Structure of the Single-lobe Myosin Light Chain C in Complex with the Light Chain-binding Domains of Myosin-1C Provides Insights into Divergent IQ Motif Recognition

Abstract

Myosin light chains are key regulators of class 1 myosins and typically comprise two domains, with calmodulin being the archetypal example. They bind IQ motifs within the myosin neck region and amplify conformational changes in the motor domain. A single lobe light chain, myosin light chain C (MlcC), was recently identified and shown to specifically bind to two sequentially divergent IQ motifs of the Dictyostelium myosin-1C. To provide a molecular basis of this interaction, the structures of apo-MlcC and a 2:1 MlcC·myosin-1C neck complex were determined. The two non-functional EF-hand motifs of MlcC pack together to form a globular four-helix bundle that opens up to expose a central hydrophobic groove, which interacts with the N-terminal portion of the divergent IQ1 and IQ2 motifs. The N- and C-terminal regions of MlcC make critical contacts that contribute to its specific interactions with the myosin-1C divergent IQ motifs, which are contacts that deviate from the traditional mode of calmodulin-IQ recognition.

Keywords: EF-hand; IQ motif; light chain; myosin; nuclear magnetic resonance (NMR); protein motif; protein structure; protein-protein interaction; structural biology; x-ray crystallography.

FIGURE 1.

NMR structure of MlcC. A, backbone superposition (N, C^α, and C′ atoms) of the 20 lowest-energy NMR-derived structures of the ordered core region (Lys-7–Leu-72) of MlcC. B, backbone ribbon representation of the energy-minimized average NMR solution structure of MlcC, with the first EF-hand, inter-EF-hand linker and second EF-hand colored light blue, blue, and dark blue, respectively. The four α-helices are labeled A to D, and the two non-functional Ca²⁺-binding loops are labeled L1 and L2, and the N and C termini are labeled accordingly. C, plot of {¹H}-¹⁵N NOE values as a function of residue number for MlcC at 14.1 tesla. D, amino acid sequence of MlcC colored as in B, with the disordered N- and C-terminal segments colored red. The underlined residues indicate the +12 position of the Ca²⁺-binding loop, which in functional EF-hands is normally glutamic acid. E, electrostatic surface potential representation of MlcC with red and blue representing negatively and positively charged regions, respectively. The image is oriented to show the negatively charged surface corresponding to the linker and C helix. F, overlay of the NMR solution structures of MlcC (dark blue) and the C-lobe of apo-CaM (cyan) (PDB 1DMO).

FIGURE 2.

NMR-based identification of MlcC residues affected by the myosin-1C IQ2 motif. A, plot of normalized backbone amide chemical shift changes (Δδ) along the MlcC sequence induced by addition of the GB1-M1C-IQ2 fusion protein. The normalized chemical shift changes were calculated using the formula: Δδ = ((0.17 Δδ_N)² + (Δδ_HN)²)^0.5. Red bars identify residues displaying backbone chemical shift changes exceeding the mean chemical shift change, represented by the horizontal line, by greater than 0.5 S.D. Blue bars identify those residues exhibiting backbone amide chemical shift changes greater than the average chemical shift change but less than 0.5 S.D. B, backbone amide chemical shift changes that exceed the mean chemical shift by greater than 0.5 S.D. are mapped onto a backbone ribbon representation of MlcC.

FIGURE 3.

Conformational analysis of recombinant myosin-1C IQ1, IQ2, and IQ1.IQ2 peptides. Far-UV circular dichroism spectra were measured for 24 μm myosin-1C-IQ1 (- - -), 70 μm myosin-1C-IQ2 (–·–·–), and 45 μm myosin-1C-IQ1.2 (—) in 15 mm Tris-HCl, pH 7.5, 50 mm NaF, 2 mm β-mercaptoethanol.

FIGURE 4.

MlcC forms a 2:1 complex with M1C-IQ1.2. A, Superdex 75 size exclusion chromatographic profile of the complex formed by MlcC and the GB1-M1C-IQ1.2 fusion protein. Three peaks were observed (left panel) and were analyzed by SDS-PAGE (right panel). Peak 1 represents aggregated material that eluted in the void volume; peak 2 represents the complex of MlcC with GB1-M1C-IQ1.2; and peak 3 represents free MlcC. B, ITC analysis of the binding of MlcC (black) and apo-CaM (blue) to M1C-IQ1.2. The binding isotherms show the total heat per injection (kcal/mol of ligand injected) plotted against the molar ratio of light chain to M1C-IQ1.2. No binding interaction was detected for apo-CaM. The solid red line represents the best fit to the MlcC data using a two-site model. The K_d values calculated from the two-site model are shown. ITC experiments were performed as described under “Experimental Procedures.” The data shown are representative of three independent experiments.

FIGURE 5.

X-ray crystal structure of the 2:1 MlcC·M1C-IQ1.2 complex. A, backbone ribbon representation shows the helical myosin-1C IQ1.2 fragment colored green and the two MlcC molecules colored red, light blue, blue, and dark blue to indicate the disordered N- and C-terminal segments, the first EF-hand, the inter-EF-hand linker, and the second EF-hand, respectively. The side chains of Trp-701, Gln-719, and Arg-737, which are the first residues in the IQ1, IQ2, and IQ3 motifs, and Lys-709 and Lys-727, which replace the conserved glutamine in the IQ1 and IQ2 motifs, are shown as sticks. The N and C termini for the three molecules are labeled accordingly. Note that the two MlcC molecules are rotated by ∼20° around the helix axis, and that the N-terminal segment of MlcC is attached to the IQ1 but not the IQ2 motif. B, ribbon diagram rotated 90° around the x axis relative to the view in A. C, surface view of the complex rotated 180° around the x axis relative to the view in B. The binding of MlcC to the convex side of the M1C-IQ1.2 helix leaves the opposite concave surface largely exposed. Exposed residues of the helical myosin-1C IQ1.2 fragment are labeled accordingly.

FIGURE 6.

IQ motif-bound MlcC adopts a conformation very similar to the C-lobe of apo-CaM. A, overlay of the NMR structure of free MlcC (light blue) and the x-ray crystal structure of the M1C-IQ1 motif-bound MlcC (dark blue). B, overlay of the structures of the IQ-motif bound MlcC and the C-lobe of apo-CaM bound to the first IQ motif in human myosin 1C (PBD 4BYF).

FIGURE 7.

Interaction of MlcC with the myosin-1C IQ1 and IQ2 motifs. A, amino acid sequence alignment of the myosin-1C IQ1 and IQ2 motifs and two murine myosin-V apo-CaM-binding IQ motifs. The core residues of the IQXXXRGXXXR sequence are shown in bold. B, expanded view of MlcC from the complex x-ray crystal structure showing hydrophobic residues in the central groove of MlcC (left panel, blue sticks) and the M1C-IQ1 and M1C-IQ2 motifs (right panels, green sticks) that are involved in binding interactions. C, close-up view of the hydrophobic interactions that anchor the flexible N-terminal region of MlcC to the M1C-IQ1 motif but not the M1C-IQ2 motif. A hydrophobic cluster brings together Met-1 and Trp-73 of MlcC, Phe-718 from the M1C-IQ1 motif, and Val-16 from the M1C-IQ2-bound MlcC. Interactions mediated by His-3 and Ile-4 help to stabilize the interaction between the N-terminal region of the M1C-IQ1 motif and the C terminus of MlcC. The side chain of Tyr-736 in the IQ2 motif is not properly oriented to form a binding site for Met-1. Residues are colored as in Fig. 4.

FIGURE 8.

Interaction of MlcC with conserved lysine residues in the myosin-1C IQ1 and IQ2 motifs. A, close-up view of the binding interactions between Lys-709 and Lys-727 in the M1C-IQ1 and M1C-IQ2 motifs (green) and the inter-EF-hand linker of MlcC (light blue). B, interactions between the Lys-716 and Lys-734 residues in the M1C-IQ1 and M1C-IQ2 motifs (green) and glutamic acid residues in helix D of MlcC (light blue).

FIGURE 9.

Comparison of MlcC and the C-terminal lobe of CaM. A, C-lobe of apo-CaM was modeled onto the IQ1 and IQ2 motifs of MC-IQ1.2 in place of MlcC. The N- and C-lobes of the apo-CaM bound to the M1C-IQ1 motif are colored blue and light blue, respectively, and the N- and C-lobes of the apo-CaM bound to the M1C-IQ2 motif are colored red and light red, respectively. B, alignment of the amino acid sequences of MlcC and the C-lobe of apo-CaM. Colored boxes highlight the residues involved in anchoring the N-terminal region of MlcC to the M1C-IQ1 motif (red), hydrophobic residues in the central groove (gray), the inter-EF-hand linker (white), and the glutamic acid residues in the D helix (blue) of MlcC.