion of the structural unit with carbonyl oxygen atoms, while its correlation to the mechanical and electrochemical stability of the electrolyte is also discussed. Two linear alkenyl carbonate monomers are designed by molecular engineering to combine methyl acrylate (MA) and the commonly used ethylene carbonate (EC), w/o dimethyl carbonate (DMC) in the structure. The integration of the DMC structural unit in the side chain of the in situ constructed polymer (p-MDE) releases the free motion of the terminal EC units, which leads to a lower glass-transition temperature and higher ionic conductivity. While pure polycarbonates are normally fragile with high Young’s modulus, such a prolonged side chain also manipulates the flexibility of the polymer to provide a mechanical stable interface for Li-metal anode. Stable long-term cycling performance is achieved at room temperature for both LiFePO4 and LiCoO2 electrodes based on the p-MDE electrolyte incorporated with a solid plasticizer

Single‐Atom Cu Stabilized on Ultrathin WO2.72 Nanowire for Highly Selective and Ultrasensitive ppb‐Level Toluene Detection

Abstract Various catalysts are developed to improve the performance of metal oxide semiconductor gas sensors, but achieving high selectivity and response intensity in chemiresistive gas sensors (CGSs) remains a significant challenge. In this study, an in situ‐annealing approach to synthesize Cu catalytic sites on ultrathin WO2.72 nanowires for detecting toluene at ultralow concentrations (Ra/Rg = 1.9 at 10 ppb) with high selectivity is developed. Experimental and molecular dynamic studies reveal that the Cu single atoms (SAs) act as active sites, promoting the oxidation of toluene and increasing the affinity of Cu single‐atom catalysts (SACs)‐containing sensing materials for toluene while weakening the association with carbon dioxide or water vapor. Density functional theory studies show that the selective binding of toluene to Cu SAs is due to the favorable binding sites provided by Cu SAs for toluene molecules over other gaseous species, which aids the adsorption of toluene on WO2.72 nanowires. This study demonstrates the successful atomic‐level interface regulation engineering of WO2.72 nanowire‐supported Cu SAs, providing a potential strategy for the development of highly active and durable CGSs

In Situ Structural Observation of a Substrate- and Peroxide-Bound High-Spin Ferric-Hydroperoxo Intermediate in the P450 Enzyme CYP121

The P450 enzyme CYP121 from Mycobacterium tuberculosis catalyzes a carbon-carbon (C-C) bond coupling cyclization of the dityrosine substrate containing a diketopiperazine ring, cyclo(l-tyrosine-l-tyrosine) (cYY). An unusual high-spin (S = 5/2) ferric intermediate maximizes its population in less than 5 ms in the rapid freeze-quenching study of CYP121 during the shunt reaction with peracetic acid or hydrogen peroxide in acetic acid solution. We show that this intermediate can also be observed in the crystalline state by EPR spectroscopy. By developing an on-demand-rapid-mixing method for time-resolved serial femtosecond crystallography with X-ray free-electron laser (tr-SFX-XFEL) technology covering the millisecond time domain and without freezing, we structurally monitored the reaction in situ at room temperature. After a 200 ms peracetic acid reaction with the cocrystallized enzyme-substrate microcrystal slurry, a ferric-hydroperoxo intermediate is observed, and its structure is determined at 1.85 Å resolution. The structure shows a hydroperoxyl ligand between the heme and the native substrate, cYY. The oxygen atoms of the hydroperoxo are 2.5 and 3.2 Å from the iron ion. The end-on binding ligand adopts a near-side-on geometry and is weakly associated with the iron ion, causing the unusual high-spin state. This compound 0 intermediate, spectroscopically and structurally observed during the catalytic shunt pathway, reveals a unique binding mode that deviates from the end-on compound 0 intermediates in other heme enzymes. The hydroperoxyl ligand is only 2.9 Å from the bound cYY, suggesting an active oxidant role of the intermediate for direct substrate oxidation in the nonhydroxylation C-C bond coupling chemistry