Dissecting the molecular assembly of the Toxoplasma gondii MyoA motility complex

Abstract

Apicomplexan parasites such as Toxoplasma gondii rely on a unique form of locomotion known as gliding motility. Generating the mechanical forces to support motility are divergent class XIV myosins (MyoA) coordinated by accessory proteins known as light chains. Although the importance of the MyoA-light chain complex is well-established, the detailed mechanisms governing its assembly and regulation are relatively unknown. To establish a molecular blueprint of this dynamic complex, we first mapped the adjacent binding sites of light chains MLC1 and ELC1 on the MyoA neck (residues 775-818) using a combination of hydrogen-deuterium exchange mass spectrometry and isothermal titration calorimetry. We then determined the 1.85 Å resolution crystal structure of MLC1 in complex with its cognate MyoA peptide. Structural analysis revealed a bilobed architecture with MLC1 clamping tightly around the helical MyoA peptide, consistent with the stable 10 nm K_d measured by isothermal titration calorimetry. We next showed that coordination of calcium by an EF-hand in ELC1 and prebinding of MLC1 to the MyoA neck enhanced the affinity of ELC1 for the MyoA neck 7- and 8-fold, respectively. When combined, these factors enhanced ELC1 binding 49-fold (to a K_d of 12 nm). Using the full-length MyoA motor (residues 1-831), we then showed that, in addition to coordinating the neck region, ELC1 appears to engage the MyoA converter subdomain, which couples the motor domain to the neck. These data support an assembly model where staged binding events cooperate to yield high-affinity complexes that are able to maximize force transduction.

Keywords: Toxoplasma gondii; X-ray crystallography; cell motility; crystal structure; host cell invasion; isothermal titration calorimetry (ITC); myosin; protein structure.

Figure 1.

Leading model describing the general architecture of MyoA (motor domain, light purple circle; converter domain, dark purple oval), with ELC1 (orange) and MLC1 (teal) bound to the neck region (dark purple cylinder), and accessory proteins comprising the glideosome macromolecular complex.

Figure 2.

MLC1-binding region on MyoA neck analyzed by HDX, ITC, and X-ray crystallography reveals several key interactions crucial to complex formation. a, HDX heat maps showing difference in percentage of backbone amide deuterium incorporation of MyoA neck induced by binding of light chains at two time points of exchange. Putative directly ordered regions are marked with solid lines, and putative indirectly ordered regions are marked with dashed lines. Sequence of the MyoA peptide is shown. Red coloring of terminal residues indicates N-terminal acetylation or C-terminal amidation. Refer to supplemental Table S1 for the full HDX-MS data set relating to this figure. b, representative ITC binding isotherms following the titration of MLC1 into a solution of MyoA (775–818) or MyoA (801–818). c, left panel, 1.85 Å resolution crystal structure of MLC1 in complex with MyoA (801–831) with close-ups of the apical salt bridge (inset 1) formed between MLC1 Asp¹⁴⁵ and MyoA Arg⁸⁰⁸ and MLC1 “clamp” (inset 2) formed via two hydrogen bonds between Glu¹⁷⁹ and backbone amides of Tyr¹¹⁴ and Ala¹¹⁵. Right panel, electrostatic surface maps of MLC1 and the MyoA neck peptide. In this view, the MyoA neck peptide has been extracted from the surrounding MLC1 and rotated to reveal the basic surface along its helical longitudinal axis.

Figure 3.

ELC1 binds calcium using a classical EF-hand motif and interacts with MLC1, increasing its affinity for MyoA. a, putative EF-hands 1 and 2 of ELC1, based on consensus sequences. Ca²⁺-interacting residues are numbered, and mutated aspartates are boxed in red. b, bar graphs of representative peptides from ELC1 WT and mutants, showing changes in backbone deuterium exchange induced by Ca²⁺ binding. Sequences of peptides are shown above corresponding graphs. The data are from a single time point of exchange (300 s at 23 °C). The error bars (S.D.) are from independent triplicate experiments. Refer to supplemental Table S2 for the full HDX-MS data set relating to this figure. c, representative ITC binding isotherms of CaCl₂ titrated into ELC1 wild-type (left panel), ELC1 D15A (middle panel), or ELC1 D80A (right panel). d, representative ITC binding isotherms of ELC1 titrated into MyoA (775–818) without (left panel) and with (right panel) calcium. e, representative ITC binding isotherms ELC1 titrated into MyoA (775–818), prebound with MLC1 without (left panel) or with (right panel) calcium.

Figure 4.

ELC1 interacts with the MyoA converter subdomain, increasing its affinity for the MyoA neck and stabilizing the myosin lever arm. Schematics shown are colored as previously. a, representative ITC binding isotherm of ELC1 titrated into MyoA (1–831) prebound with MLC1. b, HDX difference map showing reduction in total deuteron incorporation of full-length MyoA, prebound with MLC1, when ELC1 is added. The decreases in amide exchange between conditions are summed over three time points of HDX (3, 30, and 300 s at 23 °C), and the error bars (S.D.) represent independent triplicate. Peptide # corresponds to the centroid amino acid of the peptide from which a data point is obtained. Inset, expanded view of HDX difference map showing order induced in MyoA converter domain (residues ∼710–740) by ELC1 binding. c, representative deuterium incorporation time course for MyoA peptide in the converter subdomain (716–720). Refer to supplemental Table S3 for the full HDX-MS data set relating to this figure.