Solution NMR Structure of Titin N2A Region Ig Domain I83 and Its Interaction with Metal Ions

Abstract

Titin, the largest single chain protein known so far, has long been known to play a critical role in passive muscle function but recent studies have highlighted titin's role in active muscle function. One of the key elements in this role is the Ca²⁺-dependent interaction between titin's N2A region and the thin filament. An important element in this interaction is I83, the terminal immunoglobulin domain in the N2A region. There is limited structural information about this domain, but experimental evidence suggests that it plays a critical role in the N2A-actin binding interaction. We now report the solution NMR structure of I83 and characterize its dynamics and metal binding properties in detail. Its structure shows interesting relationships to other I-band Ig domains. Metal binding and dynamics data point towards the way the domain is evolutionarily optimized to interact with neighbouring domains. We also identify a calcium binding site on the N-terminal side of I83, which is expected to impact the interdomain interaction with the I82 domain. Together these results provide a first step towards a better understanding of the physiological effects associated with deletion of most of the I83 domain, as occurs in the mdm mouse model, as well as for future investigations of the N2A region.

Keywords: cardiomyopathy; contraction; muscle; protein dynamics; protein stability.

Figure 1.

Overview of I-band titin layout, isoforms and phosphorylation sites. Red boxes: Ig domains; blue boxes: unique, non-repetitive sequences; yellow boxes: PEVK region; pink spheres: phosphorylation sites. All three isoforms of I-band titin in skeletal and cardiac muscle are shown. As the isoform name suggests, the N2A region is found in the N2A and the N2BA isoforms. The N2A region is shown in more detail with individual domains labelled at the bottom. The green bar below indicates the N2A construct used in previous work to investigate I-band titin binding to thin filaments. The Purple bar indicates the portion of N2A deleted in the mdm mouse.

Figure 2.. The structure of I83 is well-defined with unique features.

(A) Multiple superposition of the family of 20 structures. Only the backbone atoms (N, HN, Ca, C, O) are shown. Atoms are coloured in rainbow mode following the polypeptide chain from blue at the N terminus to red at the C terminus. (B) Cartoon view of the best member of the structure family in the same orientation and colours as in A. The positions of the β-strands are indicated. (C) The hydrophobic core and key conserved residues. All non-hydrogen atoms are shown as sticks. Aromatic side chains are coloured in yellow, aliphatic side chains are coloured in green, negatively charged side chains are coloured in red, positively charged side chains are coloured in blue, hydrophilic side chains are coloured in magenta, special residues (Gly, Cys) are coloured in orange, backbone atoms are coloured in grey. Selected residues in the hydrophobic core are labelled. Both figures created with PyMOL.

Figure 3.. Comparison of I83 to similar IgI domains.

(A) Sequence alignment of I83 to a selected set of 26 IgI domains obtained either from structure similarity or sequence similarity searches. Note that the four IgI domains of the N2A region are at the top in inverse order. Conservation is indicated by colours of different intensity based on the level of conservation: strong colour = high level of conservation; faint colour = low level of conservation; threshold for colouring is 25% similarity. Green: hydrophobic; Magenta: polar; Grey: glycine; Yellow: cysteine; Blue: positively charged. Residue numbering is given for I83. The position of β-strands and longer loops is indicated by grey bars and lines. The three negatively charged residues in the N-terminal calcium binding site are indicated by red stars. The unsual P87 is indicated by a blue bar. (B) Superposition of similar structures identified by DALI on the structure of I83. Blue: Ti-I83 ; Red: Tw-Ig26 (3UTO); Green: Ti-I27 (1WAA); Magenta: Ti-I1 (1G1C); Cyan: Ti-M4 (3QP3); Orange: Ti-A168 (2J8H); Pink: Ti-M10 (2WWK); Light Blue: Ti-I1 (2A38); Hot-Pink: Ti-I9 (5JDD); Pale blue: Ti-I11 (5JDE); Yellow: Ti-M7 (3PUC); Gray: Ti-I10 (5JDJ); Dark green: Ti-I66 (3B43); White: Tlk (1FHG); Purple: APEP (1U2H); Pale green: Ti-81 (5JOE). Selected amino acids in I83 are labelled and β-strands are shown with labels in boxes. The focus is on the FG-loop on the left and on the BC-loop on the right. Structures were superposed with the matchmaker function in UCSF Chimera and the figure was created with PyMOL.

Figure 4.. Metal binding of I83.

(A) Plot of calcium induced chemical shift perturbations (CSP) against the sequence. Thresholds for mapping on the structure are indicated by red lines. Stars indicate residues for which no data could be collected in this experiment. (B) Mapping of calcium induced CSPs on the structure of I83. Cβ atoms are shown for all residues as vdW spheres. Residues coloured in orange are between 2 and 4 sigma, residues coloured in red are >4 sigma, residues coloured in pale cyan are <2 sigma, residues coloured in grey have no values. Figure created with PyMOL. (C) NMR Titration curves for two selected residues for calcium and magnesium titrations. (D) CD titration curves over a broad concentration range of calcium. Curves are shown for wt I83 (squares) and the binding site mutant of I83 (crosses).

Figure 5.. ¹⁵N relaxation data for I83.

(A) NMR dynamics data with top to bottom ¹H-¹⁵N heteronuclear NOE, ¹⁵N T₁. and ¹⁵N T₂. Secondary structure elements are indicated by black boxes. (B) Dynamics mapped on the structure of I83 using UCSF Chimera. Heteronuclear NOE values are mapped on the top, colour coded as Blue: 0.8, Yellow: 0.4, Red: 0.1. On the bottom the T2 values are mapped on the structure, colour coded as Blue: 100 ms, Yellow: 55 ms, Red: 35 ms.

Figure 6.. Denaturation curves for I83 WT and I83 with substitutions yield similar unfolding curves.

(A) Overlapping unfolding curves from thermal melts for I83 WT (●), D36N (∎), D37N (◆), E86Q (▲), and Ca-site (x) show similar unfolding despite substitutions. E86 demonstrated a slight decrease in stability, unfolding at lower temperatures with a more gradual transition from the folded to the unfolded state. (B) Unfolding curves from urea denaturation for I83 WT (●), D36N (∎), D37N (◆), E86Q (▲), and Ca-site (x) show similar pattern of unfolding, though E86Q exhibits a slight decrease in stability.

Figure 7.. Ca-site mutant I83 is not stabilized at pCa4.3 during chemical denaturation.

The fraction folded as determined by chemical denaturation and measured by tryptophan fluorescence shows a calcium-dependent stabilization for I83 WT. (A) I83 WT exhibits minor stabilization at pCa 6 (open squares) as compared to a Ca-free environment (open circles) by a small increase in the fraction folded at [urea] < 3.5 M. Stabilization at pCa 4.3 (filled circles) is similar to the stabilization at pCa 2 (filled squares), with a larger fraction folded at [urea] ≤ 4 M than a Ca-free environment. An arrow indicates the impact of calcium on I83 WT. (B) The calcium-site mutant demonstrates very small changes to the fraction folded at different calcium concentrations.