The closed MTIP-myosin A-tail complex from the malaria parasite invasion machinery

Abstract

The Myosin A-tail interacting protein (MTIP) of the malaria parasite links the actomyosin motor of the host cell invasion machinery to its inner membrane complex. We report here that at neutral pH Plasmodium falciparum MTIP in complex with Myosin A adopts a compact conformation, with its two domains completely surrounding the Myosin A-tail helix, dramatically different from previously observed extended MTIP structures. Crystallographic and mutagenesis studies show that H810 and K813 of Myosin A are key players in the formation of the compact MTIP:Myosin A complex. Only the unprotonated state of Myosin A-H810 is compatible with the compact complex. Most surprisingly, every side-chain atom of Myosin A-K813 is engaged in contacts with MTIP. While this side-chain was previously considered to prevent a compact conformation of MTIP with Myosin A, it actually appears to be essential for the formation of the compact complex. The hydrophobic pockets and adaptability seen in the available series of MTIP structures bodes well for the discovery of inhibitors of cell invasion by malaria parasites.

Fig. 1. Sequence alignments of P. falciparum, P. vivax, P. knowlesi and P. yoelii MTIP and MyoA

A) Secondary structure elements of the PfMTIP structure are depicted in the top rows of the alignment and the corresponding secondary structure elements of PkMTIP are below each row. The region “α4-hinge-α5 of PfMTIP” is depicted as a bar colored according to the domains involved as shown in Fig. 3A. This region corresponds to the continuous “central helix” of PkMTIP indicated with a bar in shades of blue. Conserved residues are in light red, completely conserved residues in dark red. The 29 crucial residues of PfMTIP for interaction with MyoA-tail are indicated with red asterisks above the PfMTIP sequence; the 17 previously identified residues of PkMTIP contacting MyoA-tail are show in blue asterisks below the sequence. The P. yoelii MTIP sequence is added to facilitate the description of the Yeast Two-hybrid studies. B) MyoA-tail sequences in plasmodial pathogenic protozoa, Toxoplasma gondii and Cryptosporidium hominis. The canonical IQ motif is shown above the alignment, with the critical 7^th position highlighted in green. Residues involved in interactions with PfMTIP are indicated with black asterisks above the alignment.

Figure 2. Stereo views of the P. yoelii MyoA-tail

A) (2F_o-F_c) σA-weighted omit map after final refinement contoured at the 1σ level. All residues of the MyoA-tail used for co-crystallization are well defined. B) Stereo diagram of the P. yoelii MyoA-tail helices in the PfMTIP:MyoA complex, in cyan, and in the PkMTIP:MyoA complex, in grey, after superposition of the “framework” Y158 to D194 of α6-loop-α7 in the C-domains. Only residues of the MyoA-tail bound to PfMTIP are labeled. Highlighted, with shifts in Å, are crucial conformational changes in the MyoA-tail side chains upon binding to PkMTIP at pH 5.3 or PfMTIP pH 7.5.

Fig. 3. Structural overview of the MyoA-tail bound to Plasmodium MTIP

A) The dramatic conformation difference of MTIP observed in the complexes with the MyoA-tail of P. falciparum MTIP and P. knowlesi MTIP. View along the MyoA-tail helix axis in red, after superposition of the “framework” Y158 to D194 of α6-loop-α7 in the C-domains of PfMTIP and PkMTIP. PkMTIP is kept in blue shades, where the N-terminal domain is in lighter colors connected via the hinge region to the C-terminal domain in darker colors. The PfMTIP N-terminal domain is shown in yellow, the hinge region in sage and the C-terminal domain in orange. The center of mass of the N-terminal domain moves by ~24 Å, while rotating by ~140° when comparing the extended and compact complexes. B) Stereo diagrams depicting the surface of the N-terminal domain (yellow; upper) and the C-terminal domain (orange; lower) of PfMTIP with the bound MyoA-tail helix as sticks. MTIP hinge region atoms are shown as sage. Regions of PfMTIP within 4 Å of the MyoA-helix are colored with hydrophobic atoms in green, oxygens in red and nitrogens in blue.

Fig. 4. Intra-MTIP contacts stabilizing the closure of P. falciparum MTIP in the compact PfMTIP:MyoA complex

A) View along the MyoA-tail helix (red) bound between the N-terminal (yellow) and C-terminal (orange) domain of PfMTIP. Some important contacting residues are highlighted as green sticks. The hinge region stretching from C134 to N140 is colored in sage. B) Contacts between residues from the N-terminal and C-terminal domain of PfMTIP stabilizing the “clamp” of the N- and C-domains gripping the MyoA-tail. C) Salt bridge between hinge region residue D137 and α0 residue K71, thereby stabilizing hydrophobic contacts between helix α0 from the N-terminal domain and the hinged region in the current PfMTIP:MyoA complex. Distances are shown in Å.

Fig. 5. Critical contacts of MyoA with Plasmodium falciparum MTIP

A) The hydrophilic network involving MyoA-His810. The MyoA-tail is shown as stick representation in grey, contacting residues of PfMTIP are colored according to their location, N-terminal residues are in yellow, hinge region residues in sage, and C-terminal residues in orange. MyoA-tail residues are labeled in red, PfMTIP residues in black, distances shown are in Å. B) Each and every side chain atom of MyoA-K813 is interacting with PfMTIP residues in addition to a key interaction with unprotonated MyoA-H810. Stereodiagram depicting hydrophobic (magenta) and hydrophilic (black) interactions of the crucial residue MyoA-K813. Part of the MyoA-tail (grey) is shown with its interacting partners of the N-terminal domain (yellow) and C-terminal domain (orange). MyoA-tail residues are labeled in red and PfMTIP residues in black. For clarity distances were omitted.

Fig. 6. MyoA-MTIP interaction studies using the yeast two-hybrid system

Mutagenesis of key residues of the P. yoelii MyoA-tail or P. yoelii MTIP protein eliminate or reduce the interaction between these molecules, supporting the structural results. Yeast cells were transformed with either wildtype or mutant MyoA fused to the Gal4 DNA binding domain or either wildtype or mutant MTIP fused to the Gal4 activation domain. Values are represented as % wildtype activity. Experimental procedures are described in the materials and methods section.