Regulation of the Plasmodium motor complex: phosphorylation of myosin A tail-interacting protein (MTIP) loosens its grip on MyoA

Abstract

The interaction between the C-terminal tail of myosin A (MyoA) and its light chain, myosin A tail domain interacting protein (MTIP), is an essential feature of the conserved molecular machinery required for gliding motility and cell invasion by apicomplexan parasites. Recent data indicate that MTIP Ser-107 and/or Ser-108 are targeted for intracellular phosphorylation. Using an optimized MyoA tail peptide to reconstitute the complex, we show that this region of MTIP is an interaction hotspot using x-ray crystallography and NMR, and S107E and S108E mutants were generated to mimic the effect of phosphorylation. NMR relaxation experiments and other biophysical measurements indicate that the S108E mutation serves to break the tight clamp around the MyoA tail, whereas S107E has a smaller but measurable impact. These data are consistent with physical interactions observed between recombinant MTIP and native MyoA from Plasmodium falciparum lysates. Taken together these data support the notion that the conserved interactions between MTIP and MyoA may be specifically modulated by this post-translational modification.

FIGURE 1.

Thermodynamics of MTIP-MyoA analyzed by ITC, DSF, and parasite pulldown experiments. a, shown are binding isotherms after the titration of PfMyoA tail 799–818 into a solution of MTIP (left) and MTIP S108E (center). Thermodynamic parameters fitted using Origin software (Microcal) are described in the table below. Other ITC data are shown in supplemental Fig. S1. Titrations were carried out at 303 K in 20 mm HEPES (pH 7.5), 50 mm NaCl, and 1 mm TCEP. b, DSF thermograms for MTIP and MTIP mutants in free and bound forms using MyoA peptides 799–818 (799) and 803–818 (803) (right). Analysis of transition slopes is presented in supplemental Fig. S1. Changes in melting temperature (ΔT_m) between free and bound forms of each protein are described in the table below. RFU, relative fluorescence units. c, shown is a Western blot analysis of MyoA pulldown assays from P. falciparum schizont lysate using a range of MTIP concentrations for the wild type protein and the phosphomimetic mutant S108E. MyoA pulldown data for S108E are shown in supplemental Fig. S1. Lysate, 6 μg of schizont lysate; represents 2% of the amount of lysate used in each sample. −C, reaction to which no recombinant MTIP was added. The histogram shows the amounts of MyoA pulled out by 1 μg of MTIP WT or the S108E mutant. Data are from the average of three independent experiments; error bars are the mean ± S.D.

FIGURE 2.

Structure and NMR-based interaction hotspots of MTIP/PfMyoA-(799–818). a, shown is the crystal structure of MTIP (surface representation; white) in complex with PfMyoA-(799–818) (schematic representation; black), highlighting hotspots of peptide binding (red) as derived from chemical shift differences in the ¹H,¹⁵N HSQC spectra of the free and bound forms of MTIP. b, shown are ¹H,⁵N HSQC spectra of 400 μm uniformly ¹³C,¹⁵N-labeled MTIP recorded at neutral pH and 303 K in the absence (black) and presence (red) of 3 mol eq of PfMyoA-(799–818). The backbone amides are almost completely assigned in both cases; some of these assignments are shown for the bound form.

FIGURE 3.

NMR analysis of the MTIP S108E mutant. a, shown are overlays of ¹H,¹⁵N HSQC spectra of MTIP (black and red) and MTIP S108E (green and blue) in their free state (left) and in complex with PfMyoA-(799–818) (center). b, a zoomed-in section of the titration of the MyoA tail into MTIP S108E shows some amide resonances in fast exchange (Ser-162 and Gly-119) and some in slow exchange (Thr-160 and Thr-176) with the bound state on the NMR chemical shift timescale. The overlaid spectra represent 0 (black), 0.2 (red), 0.4 (green) 0.8 (yellow), 1.3 (pink), and 3.0 (cyan) mol eq of PfMyoA-(799–818); the protein concentration was 100 μm. Those in the fast exchange regime were used to determine dissociation constants for the binding equilibrium; four curves are shown. δΔ represents the change in chemical shifts for the selected amide atoms.

FIGURE 4.

¹⁵N CPMG relaxation dispersion NMR of free MTIP. a, shown are relaxation dispersion trajectories (R₂^eff versus CPMG refocusing pulse frequency) for residues from different parts of MTIP in its free state. The unstructured part of the N terminus (represented by Asp-64 and Gln-66) is highly dynamic on the ns to ps timescale but shows negligible dynamics on the slower timescales probed by the relaxation dispersion experiments, whereas the linker (represented by Asp-139 and Asn-140) and C-terminal helix α8 (represented by Cys-199 and Ile-202) appear to be undergoing exchange processes. Experimental data were obtained at neutral pH at static field strengths of 14.1 T (¹⁵N frequency of 60.77 MHz, blue) and 18.8 T (¹⁵N frequency of 81.16 MHz, red), and for those residues that exhibited exchange dynamics, data were fitted globally to models for two site exchange. b, R_ex values from experiments recorded at 18.8 T are mapped onto the crystal structure of MTIP (schematic representation; white) in complex with PfMyoA-(799–818) (not included) to reveal regions of elevated ms timescale dynamics in free MTIP (white to orange gradient). Where data are missing due to overlap or excessive line broadening, residues are colored in blue.

FIGURE 5.

Relaxation data for MTIP, MTIP S108E, and their complexes with the MyoA tail. Shown is variation of backbone ¹⁵N T₁ and T₂ relaxation times and ¹H,¹⁵N heteronuclear NOEs for wild type MTIP bound to PfMyoA-(799–818) and in the free state (left) and (right) followed by data for MTIP S108E. Below each relaxation dataset, schematic models represent the effect of amino acid substitutions and ligand binding on the orientation of MTIP domains. The average values of T₁/T₂ are given for each system; these are reported as the mean ± S.D., removing values for residues where T₂ < 50 ms or NOE < 0.6. These residues were judged to be undergoing motions separate from the overall tumbling of the MTIP molecules.