Fig 1.
KAHRP associates with spectrin.
(A,B) Schematic representations of KAHRP (A) and the spectrin α and β chains (B). KAHRP and spectrin fragments used in this study are denoted. The spectrin hetero-dimerization region is indicated in blue and the tetramerization region in red. (C) FP binding assay of KAHRP showing polarization differences (mP) upon incubation of fluoresceine-labeled K2 or K3 fragments with 50 μM of unlabeled spectrin constructs. Higher polarization differences correspond to slower tumbling rates of labeled KAHRP due to complex formation. The dotted line indicates polarization differences upon incubation with glutathione-S-transferase (GST, negative control). (D) FP titration of labeled K2 with unlabeled β10–14 and α12–16. Solid lines are fits of single site binding models; FP values were normalized to predicted maxima from the fits. The calculated interaction affinities (Kd) are shown. Panels C and D show representative data from two independent experiments, while error bars indicate one standard deviation derived from four technical repeats.
Table 1.
List of recombinant protein constructs.
Fig 2.
NMR-monitored titrations of KAHRP with spectrin.
(A,B) Overlays of regions from 13C-HSQC NMR spectra of 50 μM of 13C-labeled KAHRP K2 alone (red) and in the presence (blue) of 150 μM unlabeled spectrin β10–14 (A) or α12–16 (B). Spectra were recorded at 10°C. Panels i and ii in the middle present magnifications of areas from the panels A and B, respectively. (C) The relative intensity of all discrete NMR peaks corresponding to Cα atoms of KAHRP K2 in the presence of β10–14 (top) or α12–16 (bottom), derived from panels A and B, respectively. Reduction of peak intensities suggests association between proteins. Error bars correspond to one standard deviation and derive from the signal / noise ratio of spectra.
Fig 3.
Electrostatic docking of KAHRP and spectrin.
(A) Schematic representation of KAHRP 5´ repeat conformations upon binding to β10–14. The spectrin fragment is shown in cartoon representation, and its N- and C-terminus are denoted. The red KAHRP conformation (path, top panel) achieved the lowest (most favorable) docking energy (score). An overlay of the five lowest energy KAHRP paths is shown at the bottom panel. (B) Histogram of the docking (charge complementation) score calculated for all putative KAHRP 5´ repeat paths along β10–14. Filled bars correspond to docking scores calculated for antiparallel orientations of KAHRP with spectrin; a black line denotes docking scores from parallel orientations. (C) Graph of the electrostatic potential of the spectrin β10–14 surface along the length of the lowest scoring path (kT/e, green), overlaid with the formal charge of the KAHRP 5´ repeat (orange). The relative KAHRP and spectrin positions along the docking path are denoted using a distance calculated from the β10–14 N-terminus.
Fig 4.
(A) Schematic representation of PfEMP1 and its intracellular ATS domain. The ATS fragments are denoted; the ATS-Core is composed of two discontinuous sections that fold into a single globular unit. (B) Details from NMR 15N-HSQC spectra (S9 Fig) of 100 μM 15N-labeled ATS fragments in the absence (red) and presence (blue) of equimolar unlabeled α17-C. (C) 1D traces of NMR spectra showing ATS-Core peaks alone (black) or in the presence (red) of α17-C sub-fragments. Traces derive from 15N-HSQC spectra of 100 μM 15N-labeled ATS-NCore with two-fold excess of unlabeled spectrin sub-fragments. The ATS atoms giving rise to these NMR peaks are indicated. Reduction of peak intensities suggests association between proteins.
Fig 5.
Mapping the ATS-Core–spectrin α17 binding on the domain structures.
(A) Detail from NMR 15N-HSQC spectra of 50 μM 15N-labeled ATS-Core PFF0845c alone (red) or with two-fold excess of unlabeled α17 (blue). Spectra were recorded at 300 mM NaCl and 40°C. (B) Combined perturbations of 1H, 15N and 13C´ NMR peak positions upon ATS-Core–spectrin α17 binding under the conditions of (A), as function of ATS-Core PFF0845c (blue) and spectrin α17 (green) amino acids. Combined perturbations of 1H and 15N NMR peak positions for α17 upon ATS-Core binding at 50 mM NaCl and 25°C are also shown (grey); NMR peak perturbations are broadly similar under both experimental conditions. Mean perturbations in NMR peak positions are shown as dashed lines. The position at which PfEMP1 segments are joined to form the ATS-Core is indicated by a dashed line in the top graph. (C,D) Visualization of NMR peak position perturbations upon ATS-Core–α17 binding on (C) the ATS-Core and (D) the α17 structure. The color gradient indicates the magnitude of perturbations observed; thus, it is proportional to structural changes upon complex formation allowing the visualization of the direct interaction interface. The ATS-Core PFF0845c structure was derived by homology modeling using the ATS-Core PF08_0141 structure [31] as template. The two constructs feature 70% sequence identity and 92% sequence similarity. The α17 structure derives from a 1.54 Å resolution crystallographic model of spectrin repeats α16–17 (See S10 Fig and Table 2 for an analysis of α16–17 structure).
Fig 6.
Model of the ATS-Core–α17 complex.
(A) Overview of the ATS-Core–α17 complex following refinement by MD simulations. The protein N- and C-termini, and interfacial residues are indicated. Right panels correspond to hydrophobic (i) and electrostatic and hydrogen bonding (ii) features observed in the complex structure. Panel ii is rotated 180° compared to panel i. (B) NMR-monitored titrations of 50 μM 15N-labeled α17 with unlabeled ATS-Core PFF0845c variants that carry amino acid substitutions at the binding interface. Experiments were performed at 50 mM NaCl and 25°C. Each data point represents the average fractional perturbation in the position of the eight most affected α17 NMR peaks. Error bars indicate one standard deviation and derive from variances in fractional changes across the eight resonances. Solid lines represent fits to single site binding models. Interaction affinities are indicated. (C) FP titrations of fluoresceine-labeled ATS PFF0845c with α17 variants carrying amino acid substitutions at the binding interface. Error bars indicate one standard deviation and derive from three replicates each with four technical repeats. Solid lines represent fits to single site binding models. (D) Consensus sequence of ATS-Core domains from PfEMP1 variants in the P. falciparum 3D7 strain. Red asterisks above residues indicate ATS-Core amino acids whose substitution affects α17 binding affinity. Residue numbers correspond to the sequence of the ATS-Core structured domain after removal of loop insertions [31].
Fig 7.
Model of knob formation structure.
Panel (A) depicts an initial step in knob assembly when PfEMP1 and KAHRP have localized close to the junctional and ankyrin complexes, respectively. PHIST proteins, such as PFE1605w [38, 39, 44], may further connect PfEMP1 and the cytoskeleton. Panel (B) depicts a model of the final knob structure, characterized by high protein density as a result of KAHRP self-association [23] and the formation of spiral assemblies [40].
Table 2.
Crystallographic data and refinement statistics for α16–17.